Methods and agents for modulating and controlling GLP-1 / GLP-1 1r

Abstract

To provide a method for treating a GLP-1 / GLP-1 1R related disease such as a carbohydrate metabolism disorder-related disease, a fat metabolism disorder-related disease, or a GLP-1 / GLP-1 1R related nervous system disease, and to provide a medicament.SOLUTION: Provided is a method for treating a disease by regulating GLP-1 / GLP-1 1R, comprising administering an effective amount of GLP-1 to a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of plasminogen in the treatment of GLP-1 / GLP-1R related diseases by regulating GLP-1 / GLP-1R. [Background technology]

[0002] GLP-1 is an endogenous hormone that stimulates insulin secretion and is primarily secreted by intestinal L-cells. Expression of the proglucagon gene in small intestinal L-cells generates proglucagon (PG), which, after processing by prohormone convertase 1 / 3 (PC1 / 3), releases the GLP-1 peptide precursor. Endopeptidase catalyzes the degradation of GLP-1(1-37) into two peptide fragments, and GLP-1(7-37) is then processed by amidase to form GLP-1(7-36)amide. Expression of the glucagon gene in α-cells can also generate PG, but prohormone convertase 2 (PC2) in α-cells preferentially converts PG to glucagon, preventing α-cells from synthesizing GLP-1 under normal conditions. However, under stress or pathophysiological conditions (e.g., type 2 diabetes), α-cells can adaptively produce GLP-1.

[0003] GLP-1 promotes insulin secretion and blood glucose normalization in pancreatic islet β cells without causing hypoglycemia, inhibits glucagon production in α cells, delays gastric emptying, suppresses appetite, reduces body weight, promotes β cell proliferation, and inhibits apoptosis, thus playing an important role in regulating pancreatic islet cell function. [1] .

[0004] Diabetes tends to develop in parallel with obesity, and it is clinically accepted that a patient's weight should be included in the treatment guidelines for type 2 diabetes. Research has found that there is a negative feedback regulation between GLP-1 and insulin secretion during the progression of diabetes, and that type 2 diabetes patients have damage to the enteroinsular axis, which is accompanied by an increase in circulating lipids after meals. [2] In experimental diabetic mouse models, it was found that lipotoxic damage to β-cells affected GLP-1 function, and treatment of hyperlipidemia could enhance GLP-1-induced insulin secretion. [3] Currently, among the proven therapeutic strategies, only glucagon-like peptide-1 receptor agonists (GLP-1 RAs) can achieve simultaneous regulation of patient weight and blood glucose levels. GLP-1 plays a role in promoting insulin secretion in a blood glucose-dependent manner, suppressing appetite, and slowing gastric emptying, thereby promoting weight loss, which forms the basis of research into GLP-1 receptor agonists. [4] .

[0005] GLP-1 not only reduces peripheral blood glucose levels, protects pancreatic islet cells, and improves symptoms, but also plays a trophic role in the central nervous system as a neurotransmitter, affecting cell proliferation, neurogenesis, and cell apoptosis. GLP-1R is widely distributed in the brains of rodents and humans. [5] GLP-1 is expressed in the thalamus, cerebellum, brainstem, fornix, posterior hypothalamus, lateral phrenic nucleus, caudate putamen, hippocampus, and cerebral cortex. GLP-1 can bind to its receptors in the corresponding brain regions through the blood-brain barrier and play a role. GLP-1 can regulate multiple physiological processes in neurons, such as regulating cell survival and axonal growth, resisting excitability, oxidative damage, and death of cultured neurons in vitro, reducing neuronal beta precursor protein (βAPP), reducing endogenous Aβ levels, resisting multiple apoptotic stimuli, and inducing differentiation of cultured neurons in vitro, protecting neuronal function.[6] Animal experimental studies on Aβ toxic damage Therefore, Aβ may induce severe long-term potentiation (LTP) inhibition, and this damage can be reversed by GLP-1 analogs. [7] They found that after injection of Aβ into the ventricles of rodents, spatial learning and memory abilities were deficient when assessed via the Morris water maze, but treatment with a GLP-1 analogue improved the animals' performance in spatial learning and memory. [8] Scientists have also found that GLP-1 and its analogs can improve memory and synaptic plasticity in the brain. [9] .

[0006] The regulation of GLP-1 receptor function is highly complex, interacting with various endogenous and exogenous polypeptides and resulting in the cascade activation of multiple downstream signaling pathways. GLP-1 receptor gene polymorphisms have long been identified and may be associated with the development of obesity and diabetes.

[10] .

[0007] The present inventors have discovered the use of plasminogen in the regulation of the GLP-1 / GLP-1 receptor pathway and in the treatment of diseases associated with the GLP-1 / GLP-1 receptor pathway. Summary of the Invention

[0008] The present invention relates to the following items.

[0009] 1. A method for treating a disease by regulating GLP-1 / GLP-1R, comprising administering to a subject an effective amount of plasminogen.

[0010] 2. The method according to Item 1, wherein the disease is a disease associated with a carbohydrate metabolism disorder, a disease associated with a fat metabolism disorder, or a GLP-1 / GLP-1R-related nervous system disease.

[0011] 3. The method according to Item 2, wherein the disease is one or more diseases selected from the group consisting of diabetes, diabetic nephropathy, diabetic neuralgia, diabetic retinopathy, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral column sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

[0012] 4. A method for regulating the function of GLP-1 / GLP-1R, comprising administering to a subject an effective amount of plasminogen.

[0013] 5. The method according to Item 4, wherein the plasminogen promotes the expression of GLP-1 and / or GLP-1R.

[0014] 6. A method for treating a GLP-1 / GLP-1R-associated disease, comprising administering to a subject an effective amount of plasminogen.

[0015] 7. The method according to Item 6, wherein the GLP-1 / GLP-1R-associated disease includes one or more selected from the group consisting of elevated blood glucose levels, impaired glucose tolerance, elevated blood lipids, obesity, fatty liver, and cognitive impairment.

[0016] 8. The method according to Item 6, wherein the GLP-1 / GLP-1R-related disease comprises one or more selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

[0017] 9. The method according to any one of items 1 to 8, wherein the plasminogen is a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence 2, 6, 8, 10, or 12.

[0018] 10. The method according to any one of Items 1 to 9, wherein the plasminogen is a protein that contains a plasminogen activity fragment and still has plasminogen activity.

[0019] 11. The plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, γ-plasminogen or the like. Item 11. The method according to Item 10, wherein the plasminogen is a mutant that retains the plasminogen activity.

[0020] 12. The method according to any one of items 1 to 11, wherein the plasminogen can be used in combination with one or more other drugs or therapeutic methods.

[0021] 13. The method according to Item 12, wherein the plasminogen can be used in combination with one or more drugs or therapeutic methods selected from the group consisting of drugs or therapeutic methods for treating diabetes, drugs or therapeutic methods for treating atherosclerosis, drugs or therapeutic methods for treating cardiocerebrovascular disease, drugs or therapeutic methods for treating thrombosis, drugs or therapeutic methods for treating hypertension, drugs or therapeutic methods for lowering blood lipids, drugs or therapeutic methods for treating fatty liver, drugs or therapeutic methods for treating Parkinson's disease, drugs or therapeutic methods for treating Alzheimer's disease, and anti-infective drugs or therapeutic methods.

[0022] 14. A drug for treating a GLP-1 / GLP-1R-associated disease, comprising an effective amount of plasminogen.

[0023] 15. A product or kit for treating a GLP-1 / GLP-1R-associated disease, comprising a container containing an effective amount of plasminogen and a protocol describing the use of plasminogen to treat a GLP-1 / GLP-1R-associated disease.

[0024] 16. The drug, product, or kit according to item 14 or 15, wherein the GLP-1 / GLP-1R-related disease includes one or more selected from the group consisting of elevated blood glucose levels, impaired glucose tolerance, elevated blood lipids, obesity, fatty liver, and cognitive impairment.

[0025] 17. The drug, product, or kit according to item 14 or 15, wherein the GLP-1 / GLP-1R-related disease comprises one or more selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

[0026] 18. Use of plasminogen in the manufacture of a medicament for treating a disease by regulating GLP-1 / GLP-1R.

[0027] 19. The use according to item 18, wherein the disease is a disease associated with a carbohydrate metabolism disorder, a disease associated with a fat metabolism disorder, or a GLP-1 / GLP-1R-related nervous system disease.

[0028] 20. The diseases include diabetes, diabetic nephropathy, diabetic neuralgia, diabetic retinopathy, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, and lateral column sclerosis. Item 20. The use according to Item 19, wherein the disease is one or more diseases selected from the group consisting of inflammatory bowel disease, dyspepsia, and gastrointestinal ulcers.

[0029] 21. Use of plasminogen in the manufacture of a drug for regulating the function of GLP-1 / GLP-1R.

[0030] 22. The use according to item 21, wherein the plasminogen promotes the expression of GLP-1 / GLP-1R.

[0031] 23. Use of plasminogen in the manufacture of a medicament for treating a GLP-1 / GLP-1R-related disease.

[0032] 24. The use according to Item 23, wherein the GLP-1 / GLP-1R-related disease includes one or more selected from the group consisting of elevated blood glucose levels, impaired glucose tolerance, elevated blood lipids, obesity, fatty liver, and cognitive impairment.

[0033] 25. The use according to Item 23, wherein the GLP-1 / GLP-1R-related disease comprises one or more selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

[0034] 26. The use of any one of clauses 18 to 25, wherein the plasminogen is a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to sequence 2, 6, 8, 10, or 12.

[0035] 27. The use according to any one of items 18 to 26, wherein the plasminogen is a protein that contains a plasminogen activity fragment and still has plasminogen activity.

[0036] 28. The plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, γ-plasminogen or the like. Item 28. The use according to Item 27, wherein the plasminogen is a mutant that retains the plasminogen activity.

[0037] 29. The use according to any one of items 18 to 28, wherein the plasminogen can be used in combination with one or more other drugs or therapeutic methods.

[0038] 30. The use according to Item 29, wherein the plasminogen can be used in combination with one or more drugs or therapeutic methods selected from the group consisting of drugs or therapeutic methods for treating diabetes, drugs or therapeutic methods for treating atherosclerosis, drugs or therapeutic methods for treating cardiocerebrovascular disease, drugs or therapeutic methods for treating thrombosis, drugs or therapeutic methods for treating hypertension, drugs or therapeutic methods for lowering blood lipids, drugs or therapeutic methods for treating fatty liver, drugs or therapeutic methods for treating Parkinson's disease, drugs or therapeutic methods for treating Alzheimer's disease, and anti-infective drugs or therapeutic methods.

[0039] Definition: "Diabetes" is a condition caused by the breakdown of sugar, protein, fat, water, and electrolytes, etc., which is caused by insulin deficiency and insulin resistance, which are caused by various disease-inducing factors such as genetic factors, impaired immune function, microbial infection and its toxins, free radical toxins, and psychological factors acting on the body. It is a syndrome in which the metabolism of the blood is disturbed, and its main clinical feature is hyperglycemia.

[0040] "Diabetes complications" are damage or dysfunction of other organs or tissues in the body caused by poor blood sugar control during the diabetic process, including damage or dysfunction of the liver, kidneys, heart, retina, nervous system, etc. According to statistics from the World Health Organization (WHO), there are more than 100 types of diabetes complications, making it the most common type of complication currently.

[0041] "Plasmin" is a very important enzyme present in the blood, which can decompose fibrin polymers.

[0042] "Plasminogen (plg)" is the proenzyme form of plasmin, and is calculated to consist of 810 amino acids based on the sequence in swiss prot as the amino acid sequence of natural human plasminogen (sequence 4) of the signal peptide, and has a molecular weight of approximately 90 kD. It is a glycoprotein that is synthesized mainly in the liver and can circulate in the blood, and the cDNA sequence encoding this amino acid sequence is shown in sequence 3. Full-sized PLG (plasminogen) contains seven domains: a serine protease domain located at the C-terminus, a Pan Apple (PAp) domain located at the N-terminus, and five Kringle domains (Kringle 1-5). swiss According to the sequences in [linked to] nucleotide ...

[0043] Glu-plasminogen is a natural, full-size plasminogen, consisting of 791 amino acids (not including a signal peptide consisting of 19 amino acids). The cDNA sequence encoding this sequence is shown in Sequence 1, and its amino acid sequence is shown in Sequence 2. In vivo, Lys-plasminogen, formed by hydrolysis of the 76th-77th amino acid positions of Glu-plasminogen, exists, for example, as shown in Sequence 6, and the cDNA sequence encoding this amino acid sequence is shown in Sequence 5. δ-plasminogen is a fragment of full-size plasminogen lacking the Kringle2-Kringle5 structure, and contains only Kringle1 and the serine protease domain. [11、12]There is a literature that reports the amino acid sequence of delta-plasminogen (sequence 8).

[12] The cDNA sequence encoding the amino acid sequence is, for example, Sequence 7. Mini-plasminogen consists of Kringle 5 and a serine protease domain, and the literature reports residues Val443-Asn791 (the starting amino acid is a Glu residue that does not include the signal peptide Glu-plasminogen sequence).

[13] The amino acid sequence is shown in Sequence 10, and the cDNA sequence encoding the amino acid sequence is shown in Sequence 9. However, micro-plasminogen contains only a serine protease domain, and the literature reports that its amino acid sequence is residues Ala543-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the initiating amino acid).

[14] Patent document CN102154253A discloses that the sequence contains residues Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and the sequence of this patent can be referred to Patent document CN102154253A, whose amino acid sequence is as shown in Sequence 12, and the cDNA sequence encoding the amino acid sequence is as shown in Sequence 11.

[0044] In the present invention, "plasmin", "fibrin plasmin", and "fibrous protein plasmin" can be used interchangeably and have the same meaning. "Plasminogen", "fibrin plasminogen", and "fibrous protein plasminogen" can be used interchangeably and have the same meaning.

[0045] Those skilled in the art can understand that all the technical configurations of plasminogen in the present invention are applicable to plasmin, so the technical configurations described in the present invention cover plasminogen and plasmin.

[0046] During circulation, plasminogen is in a closed, inactive conformation. Upon binding to a clot or cell surface, it converts to an open, active plasmin through the interaction of plasminogen activators (PAs). Active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the clot. The PAp domain of plasminogen is the key epitope that maintains plasminogen in the closed, inactive conformation, while the KR domain can bind to lysine residues on receptors and substrates. Several enzymes are known to act as plasminogen activators, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and clotting factor XII (Hagemann factor).

[0047] A "plasminogen active fragment" refers to an active fragment of plasminogen protein that can bind to a target sequence in a substrate and exert its proteolytic function. The technical configuration related to plasminogen of the present invention includes a technical configuration in which a plasminogen active fragment is used to replace plasminogen. The plasminogen active fragment described in the present invention is a protein containing the serine protease domain of plasminogen. Preferably, the plasminogen active fragment described in the present invention includes a protein containing sequence 14 or an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to sequence 14. Therefore, the plasminogen described in the present invention includes a protein that contains the plasminogen active fragment and still has the plasminogen activity.

[0048] Currently, methods for measuring fibrin plasminogen and its activity in blood include: assay for tissue fibrin plasminogen activator activity (t-PAA), assay for plasma tissue fibrin plasminogen activator antigen (t-PAAg), assay for plasma tissue plasminogen activity (plgA), assay for plasma tissue plasminogen antigen (plgAg), assay for plasma tissue fibrin plasminogen activator inhibitor activity, assay for plasma tissue fibrin plasminogen activator inhibitor antigen, and assay for plasma fibrin plasmin-antiplasmin complex (PAP). The most common assay method is the chromogenic substrate method: streptokinase (SK) and a chromogenic substrate are added to the plasma of the subject, and PLG in the subject's plasma is converted to plasmin under the action of SK, which then acts on the chromogenic substrate, which is then measured using a spectrophotometer. The increase in absorbance is directly proportional to the activity of fibrin plasminogen. In addition, fibrin plasminogen activity in blood can be measured using immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, and the like.

[0049] "Ortholog" refers to a homologue between different species, including both protein homologues and DNA homologues. It specifically refers to a protein or gene obtained by evolution from the same ancestral gene between different species. The plasminogen of the present invention includes human natural plasminogen, and further includes plasminogens having plasminogen activity derived from different species. Contains plasminogen orthologs.

[0050] A "conservative substitution variant" is one in which a single specified amino acid residue has been altered without altering the overall conformation and function of a protein or enzyme. This includes, but is not limited to, substitutions of an amino acid in the amino acid sequence of a parent protein with an amino acid of similar properties (e.g., acidic, basic, hydrophobic, etc.). Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be substituted for each other. Similarly, isoleucine is a hydrophobic amino acid and can be substituted by leucine, methionine, or valine. Therefore, the similarity between two proteins or amino acid sequences with similar functions may vary. For example, they may have 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes that share 60% or more amino acid identity based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and even more preferably 90% or more, and that have the same or essentially similar properties or functions as compared to the native or parent protein or enzyme.

[0051] "Isolated" plasminogen refers to plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is (1) purified to greater than 90%, greater than 95%, or greater than 98% purity (by weight), e.g., greater than 99% purity (by weight), as determined by, e.g., the Lowry method; (2) purified to at least 15 residues of N-terminal or internal amino acid sequence, as determined by a spinning cup sequencer; or (3) purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with Coomassie brilliant blue or silver staining. Isolated plasminogen can be produced from recombinant cells by bioengineering techniques and includes plasminogen that has been further isolated by at least one purification step.

[0052] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbones. The term also encompasses fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions containing leader sequences of heterologous and homologous origin (with or without an N-terminal methionine residue); and the like.

[0053] The "percentage (%) amino acid sequence identity" of a reference peptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and excluding any conservative substitutions that are not considered part of the sequence identity. Comparisons for the purpose of determining percentage amino acid sequence identity can be accomplished by a variety of means within the skill of the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm that achieves the maximum comparison required for the full length of the sequences being compared. However, for purposes of the present invention, the percentage amino acid sequence identity is determined using the sequence comparison computer software ALIGN-2.

[0054] When comparing amino acid sequences by using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (also referred to as a given amino acid sequence A having identity to or containing an amino acid sequence for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0055] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 evaluates as identical and matching in its alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood as follows: if the lengths of amino acid sequence A and amino acid sequence B are not equal, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are as described in the preceding paragraph and are generated by the ALIGN-2 computer program.

[0056] As used herein, the terms "treatment" and "prevention" refer to achieving a desired pharmacological and / or physiological effect. The effect can be complete or partial prevention of a disease or its symptoms and / or partial or complete cure of a disease and / or its symptoms. It also includes: (a) preventing a disease from occurring in a subject, where the subject has the disease precursors but has not been diagnosed with the disease; (b) suppressing a disease, inhibiting its formation; and (c) attenuating a disease and / or its symptoms, i.e., reducing the disease and / or its symptoms.

[0057] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0058] A "therapeutically effective amount" or "effective amount" is an amount of plasminogen that, when administered to a mammal or other subject to treat a disease, is capable of achieving said prevention and / or treatment of the disease. The "therapeutically effective amount" varies depending on the plasminogen used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0059] 2. Preparation of the Plasminogen of the Present Invention For therapeutic use, plasminogen may be isolated and purified from nature, or synthesized by standard chemical peptide synthesis techniques. When chemically synthesizing polypeptides, synthesis can be performed in either liquid or solid phase. Solid-phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support and the remaining amino acids in the sequence are added sequentially, is suitable for chemically synthesizing plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. The techniques used in solid-phase synthesis are described in: Barany and Solid-Phase Peptide Synthesis; pages 3-284, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85:2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. Briefly, insoluble porous beads are treated with functional units on which the peptide chain is built. Coupling / Decoupling After repeated cycles of protection, a single N-protected amino acid unit is coupled to the free N-terminal amine of the attached solid phase. The unit is then deprotected, revealing a new N-terminal amine for linking with another amino acid. The peptide remains immobilized on the solid phase, and is then excised.

[0060] The plasminogen of the present invention is produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector and operably linked to control sequences in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Expression can be controlled by a eukaryotic promoter system in a vector that is transformed or transfected into eukaryotic host cells (e.g., COS or CHO cells). Once the vector is introduced into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of the plasminogen.

[0061] Suitable expression vectors typically replicate in the host either episomes or as an integral part of the host chromosomal DNA. Expression vectors usually contain selectable markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance, kanamycin-resistance, or neomycin-resistance) that are useful for detecting in vitro those cells transformed with the desired DNA sequences.

[0062] Escherichia coli is an example of a prokaryotic host cell that can be used to clone polynucleotides encoding the plasminogen protein of the present invention. Other suitable microbial hosts include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can be generated in these prokaryotic hosts and typically contain expression control sequences (e.g., origins of replication) compatible with the host cell. Many known promoters are available, including the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, and promoter systems derived from phage lambda. Promoters generally control expression and, if necessary, may contain ribosome binding site sequences to initiate transcription and translation of the controlled gene sequence.

[0063] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., Saccharomyces (S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which suitable carriers include expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters for alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0064] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express the plasminogen of the present invention. See, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells contain expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and required processing information sites, such as a ribosome binding site, RNase A, or RNase B. The expression control sequence may include a cleavage site for A, a polyadenosine oxidation site, and a transcription terminator sequence. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0065] Once synthesized (chemically or recombinantly), the plasminogen described in the present invention can be purified by standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. The plasminogen can be essentially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or even purer, e.g., free from contaminants, such as cellular debris, large molecules other than the plasminogen of the present invention, etc.

[0066] 3. Drug combinations Plasminogen of the desired purity is mixed with pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) as needed to form a lyophilized preparation or aqueous solution to obtain a therapeutic formulation. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects at the required doses and concentrations, and further include buffers such as phosphates, citrates, and other organic acids. Antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parahydroxybenzoate esters, such as methyl or propyl parahydroxybenzoate; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (having at least 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants.

[0067] The combination preparation of the present invention may contain one or more active compounds as needed for the specific symptoms requiring treatment, preferably with complementary activities and no adverse effects, such as drugs for treating one or more diseases selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

[0068] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, and can be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in coarse emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0069] The plasminogen of the present invention to be administered internally must be sterile, which is readily accomplished by filtration through sterile filtration membranes, before or after lyophilization and recombination.

[0070] The plasminogen of the present invention can be prepared in a buffered formulation. Suitable examples of buffered formulations include semipermeable matrices of solid hydrophobic polymers having a defined shape and containing the glycoprotein, such as membranes or microcapsules. Examples of buffer matrices include polyesters, aqueous gels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and ethyl-L-glutamic acid (Sidman et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer et al., source as above), or degradable lactic acid-hydroxyacetic acid copolymers, such as Lupron. These include Depot™ (injectable microspheres composed of lactic acid-hydroxyacetic acid copolymer and leuprolide acetate), and poly(D-(-)-3-hydroxybutyrate). Polymers such as ethylene-ethyl acetate and lactic acid-hydroxyacetic acid can release molecules sustainedly for over 100 days, whereas some aqueous gels release proteins for a relatively short period of time. Rational strategies for protein stabilization can be designed depending on the mechanism involved. For example, if the aggregation mechanism involves the exchange of sulfur disulfide bonds to form intermolecular disulfide bonds, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0071] 4. Dosage and dosage The pharmaceutical compositions of the present invention can be administered by various routes, such as intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., carotid), intramuscular, intranasal, skin or intradermal administration, or spinal or intracerebral delivery. Aerosol formulations, such as nasal spray formulations, contain purified aqueous or other solutions containing an active agent, a preservative, and an isotonic agent. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

[0072] In some cases, the plasminogen drug compositions of the present invention can be modified or formulated in the following manner to provide them with the ability to cross the blood-brain barrier.

[0073] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolysis replenishers, and the like. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0074] In some embodiments, the plasminogen of the present invention is formulated with an agent that promotes passage through the blood-brain barrier. In some cases, the plasminogen of the present invention is fused, directly or via a linker, to a carrier molecule, peptide, or protein that promotes passage through the blood-brain barrier. In some embodiments, the plasminogen of the present invention is fused to a polypeptide that binds to an endogenous blood-brain barrier (BBB) ​​receptor. The combination of plasminogen and a polypeptide that binds to an endogenous blood-brain barrier receptor promotes passage through the BBB. ) Receptor-binding polypeptides include antibodies, e.g., monoclonal antibodies, or antigen-binding fragments thereof, which specifically bind to endogenous BBB receptors. In some cases, the antibodies are encapsulated in liposomes. See, e.g., U.S. Patent Publication No. 2009 / 0156498.

[0075] A medical professional can determine dosage recommendations based on various clinical factors. For example, as is well known in the medical arts, the dosage for any given patient depends on several factors, including the patient's body size, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other concurrently administered medications. The dosage range for the plasminogen pharmaceutical composition of the present invention can be, for example, about 0.0001 to 2000 mg / kg of subject body weight daily, or about 0.001 to 500 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.). For example, dosages can be 1 mg / kg body weight or 50 mg / kg body weight, or in the range of 1-50 mg / kg, or at least 1 mg / kg. Doses higher or lower than this exemplary range are also encompassed, particularly when the aforementioned factors are taken into consideration. Intermediate doses within the above ranges are also within the scope of the present invention. Subjects can receive such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dose schedule is 1-10 mg / kg for several consecutive days. During the administration of the drugs of the present invention, therapeutic efficacy and safety should be evaluated in real time and periodically.

[0076] 5. Products or drug kits One embodiment of the present invention relates to an article of manufacture or pharmaceutical kit containing the plasminogen of the present invention. The article of manufacture preferably includes a container, label, or protocol. Suitable containers include bottles, vials, syringes, etc. The container can be made of various materials, such as glass or plastic. The container contains a composition that effectively treats a disease or condition of the present invention and has a sterile access point (e.g., the container is an intravenous infusion pack or vial and includes a stopper that can be pierced by a hypodermic needle). At least one activator in the composition is plasminogen. A label on or attached to the container describes the composition as being used to treat the disease of the present invention. The article of manufacture may further include a second container containing a pharmaceutical buffer, such as phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes. The article of manufacture may also include a protocol with instructions for use, such as instructing a user of the composition to administer the plasminogen composition and other medications associated with the treatment of a disease to a patient. [Brief explanation of the drawings]

[0077] [Figure 1] Figures 1A–C show the results of pancreatic GLP-1 immunostaining in 14–15-week-old db / db mice after 28 days of plasminogen administration. A shows the PBS-treated control group, B shows the plasminogen-treated group, and C shows the results of quantitative analysis. The results show that the expression of GLP-1 in pancreatic islets (indicated by the arrow) in the PBS-treated control group was significantly lower than that in the plasminogen-treated group, and the difference was statistically significant (* indicates P<0.05). These results indicate that plasminogen can promote the expression of GLP-1 in pancreatic islets in relatively young diabetic mice. [Figure 2]Figure 2A-B shows representative photographs of pancreatic GLP-1 immunostaining in 23- to 25-week-old db / db mice after 28 days of plasminogen administration. A represents the PBS-treated control group, and B represents the plasminogen-treated group. The results show that the expression of GLP-1 in pancreatic islets (indicated by arrows) in the PBS-treated control group was significantly lower than in the plasminogen-treated group. This result indicates that plasminogen can promote the expression of GLP-1 in pancreatic islets in relatively old diabetic mice. [Figure 3] Figures 3A-C show the results of islet GLP-1 staining in PLG+ / + mice in a T1DM model after 28 days of plasminogen administration. A shows the PBS-treated control group, B shows the plasminogen-treated group, and C shows the results of quantitative analysis. The results show that the expression of GLP-1 in the islets of mice in the PBS-treated control group was significantly lower than that of the plasminogen-treated group, and the difference was statistically significant (** indicates P<0.01). These results indicate that plasminogen can promote the expression of GLP-1 in the islets of type 1 diabetic mice. [Figure 4] Figures 4A–C show immunohistochemical observations of pancreatic islet glucagon in 24–25-week-old diabetic mice after 35 days of plasminogen administration. A represents the normal control group, B the PBS-treated control group, and C the plasminogen-treated group. Results show that glucagon was expressed in the α-cell area surrounding the islets in the normal control group. Compared with the plasminogen-treated group, the PBS-treated control group had significantly more glucagon-positive cells (indicated by arrows), with positive cells infiltrating the center of the islets. In the plasminogen-treated group, glucagon-positive cells were scattered around the islets. Compared with the PBS-treated group, the islet morphology in the plasminogen-treated group was more similar to that of normal mice. This indicates that plasminogen can significantly inhibit islet α-cell proliferation and glucagon secretion, correct the disrupted distribution of islet α-cells, and promote islet repair. [Figure 5]Figures 5A–D show the immunohistochemical observation of glucagon in pancreatic islets of 27-week-old diabetic mice after 35 days of plasminogen administration. A represents the normal control group, B the PBS-treated control group, C the plasminogen-treated group, and D the quantitative analysis results. The results showed that glucagon was expressed in the alpha cell area around the islets in the normal control group. Compared with the plasminogen-treated group, the PBS-treated control group had significantly more glucagon-positive cells (indicated by arrows), with positive cells infiltrating the center of the islets. Furthermore, the quantitative analysis of the mean optical density showed a statistically significant difference (* indicates P<0.05). In the plasminogen-treated group, glucagon-positive cells were scattered around the islets, and the islet morphology in the plasminogen-treated group was more similar to that of normal mice compared with the PBS-treated group. This indicates that plasminogen can significantly suppress the proliferation of pancreatic islet α-cells and glucagon secretion, correct the disorder of pancreatic islet α-cell distribution, and promote the repair of pancreatic islet damage. [Figure 6] Figures 6A-D show immunohistochemical observations of islet glucagon in PLG+ / + mice as a T1DM model after 28 days of plasminogen administration. A is the blank control group, B is the PBS-treated control group, C is the plasminogen-treated group, and D is the quantitative analysis result. The results show that the PBS-treated control group had significantly higher glucagon expression than the plasminogen-treated group, and the difference in mean optical density was statistically significant (* indicates P<0.05). This indicates that plasminogen can significantly reduce glucagon secretion from islet α cells in diabetic mice and promote the repair of islet damage. [Figure 7]Figure 7 shows the blood glucose measurement results on days 11 and 32 after plasminogen administration to 24- to 25-week-old diabetic mice. The results show that the blood glucose levels of the plasminogen-administered mice were significantly lower than those of the PBS-administered control group, and that this difference was statistically significant (* indicates P<0.05, ** indicates P<0.01). Furthermore, as the administration time increased, the blood glucose levels of the PBS-administered control mice tended to increase, whereas the blood glucose levels of the plasminogen-administered group gradually decreased. This indicates that plasminogen has a blood glucose lowering effect. [Figure 8] Figure 8 shows the effect of plasminogen on serum fructosamine levels in diabetic mice. The results show that serum fructosamine levels after plasminogen administration were significantly reduced compared to levels before administration, and the difference was statistically significant (** indicates P<0.01). This indicates that plasminogen can significantly reduce serum fructosamine levels in diabetic mice. [Figure 9] Figure 9 shows the serum fructosamine levels in 27-week-old diabetic mice after 35 days of plasminogen administration. The results showed that the serum fructosamine levels in the plasminogen-administered group were significantly lower than those in the PBS-administered control group, with the difference reaching statistical significance (P = 0.06). This indicates that plasminogen can significantly reduce serum fructosamine levels in diabetic mice. [Figure 10] Figure 10 shows the results of measuring plasma glycated hemoglobin in 27-week-old diabetic mice after 35 days of plasminogen administration. The results show that the OD values ​​of glycated hemoglobin in the plasminogen-administered mice were significantly lower than those in the PBS-administered control group, and the difference was statistically significant (** indicates P<0.01). This indicates that plasminogen has the effect of lowering plasma glycated hemoglobin in diabetic mice. [Figure 11]Figure 11 shows the IPGTT results after 10 days of plasminogen administration in 27-week-old diabetic mice. The results show that after intraperitoneal glucose injection, the blood glucose levels of the plasminogen-treated mice were lower than those of the PBS-treated control group, and the glucose tolerance curve of the plasminogen-treated group was closer to that of normal mice than that of the PBS-treated control group. This indicates that plasminogen can significantly improve the glucose tolerance of diabetic mice. [Figure 12] Figure 12 shows the blood glucose measurement results after fasting in T1DM model PLG+ / + mice after 10 days of plasminogen administration. The results show that the blood glucose levels of the PBS-treated control group were significantly higher than those of the plasminogen-treated group, and the difference was statistically significant (*** indicates P<0.001). This indicates that plasminogen can significantly reduce blood glucose levels in PLG+ / + mice, a T1DM model. [Figure 13] Figure 13 shows the IPGTT results after 28 days of plasminogen administration in PLG+ / + mice, a T1DM model. The results showed that the blood glucose concentration after glucose injection in the PBS-treated control group was significantly higher than that in the plasminogen-treated group, and the glucose tolerance curve of the plasminogen-treated group was closer to that of normal mice than that of the PBS-treated control group. This indicates that plasminogen can improve the glucose tolerance of PLG+ / + mice in this T1DM model. [Figure 14] Figure 14 shows the blood glucose measurement results after 20 days of plasminogen administration to T1DM model mice. The results show that the blood glucose levels of the PBS-treated control group mice were significantly higher than those of the plasminogen-treated group mice, and the difference was statistically significant (P = 0.04). This indicates that plasminogen can promote glucose degradation in T1DM mice and lower their blood glucose levels. [Figure 15]Figure 15 shows the results of measuring serum insulin in 27-week-old diabetic mice after 35 days of plasminogen administration. The results show that the serum insulin level in the plasminogen-administered group was significantly higher than that in the PBS-administered control group, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can effectively promote insulin secretion. [Figure 16] Figures 16A–E show HE-stained photographs of the pancreas and the statistical analysis of islet area ratios in 24–25-week-old diabetic mice after 31 days of plasminogen administration. A and B are from the PBS-treated control group, while C and D are from the plasminogen-treated group. E shows the quantitative analysis of islet area. Results show that in the PBS-treated control group, most islets were atrophied, with the atrophied islet cells replaced by acini (indicated by arrows), and acini proliferated at the edges of the islets, blurring the boundary between islets and acini. In the plasminogen-treated group, most islets were larger than in the control group, and there was no acinar proliferation within the islets; only a few acini remained within a few islets, demonstrating a clear boundary between islets and acini. Comparing the islet-to-pancreas area ratios between the plasminogen-treated and control groups, the plasminogen-treated group was nearly twice as large as the control group. This indicates that plasminogen can promote the repair of pancreatic islet damage in 24- to 25-week-old diabetic mice and can treat diabetes by repairing damaged pancreatic islets. [Figure 17] Figures 17A-C show the results of Sirius Red staining of pancreatic islets in 24- to 25-week-old diabetic mice after 31 days of plasminogen administration. A shows the control group treated with PBS, B shows the plasminogen-treated group, and C shows the results of quantitative analysis. The results show that collagen deposition in the pancreatic islets (indicated by arrows) in the plasminogen-treated mice was significantly less than that in the control group treated with PBS, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can ameliorate islet fibrosis in diabetic animals. [Figure 18]Figures 18A-B show the results of immunohistochemical staining of pancreatic islets with caspase-3 in 24- to 25-week-old diabetic mice after 31 days of plasminogen administration. A represents the control group treated with PBS, and B represents the plasminogen-treated group. The results show that caspase-3 expression (indicated by the arrow) in the plasminogen-treated group was significantly lower than that in the PBS-treated control group. This indicates that plasminogen can reduce apoptosis of pancreatic islet cells and protect the pancreatic tissue of diabetic mice. [Figure 19] Figures 19A-C show the results of immunohistochemical staining of pancreatic islet insulin in 17-18 week-old diabetic mice after 35 days of plasminogen administration. A shows the control group treated with PBS, B shows the plasminogen-treated group, and C shows the results of quantitative analysis. The results show that insulin expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and the difference was statistically significant (P = 0.15). This indicates that plasminogen can promote the functional restoration of pancreatic islets and enhance insulin production and secretion. [Figure 20] Figures 20A-C show the results of immunohistochemical staining for insulin in 24-25 week-old diabetic mice after 31 days of plasminogen administration. A shows the control group treated with PBS, B shows the plasminogen-treated group, and C shows the quantitative analysis results. The results show that insulin expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can promote the functional repair of pancreatic islets and enhance insulin production and secretion. [Figure 21]Figures 21A-C show the results of insulin immunohistochemical staining in 27-week-old diabetic mice after 35 days of plasminogen administration. A is the PBS-treated control group, B is the plasminogen-treated group, and C is the quantitative analysis result. The results show that insulin expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and the difference was statistically significant (** indicates P<0.01). This indicates that plasminogen can effectively promote the functional repair of pancreatic islets and enhance insulin production and secretion. [Figure 22] Figures 22A-D show the results of immunohistochemical staining of NF-kB in pancreatic tissue of 24- to 25-week-old diabetic mice after 31 days of plasminogen administration. A represents the normal control group, B represents the PBS-treated control group, and C represents the plasminogen-treated group. D represents the quantitative analysis results. The results show that the expression of NF-kB (indicated by the arrow) in the plasminogen-treated group was close to that of normal control mice and significantly higher than that in the PBS-treated control group, with the difference being statistically significant (* indicates P<0.05). This indicates that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB and promote the repair of pancreatic islet inflammation in 24- to 25-week-old diabetic mice. [Figure 23]Figures 23A-D show the results of immunohistochemical staining of pancreatic islets in 17- to 18-week-old diabetic mice after 35 days of plasminogen administration. A represents the normal control group, B represents the PBS-treated control group, C represents the plasminogen-treated group, and D represents the quantitative analysis results. The results showed that glucagon was expressed in the alpha cell area around the islets in the normal control mice. Compared with the plasminogen-treated group, the number of glucagon-positive cells (indicated by arrows) in the PBS-treated control group was significantly increased, and glucagon-positive cells infiltrated the center of the islets. The difference in mean optical density was statistically significant (** indicates P<0.01). In the plasminogen-treated group, glucagon-positive cells were scattered around the islets, and the islet morphology in the plasminogen-treated group was more similar to that of normal mice compared with the PBS-treated control group. This indicates that plasminogen can significantly suppress the proliferation of pancreatic islet α-cells and glucagon secretion, correct the disorder of pancreatic islet α-cell distribution, and promote the repair of pancreatic islet damage. [Figure 24] Figure 24A–D shows the results of immunohistochemical staining of pancreatic islets with IRS-2 in 17–18-week-old diabetic mice after 35 days of plasminogen administration. A represents the normal control group, B represents the PBS-treated control group, and C represents the plasminogen-treated group. D represents the quantitative analysis results. The results showed that the PBS-treated control group had significantly lower IRS-2 expression in pancreatic islets than the plasminogen-treated group, and the difference was statistically significant (** indicates P<0.01). The IRS-2 expression level in the plasminogen-treated group was closer to that in the normal control group than in the PBS-treated control group. This indicates that plasminogen can effectively increase islet cell IRS-2 expression, improve insulin signaling, and reduce islet β cell damage in 17–18-week-old diabetic mice. [Figure 25]Figures 25A–D show the immunohistochemical analysis of IRS-2 in pancreatic islets of 24–25-week-old diabetic mice after 31 days of plasminogen administration. A represents the normal control group, B represents the PBS-treated control group, and C represents the plasminogen-treated group. D represents the quantitative analysis results. The results showed that the positive expression of IRS-2 in pancreatic islets (indicated by arrows) in the PBS-treated control group was significantly lower than in the plasminogen-treated group, and the difference was statistically significant (* indicates P<0.05). The IRS-2 expression level in the plasminogen-treated group was closer to that in the normal control group than in the PBS-treated control group. This indicates that plasminogen can effectively increase islet cell IRS-2 expression, improve insulin signaling, and reduce islet β cell damage in diabetic mice. [Figure 26] Figures 26A-C show the immunohistochemical observation of IRS-2 in pancreatic islets of 27-week-old diabetic mice after 35 days of plasminogen administration. A represents the normal control group, B represents the PBS-treated control group, and C represents the plasminogen-treated group. The results showed that the positive IRS-2 expression (indicated by the arrow) in the pancreatic islets of the PBS-treated control group was significantly lower than that of the plasminogen-treated group; the IRS-2 expression level in the plasminogen-treated group was closer to that of the normal control group than that of the PBS-treated control group. This indicates that plasminogen can effectively increase IRS-2 expression in pancreatic islet cells, improve insulin signaling, and reduce islet β cell damage in diabetic mice. [Figure 27]Figures 27A-C show the immunohistochemical observation of IRS-2 in pancreatic islets of PLG+ / + T1DM mice after 28 days of plasminogen administration. A is the normal control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results showed that the positive IRS-2 expression (indicated by the arrow) in the pancreatic islets of the PBS-treated control group was significantly lower than that of the plasminogen-treated group; the IRS-2 expression level in the plasminogen-treated group was closer to that of the normal control group than that of the PBS-treated control group. This indicates that plasminogen can effectively increase islet cell IRS-2 expression, improve insulin signaling, and reduce islet β cell damage in PLG+ / + T1DM mice. [Figure 28] Figures 28A-C show the results of immunohistochemical staining of pancreatic islet neutrophils in 27-week-old diabetic mice after 35 days of plasminogen administration. A is the normal control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results showed that the number of positively expressing cells (indicated by arrows) in the plasminogen-treated group was lower than in the PBS-treated control group, and the plasminogen-treated group was closer to the normal control group than the PBS-treated control group. This indicates that plasminogen can reduce neutrophil infiltration. [Figure 29] Figures 29A-C show the results of immunohistochemical staining of islet neutrophils in PLG- / - mice after 28 days of plasminogen administration in a T1DM model. A is the blank control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results showed that the number of positively expressing cells (indicated by arrows) in the plasminogen-treated group was lower than in the PBS-treated control group, and the plasminogen-treated group was closer to the blank control group than the PBS-treated control group. This indicates that plasminogen can reduce islet neutrophil infiltration in the PLG- / - mouse T1DM model. [Figure 30]Figures 30A-C show the immunohistochemical staining of islet neutrophils in PLG+ / + mice after 28 days of plasminogen administration in a T1DM model. A is the blank control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results showed that the number of positively expressing cells (indicated by arrows) in the plasminogen-treated group was lower than in the PBS-treated control group, and the plasminogen-treated group was closer to the blank control group than the PBS-treated control group. This indicates that plasminogen can promote islet neutrophil infiltration in the PLG+ / + mouse T1DM model. [Figure 31] Figures 31A-C show the immunohistochemical staining of insulin in pancreatic islets of PLG- / - mice after 28 days of plasminogen administration in a T1DM model. A is the blank control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The immunohistochemical results show that the positive expression of insulin (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and the plasminogen-treated group was closer to the blank control group than to the PBS-treated control group. This indicates that plasminogen can promote insulin synthesis and secretion in the PLG- / - mouse T1DM model. [Figure 32] Figures 32A-C show the immunohistochemical staining of insulin in pancreatic islets of PLG+ / + mice after 28 days of plasminogen administration in a T1DM model. A is the blank control group, B is the PBS-treated control group, and C is the plasminogen-treated group. Immunohistochemical results showed that the insulin-positive expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and the plasminogen-treated group was closer to the blank control group than to the PBS-treated control group. This indicates that plasminogen can promote insulin synthesis and expression in the PLG+ / + mouse T1DM model. [Figure 33]Figures 33A-C show the results of immunohistochemical staining of pancreatic islets for NF-kB after 28 days of plasminogen administration in PLG- / - mice in a T1DM model. A is the blank control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results show that NF-kB expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than in the PBS-treated control group. This indicates that plasminogen can promote the expression of the inflammatory repair factor NF-kB and promote the repair of pancreatic islet inflammation. [Figure 34] Figures 34A-B show the results of immunohistochemical staining of NF-kB in pancreatic islets of 17- to 18-week-old diabetic mice after 35 days of plasminogen administration. A shows the control group treated with PBS, and B shows the plasminogen-treated group. The results show that NF-kB expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group. This indicates that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB and promote the repair of islet inflammation in relatively young (17- to 18-week-old) diabetic mice. [Figure 35] Figures 35A-C show the immunohistochemical observation results of NF-kB in pancreatic islets of 27-week-old diabetic mice after 35 days of plasminogen administration. A is the normal control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The experimental results of the present invention show that NF-kB expression (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group. This indicates that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB and promote the repair of pancreatic islet inflammation in relatively old (27-week-old) diabetic mice. [Figure 36]Figures 36A-C show the immunohistochemical observation of TNF-α in pancreatic islets in 24- to 25-week-old diabetic mice after 31 days of plasminogen administration. A is the normal control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results show that the positive expression of TNF-α (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and that the plasminogen-treated group was closer to the normal control group than the PBS-treated control group. This indicates that plasminogen can promote TNF-α expression and promote the repair of pancreatic islet damage in 24- to 25-week-old diabetic mice. [Figure 37] Figures 37A-C show the immunohistochemical staining of pancreatic islets in 27-week-old diabetic mice after 35 days of plasminogen administration. A is the normal control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results show that the positive expression of TNF-α (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group, and that the plasminogen-treated group was closer to the normal control group than the PBS-treated control group. This indicates that plasminogen can promote TNF-α expression and promote the repair of pancreatic islet damage in 27-week-old diabetic mice. [Figure 38] Figures 38A-B show the immunohistochemical staining of pancreatic islets in PLG- / - mice after 28 days of plasminogen administration in a T1DM model. A shows the PBS-treated control group, and B shows the plasminogen-treated group. The results show that the positive expression of TNF-α (indicated by the arrow) in the plasminogen-treated group was significantly higher than that in the PBS-treated control group. This indicates that plasminogen can promote TNF-α expression and promote the repair of pancreatic islet damage in PLG- / - mice in a T1DM model. [Figure 39]Figures 39A-C show the immunohistochemical observation of islet IgM in PLG- / - mice after 28 days of plasminogen administration in a T1DM model. A is the blank control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results of this experimental study showed that the positive expression of IgM (indicated by the arrow) in the plasminogen-treated group was significantly lower than that in the PBS-treated control group, and the plasminogen-treated group was closer to the normal control group than the PBS-treated control group. This indicates that plasminogen can reduce IgM expression and attenuate islet damage in PLG- / - mice in a T1DM model. [Figure 40] Figures 40A-C show the results of TUNEL staining of pancreatic islets in 24-25 week-old diabetic mice after 31 days of plasminogen administration. A is the normal control group, B is the PBS-treated control group, and C is the plasminogen-treated group. The results of this experimental study showed that the normal control group had very low TUNEL staining. The number of positive cells (indicated by arrows) in the plasminogen-treated group was significantly lower than that in the PBS-treated control group. The apoptosis rate in the normal control group was approximately 8%, the apoptosis rate in the PBS-treated control group was approximately 93%, and the apoptosis rate in the plasminogen-treated group was approximately 16%. This indicates that plasminogen can significantly reduce apoptosis of pancreatic islet cells in diabetic mice. [Figure 41] Figure 41 shows the results of measuring serum insulin levels in T1DM model mice after 20 days of plasminogen administration. The serum insulin levels in the PBS-treated control group were significantly lower than those in the plasminogen-treated group, and the difference was statistically significant (P = 0.08). This indicates that plasminogen can promote insulin secretion in T1DM mice. [Figure 42]Figure 42A-D shows the results of islet GLP-1R staining in 24- to 25-week-old diabetic mice after 31 days of plasminogen administration. A is the normal control group, B is the PBS control group, C is the plasminogen-treated group, and D is the quantitative analysis result. The results show that the expression of GLP-1R in the islets of the PBS-treated mice (indicated by the arrow) was significantly lower than that of the normal control group, while the expression of GLP-1R in the islets of the plasminogen-treated mice was lower than that of the normal control group but significantly higher than that of the PBS-treated group, and the difference was statistically significant (* indicates P<0.05, ** indicates P<0.01). These results indicate that plasminogen can promote the expression of GLP-1R in the islets of diabetic mice. [Figure 43] Figures 43A-D show the results of immunohistochemistry of pancreatic GLP-1R in hyperlipidemic mice after 30 days of plasminogen administration. A represents the blank control group, B represents the PBS control group, C represents the plasminogen-treated group, and D represents the results of quantitative analysis. The results show that the expression of GLP-1R in the pancreatic islets of mice in the PBS control group was significantly lower than that of normal control mice, while the expression of GLP-1R in the pancreatic islets of mice in the plasminogen-treated group was lower than that of the blank control group but significantly higher than that of the PBS control group, and the difference was statistically significant (** indicates P<0.01). These results indicate that plasminogen can promote the expression of GLP-1R in the pancreatic islets of hyperlipidemic mice. [Figure 44]Figure 44A-C shows the results of immunohistochemistry of pancreatic GLP-1R in 14- to 15-week-old db / db mice after 28 days of plasminogen administration. A is the PBS control group, B is the plasminogen-treated group, and C is the quantitative analysis result. The results show that the expression of GLP-1R in pancreatic islets (indicated by the arrow) in the PBS control group was significantly lower than that in the plasminogen-treated group, and the difference was statistically significant (P = 0.09). These results indicate that plasminogen can promote the expression of GLP-1R in pancreatic islets in relatively young (14- to 15-week-old) diabetic mice. [Figure 45] Figures 45A-C show the results of immunohistochemical staining of hepatic GLP-1R in atherosclerosis model mice after 30 days of plasminogen administration. A shows the control group treated with PBS, B shows the plasminogen-treated group, and C shows the results of quantitative analysis. The results show that the expression of hepatic GLP-1R (indicated by the arrow) in the plasminogen-treated mice was significantly higher than that in the control group treated with PBS, and the difference was statistically significant (*** indicates P<0.001). These results suggest that plasminogen can promote hepatic GLP-1R expression in atherosclerosis model mice, thereby promoting the synthesis, secretion, absorption, or oxidation of hepatic fat, potentially reducing blood lipid levels and improving hyperlipidemia. [Figure 46] Figures 46A-C show representative photographs of hepatic GLP-1R immunostaining in hyperlipidemia model mice after 30 days of plasminogen administration. A shows the control group treated with PBS, B shows the plasminogen-treated group, and C shows the results of quantitative analysis. The results show that hepatic GLP-1R expression (indicated by the arrow) in the plasminogen-treated mice was significantly higher than that in the PBS-treated control group, and the difference was statistically significant (P = 0.09). These results indicate that plasminogen can promote hepatic GLP-1R expression in hyperlipidemia model mice, thereby promoting the synthesis, secretion, absorption, or oxidation of liver fat, thereby reducing blood lipid levels and improving hyperlipidemia. [Figure 47]Figures 47A-C show the results of GLP-1R immunostaining in the substantia nigra of MPTP-induced Parkinson's disease model mice after 14 days of plasminogen administration. A is the control group administered with PBS, B is the plasminogen-administered group, and C is the result of quantitative analysis. The results show that the expression of GLP-1R in the substantia nigra of the plasminogen-administered mice (indicated by the arrow) was significantly higher than that of the control group administered with PBS, and the difference was statistically significant (* indicates P<0.05). These results indicate that plasminogen can promote the expression of GLP-1R in the substantia nigra of Parkinson's disease model mice. [Figure 48] Figure 48 shows the calculated results of body weight change after 28 days of plasminogen administration to obese model mice induced by a high-calorie diet. The results are calculated by subtracting the body weight on day 1 from the body weight on day 29. As a result, there was no significant change in body weight in the blank control group, while the body weight of the plasminogen-administered group decreased significantly, and the difference was statistically significant compared to the solvent PBS-administered control group (* indicates P<0.05). This indicates that plasminogen can promote weight loss in obese model mice. [Figure 49] Figure 49 shows the statistical results of body weight index after 28 days of plasminogen administration to high-calorie diet-induced obese mouse models. The results showed that the body weight index of the plasminogen-administered mice was significantly lower than that of the PBS-administered control group, and the difference was statistically significant (* indicates P<0.05, ** indicates P<0.01). Furthermore, the body weight index of the plasminogen-administered mice was closer to that of the blank control group than that of the PBS-administered control group. This indicates that plasminogen can significantly reduce the body weight index of obese mouse models and alleviate obesity. [Figure 50]Figure 50 shows the statistical results of Lee's index after 28 days of plasminogen administration to high-calorie diet-induced obese mouse models. The results showed that the Lee's index of the plasminogen-administered mice was significantly lower than that of the PBS-administered control group, and the difference was statistically significant (* indicates P<0.05). Furthermore, compared with the PBS-administered control group, the Lee's index of the plasminogen-administered mice was closer to that of the blank control group. This indicates that plasminogen can significantly reduce the Lee's index of obese mouse models and alleviate obesity. [Figure 51] Figure 51 shows the statistical results of abdominal fat index after 28 days of plasminogen administration to high-calorie diet-induced obese mouse models. The results showed that the abdominal fat index of the plasminogen-administered mice was significantly lower than that of the PBS-administered control group, and the difference was statistically significant (* indicates P<0.05). Furthermore, compared with the PBS-administered control group, the abdominal fat content of the plasminogen-administered mice was closer to that of the blank control group. This indicates that plasminogen can significantly reduce abdominal fat deposition in obese mouse models. [Figure 52] Figures 52A-D show the statistical results of H&E staining of fat vacuole area in abdominal fat of high-calorie diet-induced obese mice after 28 days of plasminogen administration. A is the blank control group, B is the PBS-treated control group, C is the plasminogen-treated group, and D is the quantitative analysis result. The results showed that the mean fat vacuole area in the plasminogen-treated group was significantly smaller than that in the PBS-treated control group, and the difference was statistically significant (** indicates P<0.01). Furthermore, compared with the PBS-treated control group, the fat vacuole area in the plasminogen-treated group was closer to that in the blank control group. This indicates that plasminogen can significantly reduce adipocyte size and abdominal fat deposition in obese mice. [Figure 53]Figure 53A-C shows Oil Red O stained photographs of the livers of 24- to 25-week-old diabetic mice after 35 days of plasminogen administration. A is the control group administered with PBS, B is the plasminogen-administered group, and C is the quantitative analysis result. As a result, the area of ​​lipid deposition in the livers of mice administered with plasminogen was significantly smaller than that of the control group administered with PBS, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can reduce fat deposition in the livers of diabetic mice. [Figure 54] Figures 54A-C show representative photographs of Oil Red O staining of livers in ApoE atherosclerosis model mice after 30 days of plasminogen administration. A is the control group administered with PBS, B is the plasminogen-administered group, and C is the quantitative analysis result. The results showed that fat deposition in the livers of mice administered with plasminogen was significantly less than that of the control group administered with PBS, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can reduce fat deposition in the livers of atherosclerosis model mice. [Figure 55] Figures 55A-C show the results of Oil Red O staining of the livers of 16-week-old hyperlipidemia model mice after 30 days of plasminogen administration. A is the control group administered with PBS, B is the plasminogen-administered group, and C is the quantitative analysis result. As a result, fat deposition in the livers of the plasminogen-administered mice was significantly less than that of the control group administered with PBS, and the difference was statistically significant in quantitative analysis (* indicates P<0.05). This indicates that plasminogen can improve fat deposition in the livers of hyperlipidemia model mice. [Figure 56]Figures 56A-D show the results of LFB staining of the corpus callosum in a mouse model of cuprizone-induced demyelination after 14 days of plasminogen administration. A represents the blank control group, B the PBS-treated control group, C the plasminogen-treated group, and D the quantitative analysis results. The morphology of the corpus callosum myelin in the blank control group was essentially normal, while the positive staining (indicated by the arrow) of the corpus callosum myelin in the plasminogen-treated group was significantly greater than that in the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can promote the regeneration of corpus callosum myelin in the mouse model of cuprizone-induced demyelination. [Figure 57] Figures 57A-D show the results of immunohistochemistry for neurofilament protein (NFP) in Cuprizone-induced demyelination model mice after 14 days of plasminogen administration. A is the blank control group, B is the PBS-treated control group, C is the plasminogen-treated group, and D is the quantitative analysis result. The results showed that the expression of NFP (indicated by the arrow) in the corpus callosum of the plasminogen-treated group was significantly higher than that of the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05). Furthermore, the expression of NFP in the corpus callosum of the plasminogen-treated group was closer to that of the blank control group than that of the PBS-treated control group. This indicates that plasminogen can promote NFP expression, thereby promoting nerve fiber regeneration. [Figure 58]Figure 58A-C shows the results of immunostaining for protein gene product 9.5 (PGP9.5) in burned skin of diabetic burn model mice after plasminogen administration. A is a representative photograph of PGP9.5 staining. A-C are representative photographs of the PBS-treated control group on days 4, 8, and 15, respectively, and d-F are representative photographs of the plasminogen-treated group on days 4, 8, and 15. B is the quantitative analysis result of immunostaining on days 4 and 8 of administration. C is the quantitative analysis result on day 15 of administration. The results showed that the positive expression of PGP9.5 in the burned skin of mice in the plasminogen-treated group was higher than that of the PBS-treated control group. Furthermore, the difference in PGP9.5 expression between the two groups of mice was close to statistical significance on day 8 and statistically significant on day 15 (* indicates P<0.05). This indicates that plasminogen can promote nerve regeneration in diabetic burn skin. [Example]

[0078] The human plasminogen used in the examples below was derived from donor plasma and was reported in the literature. [15-17] It is obtained by purifying plasminogen from plasma using an optimized process based on the method described in. The purity of plasminogen monomer is over 95%. Example 1

[0079] Example 1 relates to the promotion of plasminogen in the expression of GLP-1 in pancreatic islets of 14-15 week old diabetic mice. Twelve 14-15-week-old db / db male mice were randomly divided into two groups based on their weight: a plasminogen-treated group and a PBS-treated control group (6 mice per group). Plasminogen or PBS was administered starting on day 1. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. Mice were sacrificed on day 29, and the pancreas was removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS for 5 minutes each time. 5% normal sheep serum was added. The sections were then blocked with a solution (Vector Laboratories, Inc., USA) for 30 minutes; after the time had elapsed, the sheep serum solution was discarded, and rabbit anti-mouse GLP-1 antibody (Wuhan Boster Biological Technology, PB0742) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated for 1 hour at room temperature, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were then developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. After dehydration through graded alcohols, cleared with xylene, and mounted in neutral rubber, the sections were observed under a light microscope at 200x magnification. Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is normally expressed at low levels and whose expression can stimulate insulin secretion and inhibit glucagon secretion.

[18] . The results showed that the expression of GLP-1 (indicated by the arrow) in the pancreatic islets of mice in the PBS-treated control group (Fig. 1A) was significantly lower than that in the plasminogen-treated group (Fig. 1B), and the difference was statistically significant (Fig. 1C) (* indicates P<0.05). These results indicate that plasminogen can promote the expression of GLP-1 in the pancreatic islets of relatively young (14-15 week old) diabetic mice. Example 2

[0080] Example 2 relates to the promotion of plasminogen-induced GLP-1 expression in pancreatic islets of diabetic mice aged 23-25 ​​weeks. Thirteen 23-25 ​​week-old db / db male mice were selected and weighed on the day of the experiment (day 0). The db / db mice were randomly divided into two groups based on their weight: a plasminogen-treated group (7 mice) and a PBS-treated control group (6 mice). Plasminogen or PBS was administered starting on day 1. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These administrations were continued for 28 consecutive days. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through a graded alcohol series, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse GLP-1 antibody (Wuhan Boster Biological Technology, PB0742) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01M PBS for 5 minutes each time. The tissue was developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration with graded alcohols, clearing with xylene, and embedding in neutral rubber, the sections were observed under an optical microscope at 200x magnification. The results showed that the expression of GLP-1 (indicated by the arrow) in the pancreatic islets of mice in the PBS-treated control group (Figure 2A) was significantly lower than that in the plasminogen-treated group (Figure 2B). This result indicates that plasminogen can promote the expression of GLP-1 in the pancreatic islets of relatively old (23-25 ​​weeks old) diabetic mice. Example 3

[0081] Example 3 shows that plasminogen inhibits PLG in T1DM models. + / + Mouse pancreatic islets It is concerned with promoting the expression of GLP-1. 9-10 week old PLG + / + Eight male mice were randomly divided into two groups: a control group treated with PBS and a plasminogen-treated group (four mice in each group). After fasting for four hours, the mice in these two groups were given a single intraperitoneal injection of 200 mg / kg of STZ (Sigma S0130) to induce type 1 diabetes.

[19] Dosing began 12 days after injection. The first day of dosing was designated Day 1. The plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection. The control group received the same volume of PBS via tail vein injection for 28 consecutive days. On Day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through a graded alcohol series, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and then washed twice with 0.01 M PBS, for 5 minutes each time. The cells were blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes; after the time was up, the sheep serum was discarded and the cells were stained with rabbit anti-mouse GLP-1 antibody (Wuhan The sections were then incubated overnight at 4°C with a dropwise addition of a goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour and washed twice with 0.01M PBS for 5 minutes each time. The sections were then developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The sections were dehydrated through a graded alcohol series, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The results showed that the expression of GLP-1 in the pancreatic islets of mice in the PBS-treated control group (Fig. 3A) was significantly lower than that in the plasminogen-treated group (Fig. 3B), and the difference was statistically significant (** indicates P<0.01). These results indicate that plasminogen can promote the expression of GLP-1 in the pancreatic islets of T1DM mice. Example 4

[0082] Example 4 demonstrates that plasminogen reduces islet alpha cell proliferation, restores normal islet alpha cell distribution, and reduces glucagon secretion in 24-25 week old diabetic mice. Eleven db / db male mice and five db / m male mice aged 24–25 weeks were weighed and randomly divided into two groups: five in the plasminogen-treated group and six in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection. The PBS-treated control group received either the same volume of PBS via tail vein injection or no injection at all for 31 consecutive days. Mice in the normal control group received no treatment. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam, ab92517) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour and washed twice with 0.01 M PBS for 5 minutes each time. A DAB kit (Vector l After three rinses with water, the sections were counterstained with hematoxylin for 30 seconds and then rinsed with running water for 5 minutes. The sections were dehydrated in graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Pancreatic islet alpha cells synthesize and secrete glucagon and are distributed mainly in the peripheral areas of the islets. Compared with the plasminogen-treated group (Figure 4C), the PBS-treated group (Figure 4B) showed a significant increase in glucagon-positive cells (indicated by arrows), with the positive cells infiltrating the center of the islets. The glucagon-positive cells in the plasminogen-treated group were scattered around the periphery of the islets, and the islet morphology in the plasminogen-treated group was more similar to that of the normal control group (Figure 4A) compared with the PBS-treated group. This indicates that plasminogen significantly suppresses islet α-cell proliferation and glucagon secretion in 24-25 week-old diabetic mice, correcting the disrupted distribution of islet α-cells and suggesting that plasminogen can promote the repair of islet injury. Example 5

[0083] Example 5 relates to the fact that plasminogen suppresses the proliferation of pancreatic islet α-cells, restores normal distribution of pancreatic islet α-cells, and reduces glucagon secretion in 27-week-old diabetic mice. Nine 27-week-old db / db male mice and three db / m male mice were weighed and randomly divided into two groups: four in the plasminogen-treated group and five in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 35 consecutive days. Mice in the normal control group received no treatment. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker and incubated in 3% hydrogen peroxide for 15 minutes. Then, the sections were washed twice with 0.01 M PBS for 5 minutes each. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour. The sections were washed twice with 0.01 M PBS for 5 minutes each. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Pancreatic islet alpha cells synthesize and secrete glucagon and are distributed mainly in the peripheral areas of the islets. Compared with the plasminogen-treated group (Figure 5C), the PBS-treated group (Figure 5B) showed a significant increase in glucagon-positive cells (indicated by arrows), with the positive cells infiltrating the center of the islets, and the mean optical density quantitative analysis showed statistically significant differences (* indicates P<0.05) (Figure 5D). The glucagon-positive cells in the plasminogen-treated group were scattered around the periphery of the islets, and the islet morphology in the plasminogen-treated group was more similar to that of the normal control group (Figure 5A) compared with that in the PBS-treated group. This indicates that plasminogen significantly suppresses islet α-cell proliferation and glucagon secretion in 27-week-old diabetic mice, correcting the disrupted distribution of islet α-cells and suggesting that plasminogen can promote the repair of islet injury. Example 6

[0084] Example 6 shows the effect of plasminogen on PLG in a T1DM model. + / + Mouse Gurkha It is concerned with reducing gonorrhea. 9-10 week old PLG + / + Fifteen male mice were randomly divided into two groups based on body weight: a blank control group (5 mice) and a model group (10 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on blood glucose levels: a PBS-treated control group and a plasminogen-treated group, each consisting of five mice. Following group allocation, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker and incubated in 3% hydrogen peroxide for 15 minutes. The sections were washed twice with PBS for 5 minutes each time. They were then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes; after the time had elapsed, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam, ab92517) was added dropwise and incubated overnight at 4°C. They were then washed twice with 0.01M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were then developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The sections were dehydrated through a graded alcohol series, cleared with xylene, mounted in neutral rubber, and observed under a light microscope at 200x magnification. Pancreatic islet alpha cells synthesize and secrete glucagon and are distributed mainly in the peripheral areas of the islets. The positive expression of glucagon (indicated by the arrow) in the PBS-treated control group (Figure 6B) was significantly higher than that in the plasminogen-treated group (Figure 6C). The difference was statistically significant (* indicates P<0.05) (Figure 6D), and the plasminogen-treated group was closer to the blank control group (Figure 6A) than the PBS-treated control group. This indicates that plasminogen can significantly reduce glucagon secretion from islet α-cells in STZ-induced T1DM mice. Example 7

[0085] Example 7 relates to plasminogen lowering blood glucose in diabetic mice. Eight 24-25 week-old db / db male mice were randomly divided into two groups: five mice in the plasminogen-treated group and three mice in the PBS-treated control group. The day of the experiment (day 0) was used as the starting day for the study, and the mice were weighed and assigned to the groups. Starting on day 1, mice were administered plasminogen or PBS. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 31 consecutive days. After a 16-hour fast on days 10 and 31, blood glucose levels were measured using blood glucose test strips (Roche, Mannheim, Germany) on days 11 and 32. As a result, the blood glucose levels of the mice in the plasminogen-treated group were significantly lower than those of the control group treated with PBS, and the difference was statistically significant (* indicates P<0.05, ** indicates P<0.01). Furthermore, as the administration time increased, the blood glucose levels of the mice in the PBS-treated control group tended to increase, whereas the blood glucose levels of the plasminogen-treated group gradually decreased (Figure 7). This indicates that plasminogen has the effect of lowering blood glucose levels in diabetic animals. Example 8

[0086] Example 8 relates to the fact that plasminogen reduces fructosamine levels in diabetic mice. Five 24- to 25-week-old db / db male mice were selected. 50 μl of blood was collected from each mouse via the ocular venous plexus the day before dosing to measure serum fructosamine concentrations. The day of blood collection was designated day 0. Plasminogen administration began on day 1, and human plasminogen was administered via tail vein injection at 2 mg / 0.2 mL / mouse / day for 31 consecutive days. On day 32, the eyes were enucleated, blood was collected, and serum fructosamine concentrations were measured. Fructosamine concentrations were measured using a fructosamine assay kit (Nanjing Jiancheng, A037-2). Fructosamine levels reflect the average blood glucose level within 1 to 3 weeks. As a result, serum fructosamine levels after plasminogen administration were significantly reduced compared to before administration, and the difference was statistically significant (** indicates P<0.01) (Figure 8). This indicates that plasminogen can effectively lower serum fructosamine levels in diabetic animals. Example 9

[0087] Example 9 relates to the effect of plasminogen on serum fructosamine levels in 27-week-old diabetic mice. Nine 27-week-old db / db male mice were weighed on the first day of the experiment (day 0) and randomly divided into two groups based on weight: four mice in the plasminogen-treated group and five mice in the PBS-treated control group. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. Plasminogen or PBS was administered starting on day 1 for 35 consecutive days. Mice were sacrificed on day 36, and serum fructosamine concentrations were measured. Fructosamine concentrations were measured using a fructosamine assay kit (Nanjing Jiancheng, A037-2). The results showed that the serum fructosamine concentration in the plasminogen-treated group was significantly lower than that in the PBS-treated control group, and the difference was close to statistical significance (P = 0.06) (Figure 9). This indicates that plasminogen can lower the blood glucose fructosamine concentration in 27-week-old diabetic mice. Example 10

[0088] Example 10 relates to the fact that plasminogen reduces glycated hemoglobin levels in diabetic mice. Nine 27-week-old db / db male mice were weighed and randomly divided into two groups based on weight: four mice in the plasminogen-treated group and five mice in the PBS-treated control group. Starting on the first day of treatment, mice were administered plasminogen or PBS vehicle. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 35 consecutive days. On day 35, the mice were fasted for 16 hours. On day 36, the eyes were enucleated and blood was collected to measure plasma glycated hemoglobin levels. The content of glycated hemoglobin can usually reflect the patient's blood glucose control status within the last 8 to 12 weeks. As a result, the OD value of glycated hemoglobin in the plasminogen-treated mice was significantly lower than that in the PBS-treated control group, and the difference was statistically significant (** indicates P<0.01) (Figure 10). This indicates that plasminogen has the effect of lowering plasma glycated hemoglobin in diabetic mice. Example 11

[0089] Example 11 relates to the fact that plasminogen improves the glucose tolerance ability of diabetic mice. Nine 27-week-old db / db male mice and three db / m mice were recruited. The db / db mice were weighed and randomly divided into two groups based on body weight: four mice in the plasminogen-treated group and five mice in the PBS-treated control group. The db / m mice served as normal controls. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection for 10 consecutive days. Mice in the normal control group received no treatment. On day 11, the mice were fasted for 16 hours, and then each mouse received 5 g / kg body weight of 5% glucose via intraperitoneal injection. Blood glucose concentrations were measured using blood glucose test strips (Roche, Mannheim, Germany) at 0, 30, 60, 90, 120, and 180 minutes. The intraperitoneal glucose tolerance test (IPGTT) can test the body's ability to tolerate glucose. In the prior art, it is known that diabetes patients have impaired glucose tolerance. The experimental results showed that after intraperitoneal injection of glucose, the blood glucose levels of the plasminogen-treated mice were lower than those of the PBS-treated control group, and the glucose tolerance curve of the plasminogen-treated group was closer to that of normal mice than that of the PBS-treated control group (Figure 11), indicating that plasminogen can significantly improve the glucose tolerance of diabetic mice. Example 12

[0090] Example 12 shows the effect of plasminogen on PLG in a T1DM model. + / + It is about lowering blood sugar levels in mice. 9-10 week old PLG + / + Ten male mice were randomly divided into two groups: a control group treated with PBS and a plasminogen-treated group (five mice each). After fasting for four hours, the mice in both groups were given a single intraperitoneal injection of 200 mg / kg of streptozotocin (STZ) (Sigma S0130) to induce T1DM.

[19] Dosing began 12 days after STZ injection. The first day of dosing was counted as day 1. The plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection for 10 consecutive days. On day 11, the mice were fasted for 16 hours, after which blood glucose was measured using blood glucose test strips (Roche, Mannheim, Germany). As a result, the blood glucose level of the control mice administered with PBS was clearly higher than that of the plasminogen-administered mice, and the difference was statistically significant (*** indicates P<0.001) (FIG. 12). This indicates that plasminogen plays a key role in the PLG regulation of T1DM models. + / + This shows that it can significantly reduce blood sugar levels in mice. Example 13

[0091] Example 13 relates to the improvement of glucose tolerance in T1DM model mice by plasminogen. 9-10 week old PLG + / + Fifteen male mice were randomly divided into two groups based on their body weight: a blank control group (5 mice) and a model group (10 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with 200 mg / kg of STZ (Sigma, SO130) to induce type 1 diabetes.

[19] The blank control group was intraperitoneally injected with 0.25 ml of sodium citrate solution (pH 4.5). 12 days after STZ injection, blood glucose levels were measured with a glucometer, and the model mice were randomly divided into two groups based on blood glucose level: a PBS-administered control group and a plasminogen-administered group, each with five mice. Medication began after grouping, with the first day of administration being considered the first day. The plasminogen-administered group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, and the PBS-administered control group received the same volume of plasminogen. PBS was administered via tail vein injection for 28 consecutive days. Mice in the blank control group received no treatment. On the 28th day, the mice were fasted for 6 hours, and then 5g / kg body weight of 5% glucose solution was administered intraperitoneally. Blood glucose concentrations were measured using blood glucose test strips (Roche, Mannheim, Germany) at 0, 15, 30, 60, and 90 minutes after injection. The intraperitoneal glucose tolerance test (IPGTT) can test the body's ability to tolerate glucose. In the prior art, it is known that diabetes patients have impaired glucose tolerance. As a result, after glucose injection, the blood glucose concentration of the PBS-administered control group mice was significantly higher than that of the plasminogen-administered group, and the glucose tolerance curve of the plasminogen-administered group was closer to that of the normal mice group than that of the PBS-administered control group (Figure 13). This is because plasminogen acts as a PLG + / + It has been shown to improve glucose tolerance in a mouse T1DM model. Example 14

[0092] Example 14 relates to the effect of plasminogen on improving the glucose degradation capacity of mice in a T1DM model. Eight 9-10 week old C57 male mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group (four mice each). After fasting for four hours, the PBS-treated and plasminogen-treated mice were given a single intraperitoneal injection of 200 mg / kg body weight of streptozotocin (STZ) (Sigma S0130) to induce T1DM.

[19] Dosing began 12 days after STZ injection, with the first day of dosing counted as day 1. The plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection for 19 consecutive days. On day 20, the mice were fasted for 6 hours and then gavaged with 2 g / kg body weight of 20% glucose. After 60 minutes, blood was collected from the orbital venous plexus, centrifuged, and the supernatant was collected. Blood glucose was measured using a glucose assay kit (Shanghai Rongsheng 361500). The blood glucose levels in the PBS-treated control group were significantly higher than those in the plasminogen-treated group, and the difference was statistically significant (P = 0.04) (Figure 14). This indicates that plasminogen can improve the glucose degradation ability of mice in the T1DM model and lower blood glucose levels. Example 15

[0093] Example 15 relates to the stimulation of insulin secretion by plasminogen in diabetic mice. Nine 27-week-old db / db male mice were weighed on the first day of the experiment (day 0) and randomly divided into two groups based on weight: four mice in the plasminogen-treated group and five mice in the PBS-treated control group. Plasminogen or PBS was administered starting on day 1. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 35 consecutive days. On day 35, the mice were fasted for 16 hours. On day 36, the eyes were enucleated, blood was collected, and the supernatant was centrifuged. Serum insulin levels were measured using an insulin assay kit (Mercodia AB) according to the manufacturer's instructions. The serum insulin levels in the plasminogen-treated group were significantly higher than those in the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05) (Figure 15). This indicates that plasminogen can significantly promote insulin secretion in diabetic mice. Example 16

[0094] Example 16: Protective effect of plasminogen on the pancreas of diabetic mice. is. Seven 24- to 25-week-old db / db male mice were randomly divided into two groups based on body weight: four in the plasminogen-treated group and three in the PBS-treated control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 31 consecutive days. On day 32, the mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. The fixed pancreatic tissue was dehydrated through a graded alcohol series, cleared in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, submerged in water, stained with hematoxylin and eosin (HE staining), fractionated in 1% hydrochloric acid ethanol, blued with aqueous ammonia, and then dehydrated in a series of alcohols and mounted. The sections were then observed under an optical microscope at 200x and 400x magnification. In the PBS-treated control group (Figures 16A and 16B), most islets were atrophied, and the atrophied islet cells were replaced by acini (indicated by arrows). The acini at the edges of the islets proliferated, blurring the boundary between islets and acini. In the plasminogen-treated group (Figures 16C and 16D), most islets were larger than in the control group, and there was no acinar proliferation within the islets. Only a few acini remained within a few islets, demonstrating a clear boundary between islets and acini. The islet-to-pancreas area ratio in the plasminogen-treated group was nearly double that of the control group (Figure 16E). This indicates that plasminogen can promote the repair of islet damage in diabetic mice. Example 17

[0095] Example 17 relates to the reduction of islet collagen deposition in diabetic mice by plasminogen. Sixteen 24-25 week-old db / db male mice were randomly divided into two groups based on body weight: 10 mice in the plasminogen-treated group and 6 mice in the PBS-treated control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 31 consecutive days. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick and were deparaffinized, submerged, washed once in water, stained with 0.1% Sirius Red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, and then blued with 1% hydrochloric acid ethanol and aqueous ammonia, rinsed with running water, dried, mounted, and examined under an optical microscope at 200x magnification. Sirius Red staining can stain collagen persistently, and as a special staining method for pathological sections, Sirius Red staining can specifically reveal collagen tissue. The staining results showed that the collagen deposition (indicated by the arrow) in the islets of the plasminogen-treated mice (Fig. 17B) was significantly lower than that of the PBS-treated control group (Fig. 17A), and the difference was statistically significant (* indicates P<0.05) (Fig. 17C), indicating that plasminogen can reduce islet fibrosis in diabetic animals. Example 18

[0096] Example 18 relates to the fact that plasminogen reduces apoptosis of pancreatic islet cells in diabetic mice. Six db / db male mice aged 24-25 weeks were randomly divided into two groups based on their body weight. Mice were divided into two groups: four in the plasminogen-treated group and two in the PBS-treated control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 31 consecutive days. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once in water. They were then incubated in 3% hydrogen peroxide for 15 minutes and washed twice in water, for 5 minutes each time. The sections were blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 1 hour; after the time had elapsed, the serum was discarded, and the tissue was circled with a PAP marker. A rabbit anti-mouse caspase-3 antibody (Wuhan Boster Biological Technology, BA2142) was added dropwise and incubated overnight at 4°C, followed by two 5-minute washes with PBS. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added at room temperature for 1 hour and then washed twice with PBS for 5 minutes each time. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After stepwise dehydration, clearing, and mounting, the sections were observed under a light microscope at 400x magnification. Caspase-3 is the most important cleavage enzyme in the process of cell apoptosis, and its higher expression indicates a higher proportion of cells in a state of apoptosis.

[20] . The results of the present experiment showed that the expression of caspase-3 (indicated by the arrow) in the plasminogen-treated group (Fig. 18B) was significantly lower than that in the PBS-treated control group (Fig. 18A), indicating that plasminogen can reduce apoptosis of pancreatic islet cells. Example 19

[0097] Example 19 relates to the fact that plasminogen promotes insulin expression and secretion in 17-18 week old diabetic mice. Eight 17- to 18-week-old db / db male mice were randomly divided into two groups based on body weight: a plasminogen-treated group and a PBS-treated control group (four mice each). Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 35 consecutive days. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once in water. They were then incubated in 3% hydrogen peroxide for 15 minutes and washed twice in water, for 5 minutes each time. The sections were blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 1 hour; after the time had elapsed, the serum was discarded, and the tissue was circled with a PAP marker. Rabbit anti-mouse insulin antibody (Abcam, ab63820) was added dropwise and incubated overnight at 4°C, followed by two 5-minute washes with PBS. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added at room temperature for 1 hour and washed twice with PBS for 5 minutes each. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After stepwise dehydration, clearing, and mounting, the sections were observed under a light microscope at 200x magnification. As a result, the expression of insulin (indicated by the arrow) in the plasminogen-administered group (Fig. 19B) The plasminogen-induced islet function was significantly higher than that of the PBS-treated control group (Fig. 19A), and the difference was statistically significant (P = 0.15) (Fig. 19C). This indicates that plasminogen can promote the functional restoration of pancreatic islets and enhance insulin expression and secretion. Example 20

[0098] Example 20 relates to the fact that plasminogen promotes insulin expression and secretion in diabetic mice aged 24-25 weeks. Eight 24- to 25-week-old db / db male mice were randomly divided into two groups based on body weight: five in the plasminogen-treated group and three in the PBS-treated control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection for 31 consecutive days. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once in water. They were then incubated in 3% hydrogen peroxide for 15 minutes and washed twice in water, for 5 minutes each time. The sections were blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 1 hour; after the time had elapsed, the serum was discarded, and the tissue was circled with a PAP marker. Rabbit anti-mouse insulin antibody (Abcam, ab63820) was added dropwise and incubated overnight at 4°C, followed by two 5-minute washes with PBS. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added at room temperature for 1 hour and washed twice with PBS for 5 minutes each. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After stepwise dehydration, clearing, and mounting, the sections were observed under a light microscope at 200x magnification. The results showed that insulin expression (indicated by the arrow) in the plasminogen-treated group (Fig. 20B) was significantly higher than that in the PBS-treated control group (Fig. 20A), and the difference was statistically significant (P = 0.02) (Fig. 20C), indicating that plasminogen can effectively restore islet function and promote insulin expression and secretion. Example 21

[0099] Example 21 relates to the effect of plasminogen on the restoration of insulin synthesis and secretion function in diabetic mice. Nine 27-week-old db / db male mice were randomly divided into two groups based on body weight: four in the plasminogen-treated group and five in the PBS-treated control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 35 consecutive days. On day 35, mice were fasted for 16 hours, and then sacrificed on day 36. The pancreases were removed and fixed in 4% paraformaldehyde fixative. The fixed pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. The tissue was incubated with 3% hydrogen peroxide for 15 minutes, washed twice with water for 5 minutes each time, and then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 1 hour. After the time was up, the sheep serum was discarded, the tissue was circled with a PAP marker, and rabbit anti-mouse insulin antibody (Abcam, ab63820) was added dropwise and incubated overnight at 4°C. The tissue was then washed twice with PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature and washed twice with PBS for 5 minutes each time. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration, clearing, and mounting, the sections were observed under a light microscope at 200x magnification. The results showed that insulin expression (indicated by the arrow) in the plasminogen-treated group (Fig. 21B) was significantly higher than that in the PBS-treated control group (Fig. 21A), and the difference was statistically significant (P = 0.005) (Fig. 21C), indicating that plasminogen can effectively restore islet function and promote insulin expression and secretion in diabetic mice. Example 22

[0100] Example 22 relates to the promotion of plasminogen-mediated expression of the multi-directional nuclear transcription factor NF-kB in pancreatic islets of 24- to 25-week-old diabetic mice. Ten 24- to 25-week-old db / db male mice were randomly divided into two groups based on body weight: four in the plasminogen-treated group and six in the PBS-treated control group. Four db / m mice served as the normal control group, which remained untreated. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 31 consecutive days. On day 32, mice were sacrificed, and the pancreas was removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. The sections were incubated with 3% hydrogen peroxide for 15 minutes and washed twice with water for 5 minutes each time. They were then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 1 hour. After the time was up, the sheep serum was discarded, and the tissue was circled with a PAP marker. Rabbit anti-mouse NF-kB (Cell Signaling, 8242) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with PBS for 5 minutes each time. The secondary goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time. The sections were then stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. After graded dehydration, clearing, and mounting, the sections were examined under a light microscope at 200x magnification. NF-kB is a member of a family of transcription factor proteins that plays an important role in the process of inflammation and repair.

[21] . The results of the present study showed that the expression of NF-kB (indicated by the arrow) in the plasminogen-treated group (Figure 22C) was similar to that in normal control mice (Figure 22A) and significantly higher than that in the PBS-treated control group (Figure 22B), and the difference was statistically significant (* indicates P<0.05) (Figure 22D). This indicates that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB, thereby promoting the repair of pancreatic islet inflammation in 24-25 week-old diabetic mice. Example 23

[0101] Example 23 demonstrates that plasminogen reduces islet alpha cell proliferation, restores normal islet alpha cell distribution, and reduces glucagon secretion in 17-18 week old diabetic mice. Eight 17-18 week old db / db male mice and three db / m male mice were selected. The db / db mice were randomly divided into two groups based on their body weight: a plasminogen-treated group and a PBS-treated control group (four mice each). The db / m mice served as normal controls. Starting on day 1, mice were administered plasminogen or PBS vehicle. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 35 consecutive days. Normal control mice received no treatment. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through a graded alcohol series, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and then washed twice with 0.01 M PBS, for 5 minutes each time. The sections were blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes; after the time had elapsed, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam, ab92517) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with 0.01M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added for 1 hour at room temperature, followed by two 5-minute washes with 0.01M PBS each time. The sections were then developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The sections were dehydrated through a graded alcohol series, cleared with xylene, mounted in neutral rubber, and examined under a light microscope at 200x magnification. Pancreatic islet alpha cells synthesize and secrete glucagon and are mainly scattered in the peripheral areas of the islets. Compared with the plasminogen-treated group (Figure 23C), the PBS-treated group (Figure 23B) showed a significant increase in glucagon-positive cells (indicated by arrows), with the positive cells infiltrating the center of the islets. Quantitative analysis of the mean optical density showed statistically significant differences (** indicates P<0.01) (Figure 23D). The glucagon-positive cells in the plasminogen-treated group were scattered around the periphery of the islets. Compared with the PBS-treated group, the islet morphology in the plasminogen-treated group was more similar to that of the normal control group (Figure 23A). These results suggest that plasminogen significantly inhibits islet α-cell proliferation and glucagon secretion in 17- to 18-week-old diabetic mice, correcting the disrupted distribution of islet α-cells and promoting the repair of islet injury. Example 24

[0102] Example 24 relates to the promotion of plasminogen-induced insulin receptor substrate 2 (IRS-2) expression in pancreatic islets of 17-18 week old diabetic mice. Seven db / db male mice and three db / m male mice aged 17–18 weeks were randomly divided into two groups based on body weight: three in the plasminogen-treated group and four in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 35 consecutive days. Normal control mice received no treatment. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam, ab134101) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour and washed twice with 0.01 M PBS for 5 minutes each time. A DAB kit (Vector Laboratories, The sections were stained with hematoxylin (Bio-Rad, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated in graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under an optical microscope at 200x magnification. Insulin receptor substrate-2 (IRS-2) is a substrate for the activatable insulin receptor tyrosine kinase and is a key molecule in the insulin signaling pathway, and is crucial for the survival of pancreatic islet β cells. IRS-2 has a protective effect upon increased expression in islet β cells and is crucial for the maintenance of functional islet β cells. [22-23] . IRS-2 immunohistochemistry showed that the positive expression of IRS-2 (indicated by the arrow) in the islets of mice treated with PBS (Figure 24B) was significantly lower than that of the plasminogen-treated group (Figure 24C), and the difference was statistically significant (** indicates p<0.01) (Figure 24D). Furthermore, the plasminogen-treated group was closer to the blank control group (Figure 24A) than the PBS-treated group. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells of 17- to 18-week-old diabetic mice. Example 25

[0103] Example 25 relates to the promotion of plasminogen-mediated IRS-2 expression in pancreatic islets of 24- to 25-week-old diabetic mice. Eleven db / db male mice and five db / m male mice aged 24–25 weeks were randomly divided into two groups based on body weight: five in the plasminogen-treated group and six in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, designated as day 1, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 31 consecutive days. Normal control mice received no treatment. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam, ab134101) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. IRS-2 immunohistochemistry showed that the positive expression of IRS-2 (indicated by the arrow) in the islets of mice treated with PBS (Figure 25B) was significantly lower than that of the plasminogen-treated group (Figure 25C), and the difference was statistically significant (* indicates P<0.05) (Figure 25D). Furthermore, the plasminogen-treated group was closer to the normal control group (Figure 25A) than the PBS-treated group. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells of 24- to 25-week-old diabetic mice. Example 26

[0104] Example 26 relates to the promotion of plasminogen-mediated islet IRS-2 expression in 27-week-old diabetic mice. Nine 27-week-old db / db male mice and three db / m male mice were randomly divided into two groups based on body weight: four in the plasminogen-treated group and five in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, designated as day 1, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. Normal control mice received no treatment. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam, ab134101) was added dropwise and incubated overnight at 4°C. 0.01 M The sections were washed twice with PBS for 5 minutes each time. The secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), was incubated at room temperature for 1 hour, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were developed using a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. The sections were dehydrated through a graded alcohol series, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The IRS-2 immunohistochemistry results showed that the positive expression of IRS-2 (indicated by the arrow) in the pancreatic islets of the PBS-treated control mice (Fig. 26B) was significantly lower than that of the plasminogen-treated group (Fig. 26C), whereas the IRS-2 expression level in the plasminogen-treated group was similar to that of the normal control mice (Fig. 26A). This indicates that plasminogen can effectively increase the expression of IRS-2 in the pancreatic islet cells of 27-week-old diabetic mice. Example 27

[0105] Example 27 shows a method for preparing a plasminogen-containing plasminogen ion-transferase (PLG) + / + This study aims to promote the expression of IRS-2 in the pancreatic islets of T1DM mice. 9-10 week old PLG + / + Fifteen male mice were randomly divided into two groups based on body weight: a blank control group (5 mice) and a model group (10 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. Model mice were randomly divided into two groups based on blood glucose levels: a PBS-treated control group and a plasminogen-treated group, each consisting of five mice. Following group assignment, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. They were then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam, ab134101) was added dropwise and incubated overnight at 4°C. They were then washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added at room temperature for 1 hour. The sections were incubated for 1 h and then washed twice with 0.01 M PBS for 5 min each time. They were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 s, and then rinsed with running water for 5 min. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The IRS-2 immunohistochemistry results showed that the positive expression of IRS-2 (indicated by the arrow) in the islets of mice treated with PBS (Fig. 27B) was significantly lower than that of the plasminogen-treated group (Fig. 27C), and the plasminogen-treated group was closer to the blank control group (Fig. 27A) than the PBS-treated group. This suggests that plasminogen effectively increases the expression of IRS-2 in islet cells, improving insulin signaling and promoting PLG. + / + This indicates that damage to pancreatic islet beta cells in T1DM mice can be reduced. Example 28

[0106] Example 28 relates to the effect of plasminogen on the reduction of neutrophil infiltration into pancreatic islets in diabetic mice aged 24-26 weeks. Nine db / db male mice and three db / m male mice aged 24–26 weeks were randomly divided into two groups: four in the plasminogen-treated group and five in the PBS-treated control group. The db / db mice served as normal controls. The first day of the experiment was designated Day 0, and the mice were weighed and divided into groups. Starting on Day 2, plasminogen or PBS was administered, designated Day 1. The plasminogen-treated group received 2 mg / 0.2 mL of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection for 35 consecutive days. On Day 36, the mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. They were fixed with EDTA for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed with water. They were then incubated with 3% hydrogen peroxide for 15 minutes. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each. They were then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rat anti-mouse neutrophil antibody (cedarlane, CL8993AP) was added dropwise and incubated overnight at 4°C. They were then washed twice with 0.01 M PBS for 5 minutes each. The sections were incubated with goat anti-rat IgG (HRP) antibody (Abcam, ab97057) secondary antibody at room temperature for 1 hour, then washed twice with 0.01 M PBS for 5 minutes each time. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. The sections were dehydrated through a graded alcohol series, cleared with xylene, and mounted in neutral rubber. The sections were then examined under a light microscope at 400x magnification. Neutrophils are important members of the non-specific cellular immune system, and when inflammation occurs, neutrophils are attracted to the site of inflammation by chemotactic substances. Neutrophil immunohistochemistry showed that the number of positively expressing cells in the plasminogen-treated group (Fig. 28C) was lower than that in the PBS-treated control group (Fig. 28B), and the plasminogen-treated group was closer to the normal control group (Fig. 28A) compared with the PBS-treated control group, indicating that plasminogen can reduce neutrophil infiltration into the islets of diabetic mice. Example 29

[0107] Example 29 shows a method for preparing a plasminogen-containing plasminogen ion-transferase (PLG) - / - It is related to reducing islet neutrophil infiltration in a mouse model of T1DM. 9-10 week old PLG - / - Ten male mice were taken and randomly divided into two groups based on their weight. The mice were divided into a blank control group (3 mice) and a model group (7 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on their blood glucose levels: a PBS-treated control group (3 mice) and a plasminogen-treated group (4 mice). Following group assignment, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. They were fixed with EDTA for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed with water. They were then incubated with 3% hydrogen peroxide for 15 minutes. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each. They were then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rat anti-mouse neutrophil antibody (cedarlane, CL8993A) was added dropwise and incubated overnight at 4°C. They were then washed twice with 0.01 M PBS for 5 minutes each. The sections were incubated with goat anti-rat IgG (HRP) antibody (Abcam, ab97057) secondary antibody at room temperature for 1 hour, then washed twice with 0.01 M PBS for 5 minutes each time. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. The sections were dehydrated through a graded alcohol series, cleared with xylene, and mounted in neutral rubber. The sections were then examined under a light microscope at 400x magnification. The immunohistochemistry results of neutrophils showed that the number of positively expressing cells (indicated by arrows) in the plasminogen-treated group (Fig. 29C) was less than that in the PBS-treated control group (Fig. 29B), and the plasminogen-treated group was closer to the blank control group (Fig. 29A) than the PBS-treated control group. This suggests that plasminogen is a key regulator of PLG. - / - We have shown that it can reduce neutrophil infiltration into pancreatic islets in a mouse T1DM model. Example 30

[0108] Example 30 shows a method for preparing a plasminogen-containing plasminogen ion-transferase (PLG) + / + It is related to reducing islet neutrophil infiltration in a mouse model of T1DM. 9-10 week old PLG + / + Fifteen male mice were randomly divided into two groups based on body weight: a blank control group (5 mice) and a model group (10 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on blood glucose levels: a PBS-treated control group and a plasminogen-treated group, each consisting of five mice. Following group allocation, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once in water. They were fixed with EDTA for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed in water. 3% hydrogen peroxide was added. The tissue was then incubated for 15 minutes in 0.01M PBS, circled with a PAP marker, and incubated for 15 minutes in 3% hydrogen peroxide. The tissue was then washed twice with 0.01M PBS, for 5 minutes each time. Blocking was performed for 30 minutes in 5% normal sheep serum (Vector Laboratories, Inc., USA). After the time was up, the sheep serum was discarded, and rat anti-mouse neutrophil antibody (cedarlane, CL8993A) was added dropwise and incubated overnight at 4°C. The tissue was then washed twice with 0.01M PBS, for 5 minutes each time. The secondary antibody, goat anti-rat IgG (HRP) antibody (Abcam, ab97057), was then incubated for 1 hour at room temperature, followed by two washes with 0.01M PBS, for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 400x magnification. The immunohistochemistry results of neutrophils showed that the number of positively expressing cells (indicated by arrows) in the plasminogen-treated group (Fig. 30C) was less than that in the PBS-treated control group (Fig. 30B), and the plasminogen-treated group was closer to the blank control group (Fig. 30A) than the PBS-treated control group. This suggests that plasminogen is a key regulator of PLG. + / + We have shown that it can reduce neutrophil infiltration into pancreatic islets in a mouse T1DM model. Example 31

[0109] Example 31 describes the role of plasminogen in PLG in T1DM models. - / - It is concerned with promoting insulin synthesis and secretion in mice. 9-10 week old PLG - / - Ten male mice were randomly divided into two groups based on their body weight: a blank control group (3 mice) and a model group (7 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on their blood glucose levels: a PBS-treated control group (3 mice) and a plasminogen-treated group (4 mice). Following group assignment, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse insulin antibody (Abcam, ab63820) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour and washed twice with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Immunohistochemical results showed that the positive expression of insulin (indicated by the arrow) in the plasminogen-treated group (Fig. 31C) was significantly higher than that in the PBS-treated control group (Fig. 31B), and the plasminogen-treated group was closer to the blank control group (Fig. 31A) than the PBS-treated control group. This suggests that plasminogen plays a key role in the regulation of PLG in the T1DM model. - / - Mau These results indicate that it can promote insulin synthesis and secretion in the brain. Example 32

[0110] Example 32 describes the role of plasminogen in PLG in T1DM models. + / + It is about promoting insulin synthesis and expression in mice. 9-10 week old PLG + / + Fifteen male mice were randomly divided into two groups based on body weight: a blank control group (5 mice) and a model group (10 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on blood glucose levels: a PBS-treated control group and a plasminogen-treated group, each consisting of five mice. Following group allocation, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse insulin antibody (Abcam, ab63820) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour and washed twice with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Immunohistochemical results showed that the positive expression of insulin (indicated by the arrow) in the plasminogen-treated group (Fig. 32C) was significantly higher than that in the PBS-treated control group (Fig. 32B), and the plasminogen-treated group was closer to the blank control group (Fig. 32A) compared with the PBS-treated control group. This suggests that plasminogen plays a key role in the regulation of PLG in the T1DM model. + / + It has been shown to be able to promote insulin synthesis and expression in mice. Example 33

[0111] Example 33 shows a method for preparing a plasminogen-containing plasminogen ion-transferase (PLG) - / - It promotes the expression of the pancreatic islet multidirectional nuclear transcription factor NF-kB in a mouse model of T1DM. 9-10 week old PLG - / - Ten male mice were randomly divided into two groups based on their body weight: a blank control group (3 mice) and a model group (7 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). 12 days after STZ injection, blood glucose levels were measured using a glucometer, and the model mice were randomly divided into two groups based on blood glucose: a PBS-administered control group (3 mice) and a plasminogen-administered group (4 mice). Medication began after group division, with the first day of administration counted as day 1. The plasminogen-administered group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-administered control group received the same volume of PBS via tail vein injection. This administration continued for 28 consecutive days. No administration was given to the blank control group mice. On day 29, the mice were sacrificed, and the pancreases were removed and resuspended in 4% paraformaldehyde. The pancreatic tissue was fixed in aldehyde fixative. After fixation, the pancreatic tissue was dehydrated through a graded alcohol series, permeabilized with xylene, and then embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. The tissue was blocked for 30 minutes with 5% normal sheep serum (Vector Laboratories, Inc., USA). After the time was up, the sheep serum was discarded, and a rabbit anti-mouse NF-kB antibody (Cell Signal, 8242) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with 0.01 M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour, then washed twice with 0.01 M PBS for 5 minutes each time. They were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. Sections were observed under a light microscope at 200x magnification. The experimental results showed that the expression of NF-kB (indicated by the arrow) in the plasminogen-treated group (Figure 33C) was significantly higher than that in the PBS-treated control group (Figure 33B), indicating that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB, thereby promoting the repair of pancreatic islet inflammation. Example 34

[0112] Example 34 relates to the promotion of plasminogen-mediated expression of the multidirectional nuclear transcription factor NF-kB in pancreatic islets of 17- to 18-week-old diabetic mice. Seven 17- to 18-week-old db / db male mice were randomly divided into two groups based on body weight: three mice in the plasminogen-treated group and four mice in the PBS-treated control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 35 consecutive days. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse NF-kB antibody (Cell Signal, 8242) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01M PBS for 5 minutes each time. The tissue was developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. The sections were dehydrated in graded alcohols, cleared with xylene, and embedded in neutral rubber, and the sections were observed under an optical microscope at 200x magnification. The results of the present experiment showed that the expression of NF-kB (indicated by the arrow) in the plasminogen-treated group (Figure 34B) was significantly higher than that in the PBS-treated control group (Figure 34A). This indicates that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB, thereby promoting the repair of pancreatic islet inflammation in relatively young (17-18 week old) diabetic mice. Example 35

[0113] Example 35 shows the effect of plasminogen on the multidirectional nuclear transcription factor NF-α in 27-week-old diabetic mice. It is concerned with promoting the expression of kB. Nine 27-week-old db / db male mice and three db / m male mice were randomly divided into two groups based on body weight: four in the plasminogen-treated group and five in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, designated as day 1, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. Normal control mice received no treatment. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse NF-kB antibody (Cell Signal, 8242) was added dropwise and incubated overnight at 4°C. Washing twice with 0.01 M PBS for 5 minutes each time was performed. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated for 1 hour at room temperature, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The experimental results showed that the expression of NF-kB (indicated by the arrow) in the plasminogen-treated group (Figure 35C) was significantly higher than that in the PBS-treated control group (Figure 35B), and that the plasminogen-treated group was closer to the normal control group (Figure 35A) than the PBS-treated control group. This indicates that plasminogen can promote the expression of the multidirectional nuclear transcription factor NF-kB in relatively old (27-week-old) diabetic mice, thereby promoting the repair of pancreatic islet inflammation. Example 36

[0114] Example 36 relates to the promotion of plasminogen-induced TNF-α expression in pancreatic islets of 24- to 25-week-old diabetic mice. Eleven db / db male mice and five db / m male mice aged 24–25 weeks were randomly divided into two groups based on body weight: five in the plasminogen-treated group and six in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, designated as day 1, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection. The PBS-treated control group received either the same volume of PBS via tail vein injection or no injection at all for 31 consecutive days. Normal control mice received no treatment. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse TNF-α antibody (Abcam, ab34674) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour and washed twice with 0.01 M PBS for 5 minutes each time. A DAB kit (Vector l After three rinses with water, the sections were counterstained with hematoxylin for 30 seconds and then rinsed with running water for 5 minutes. The sections were dehydrated in graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Tumor necrosis factor-α (TNF-α) is produced primarily by activated monocytes / macrophages and is an important factor promoting inflammation.

[24] . The results of this study showed that the positive expression of TNF-α in the plasminogen-treated group (Figure 36C) was significantly higher than that in the PBS-treated control group (Figure 36B), and that the plasminogen-treated group was closer to the normal control group (Figure 36A) than the PBS-treated control group. This indicates that plasminogen can promote the expression of TNF-α in 24-25 week-old diabetic mice and promote the repair of pancreatic islet injury. Example 37

[0115] Example 37 relates to the promotion of plasminogen-induced TNF-α expression in pancreatic islets of 27-week-old diabetic mice. Nine 27-week-old db / db male mice and three db / m male mice were randomly divided into two groups based on body weight: four in the plasminogen-treated group and five in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, designated as day 1, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. Normal control mice received no treatment. On day 36, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse TNF-α antibody (Abcam, ab34674) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. They were dehydrated through graded alcohols, cleared with xylene, and mounted in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The results showed that the positive expression of TNF-α in the plasminogen-treated group (Figure 37C) was significantly higher than that in the PBS-treated control group (Figure 37B), and that the plasminogen-treated group was closer to the normal control group (Figure 37A) than the PBS-treated control group, indicating that plasminogen can promote the expression of TNF-α in 27-week-old diabetic mice and promote the repair of pancreatic islet injury. Example 38

[0116] Example 38 shows a method for preparing a plasminogen-containing plasminogen ion-transferase (PLG) - / - It is about promoting the expression of pancreatic islet TNF-α in a mouse T1DM model. 9-10 week old PLG - / - Ten male mice were randomly divided into two groups based on their body weight: a blank control group (3 mice) and a model group (7 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] , and a blank control group was administered intraperitoneally once for 0.25 m. 1 of sodium citrate solution (pH 4.5) was injected into the model mice. 12 days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on blood glucose levels: a PBS-treated control group (3 mice) and a plasminogen-treated group (4 mice). Following group division, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, the mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through a graded alcohol series, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse TNF-α antibody (Abcam, ab34674) was added dropwise and incubated overnight at 4°C. Washed twice with 0.01M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01M PBS for 5 minutes each time. The tissue was developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. The sections were dehydrated in graded alcohols, cleared with xylene, and embedded in neutral rubber, and the sections were observed under an optical microscope at 200x magnification. The results of this experimental study showed that the positive expression of TNF-α in the plasminogen-administered group (Fig. 38B) was significantly higher than that in the PBS-administered control group (Fig. 38A). This is because plasminogen acts as a - / - It has been shown that it can promote the expression of TNF-α in a mouse T1DM model and promote the repair of pancreatic islet damage. Example 39

[0117] Example 39 shows a method for preparing a plasminogen-containing plasminogen ion-transferase (PLG) - / - It is related to reducing pancreatic islet damage in a mouse model of T1DM. 9-10 week old PLG - / - Ten male mice were randomly divided into two groups based on their body weight: a blank control group (3 mice) and a model group (7 mice). After fasting for 4 hours, the model group mice were intraperitoneally injected with STZ (Sigma, SO130) at 200 mg / kg body weight to induce type 1 diabetes.

[19] The blank control group received a single intraperitoneal injection of 0.25 ml of sodium citrate solution (pH 4.5). Twelve days after STZ injection, blood glucose levels were measured using a glucometer. The model mice were randomly divided into two groups based on blood glucose levels: a PBS-treated control group (3 mice) and a plasminogen-treated group (4 mice). Following grouping, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 ml / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 28 consecutive days. Mice in the blank control group received no treatment. On day 29, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and goat anti-mouse IgM (HRP) antibody (Abcam, ab97230) was added dropwise and incubated at room temperature for 1 hour. The sections were then washed twice with 0.01 M PBS for 5 minutes each time. The sections were then stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and washed with running water for 5 minutes. The sections were dehydrated in graded alcohols, cleared in xylene, mounted in neutral rubber, and examined under an optical microscope at 200x magnification. IgM antibodies play an important role in the clearance of apoptotic and necrotic cells, and the level of IgM antibodies at the site of tissue and organ injury is positively correlated with the severity of injury. [25-26] Therefore, the level of IgM antibodies in a tissue or organ can reflect the damage status of that tissue or organ. The results of the study showed that the positive expression of IgM in the plasminogen-treated group (Fig. 39C) was significantly lower than that in the PBS-treated control group (Fig. 39B), and that the plasminogen-treated group was closer to the blank control group (Fig. 39A) compared with the PBS-treated control group. This indicates that plasminogen can reduce the expression of IgM, and that plasminogen can inhibit the expression of PLG. - / - These findings suggest that it can reduce pancreatic islet damage in a mouse T1DM model. Example 40

[0118] Example 40 relates to the effect of plasminogen on the reduction of apoptosis in pancreatic islet cells in 24-25 week old diabetic mice. Eleven db / db male mice and five db / m male mice aged 24–25 weeks were randomly divided into two groups based on body weight: five in the plasminogen-treated group and six in the PBS-treated control group. The db / db mice served as normal controls. Starting on the first day of treatment, designated as day 1, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection. The PBS-treated control group received either the same volume of PBS via tail vein injection or no injection at all for 31 consecutive days. Normal control mice received no treatment. On day 32, mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, and a drop of protease K working solution was added to cover the tissue. The sections were incubated at room temperature for 7 minutes and then diluted with 0.01 M protease K. The sections were washed three times with PBS for 3 minutes each time. A mixture of reagents 1 and 2 (5:45) from the TUNEL kit (Roche) was added dropwise and incubated at 37°C for 40 minutes. Then, three washes were made with 0.01 M PBS for 3 minutes each time. A 3% solution of hydrogen peroxide (hydrogen peroxide:methanol = 1:9) prepared with methanol was added dropwise and incubated at room temperature for 20 minutes in the dark. Then, three washes were made with 0.01 M PBS for 3 minutes each time. Reagent 3 from the TUNEL kit was added dropwise and incubated at 37°C for 30 minutes. Then, three washes were made with 0.01 M PBS. The sections were then stained with a DAB kit (Vector Laboratories, Inc., USA). After three washes, the sections were counterstained with hematoxylin for 30 seconds and rinsed with running water for 5 minutes. The sections were dehydrated in graded alcohols, cleared with xylene, mounted in neutral rubber, and examined under an optical microscope at 400x magnification. TUNEL staining can be used to detect breaks in nuclear DNA in tissue cells at the end of apoptosis. The results of this experimental study showed that the normal control group showed very low levels of TUNEL-positive staining (Figure 40A). The number of positive cells (indicated by the arrow) in the plasminogen-treated group (Figure 40C) was significantly lower than that in the PBS-treated control group (Figure 40B). The apoptosis rate in the normal control group was approximately 8%, the apoptosis rate in the PBS-treated control group was approximately 93%, and the apoptosis rate in the plasminogen-treated group was approximately 16%. This indicates that plasminogen can significantly reduce apoptosis of pancreatic islet cells in diabetic mice. Example 41

[0119] Example 41 relates to the ability of plasminogen to improve insulin secretion in mice in a T1DM model. Thirteen 9-10 week old C57 male mice were selected. After fasting for 4 hours, the mice were then streptomycin-treated. T1DM was induced by a single intraperitoneal injection of tozotocin (STZ) (Sigma S0130) at 200 mg / kg body weight.

[19] Twelve days after STZ injection, blood glucose was measured and the mice were randomly divided into two groups based on blood glucose level: a PBS-treated control group (6 mice) and a plasminogen-treated group (7 mice). After grouping, dosing began. Starting on the first day of dosing, the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 20 consecutive days. On day 21, the mice were fasted for 6 hours, after which blood was collected from the ocular venous plexus, centrifuged, and the supernatant was collected. Serum insulin concentrations were measured using an insulin assay kit (Mercodia AB) according to the manufacturer's instructions. The results showed that the insulin concentration in the PBS-treated control group was significantly lower than that in the plasminogen-treated group, and the difference was statistically significant (P = 0.08) (Figure 41). This indicates that plasminogen can promote insulin secretion in mice in the T1DM model. Example 42

[0120] Example 42 relates to the promotion of pancreatic GLP-1R expression in 24- to 25-week-old diabetic mice by plasminogen. Eleven db / db male mice and five db / m male mice aged 24–25 weeks were randomly divided into two groups based on their body weight: a plasminogen-treated group (5 mice) and a PBS-treated control group (6 mice). The db / m mice served as the normal control group. Starting on the first day of treatment, plasminogen or PBS was administered. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received either the same volume of PBS via tail vein injection or no liquid injection for 31 consecutive days. The normal control mice received no treatment. On day 32, the mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative. The fixed pancreatic tissue was dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse GLP-1R antibody (NOVUS, NBP1-97308) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration with graded alcohols, the sections were cleared with xylene, and embedded in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Glucagon-like peptide-1 receptor GLP-1R, a member of the glucagon receptor family, is a G protein-coupled receptor that can regulate blood glucose levels by promoting insulin secretion. [27-28] . The results showed that the expression of GLP-1R (indicated by the arrow) in the pancreatic islets of mice in the PBS-treated control group (Fig. 42B) was significantly lower than that in the normal control group (Fig. 42A), and that the expression of GLP-1R in the pancreatic islets of mice in the plasminogen-treated group (Fig. 42C) was lower than that in the normal control group but significantly higher than that in the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05, ** indicates P<0.01) (Fig. 42D). These results indicate that plasminogen can promote the expression of GLP-1R in the pancreatic islets of diabetic mice. Example 43

[0121] Example 43 relates to the promotion of pancreatic GLP-1R expression by plasminogen in hyperlipidemic model mice. Seventeen 9-week-old male C57 mice were fed a 3% cholesterol high-fat diet (Nantong Trophy Feed Technology Co., Ltd.) for 4 weeks to induce hyperlipidemia. [29-30]This model was designated a 3% cholesterol hyperlipidemia model. After modeling, mice were subsequently fed a 3% cholesterol high-fat diet. Five male wild-type mice of the same age served as a blank control group and were fed a normal maintenance diet throughout the experiment. Three days before dosing, 50 μL of blood was collected from each mouse to measure total cholesterol. Mice were randomly divided into two groups based on total cholesterol concentration and body weight: a plasminogen-treated group (nine mice) and a PBS-treated control group (eight mice). Starting on the first day of treatment, the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were administered for 30 consecutive days. Mice in the blank control group received no treatment. On day 31, the mice were sacrificed, and the pancreases were removed and fixed in 4% paraformaldehyde fixative for 24–48 hours. The fixed tissue was dehydrated through graded alcohols, permeabilized with xylene, and then embedded in paraffin. The tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. The tissue was then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and a rabbit anti-mouse GLP-1R antibody (NOVUS, NBP1-97308) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with 0.01 M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour, then washed twice with 0.01 M PBS for 5 minutes each time. They were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration through graded alcohols, cleared with xylene, and mounted in neutral rubber, the sections were observed under a light microscope at 200x magnification. The results showed that the expression of GLP-1R (indicated by the arrow) in the pancreatic islets of mice in the PBS-treated control group (Fig. 43B) was significantly lower than that in the normal control group (Fig. 43A), and that the expression of GLP-1R in the pancreatic islets of mice in the plasminogen-treated group (Fig. 43C) was lower than that in the blank control group but significantly higher than that in the PBS-treated group, and the difference was statistically significant (** indicates P<0.01) (Fig. 43D). These results indicate that plasminogen can promote the expression of GLP-1R in the pancreatic islets of hyperlipidemia model mice. Example 44

[0122] Example 44 relates to the promotion of plasminogen-induced GLP-1R expression in pancreatic islets of 14-15 week old diabetic mice. Twelve 14-15-week-old db / db male mice were randomly divided into two groups based on their weight: a plasminogen-treated group and a PBS-treated control group (6 mice per group). Plasminogen or PBS was administered starting on day 1. The plasminogen-treated group received 2 mg / 0.2 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. Mice were sacrificed on day 29, and the pancreas was removed and fixed in 4% paraformaldehyde fixative. After fixation, the pancreatic tissue was dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. Circle the tissue with a PAP marker and incubate in 3% hydrogen peroxide for 15 minutes. The sections were then blocked with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse GLP-1R antibody (NOVUS, NBP1-97308) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with 0.01M PBS for 5 minutes each. The sections were then incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour and washed twice with 0.01M PBS for 5 minutes each. The sections were then developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. After dehydration with graded alcohols, clearing with xylene, and embedding in neutral rubber, the sections were observed under an optical microscope at 200x magnification. The results showed that the expression of GLP-1R (indicated by the arrow) in the pancreatic islets of mice treated with PBS (Fig. 44A) was significantly lower than that of the plasminogen-treated group (Fig. 44B), and the difference was statistically significant (Fig. 44C) (P = 0.09). These results indicate that plasminogen can promote the expression of GLP-1R in the pancreatic islets of relatively young (14-15 week old) diabetic mice. Example 45

[0123] Example 45 relates to the promotion of hepatic GLP-1R expression in atherosclerosis model mice by plasminogen. Nineteen 6-week-old male APOE mice weighing 18–22 g were fed a high-fat model diet (TP2031, Nantong Trophy Feed Technology Co., Ltd.) for 16 weeks to establish an atherosclerosis model. [31-32]Three days before administration, all mice were weighed, and 50 μL of blood was collected from the ocular venous plexus. Plasma total cholesterol and HDL were measured, and the atherosclerotic index was calculated. One mouse was randomly selected, and the remaining mice were randomly divided into two groups based on the atherosclerotic index: a plasminogen-treated group and a PBS-treated control group, each consisting of nine mice. After grouping, administration began. Starting on the first day of administration, the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This administration continued for 30 consecutive days. On day 31, mice were sacrificed, and their livers were removed and fixed in 4% paraformaldehyde fixative for 24–48 hours. After fixation, the tissues were dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse GLP-1R antibody (NOVUS, NBP1-97308) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration with graded alcohols, the sections were cleared with xylene, and embedded in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The results showed that the expression of GLP-1R (indicated by the arrow) in the liver of mice in the plasminogen-treated group (Figure 45B) was significantly higher than that in the PBS-treated control group (Figure 45A), and the difference was statistically significant (Figure 45C) (*** indicates P<0.001). These results indicate that plasminogen can promote the expression of GLP-1R in the liver of atherosclerosis model mice, thereby promoting the synthesis, secretion, absorption, or oxidation of liver fat, lowering blood lipid levels, and potentially improving hyperlipidemia. Example 46

[0124] Example 46 relates to the promotion of hepatic GLP-1R expression by plasminogen in hyperlipidemic model mice. Seventeen 9-week-old male C57 mice were fed a 3% cholesterol high-fat diet (Nantong Trophy Feed Technology Co., Ltd.) for 4 weeks to induce hyperlipidemia. [29-30]This model was designated a 3% cholesterol hyperlipidemia model. After modeling, mice were subsequently fed a 3% cholesterol high-fat diet. Three days before dosing, 50 μL of blood was collected from each mouse to measure total cholesterol. Mice were randomly divided into two groups based on total cholesterol concentration and body weight: a plasminogen-treated group (nine mice) and a PBS-treated control group (eight mice). After grouping, treatment began. Starting on the first day of treatment, the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. This treatment was continued for 30 consecutive days. On day 31, mice were sacrificed, and their livers were removed and fixed in 4% paraformaldehyde fixative for 24–48 hours. After fixation, the tissues were dehydrated through graded alcohols, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse GLP-1R antibody (NOVUS, NBP1-97308) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration with graded alcohols, the sections were cleared with xylene, and embedded in neutral rubber. The sections were then observed under a light microscope at 200x magnification. The results showed that the expression of GLP-1R (indicated by the arrow) in the liver of mice treated with plasminogen (Figure 46B) was significantly higher than that of the PBS-treated control group (Figure 46A), and the difference was statistically significant (P=0.09) (Figure 46C). These results indicate that plasminogen can promote the expression of GLP-1R in the liver of hyperlipidemia model mice, thereby promoting the synthesis, secretion, absorption, or oxidation of liver fat, lowering blood lipid levels, and potentially improving hyperlipidemia. Example 47

[0125] Example 47 relates to the promotion of GLP-1R expression in the substantia nigra of Parkinson's model mice by plasminogen. Twelve 9-week-old C57 male mice were selected and weighed one day before constructing the model. The mice were then intraperitoneally injected with 5 mg / ml MPTP solution at 30 mg / kg body weight for five consecutive days to establish the Parkinson's disease model. [33-34] Preparation of MPTP solution: 10 ml of deionized water was drawn into a syringe and added to 100 mg of MPTP powder (Sigma, M0896) to make a 10 mg / ml mother solution. 1 ml of the mother solution was then drawn into an ampoule and 1 ml of deionized water was added for a final concentration of 5 mg / ml. After model construction, mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group (6 mice per group). The first day of treatment was designated Day 1. Mice in the plasminogen-treated group received human plasminogen at 1 mg / 0.1 ml / mouse / day via tail vein injection, while mice in the PBS-treated control group received the same volume of PBS via tail vein injection for 14 consecutive days. On Day 15, mice were sacrificed, and their brains were rapidly removed and fixed in 4% paraformaldehyde fixative for 24-48 hours. The fixed brain tissue was dehydrated in graded alcohols, permeabilized with xylene, and then embedded in paraffin. Sections were cut at designated locations to a thickness of 4 μm. The sections were deparaffinized, submerged, and washed once with water. The tissue was circled with a PAP marker, incubated in 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time. Blocking was performed with 5% normal sheep serum (Vector Laboratories, Inc., USA) for 30 minutes. After the time was up, the sheep serum was discarded, and rabbit anti-mouse GLP-1R antibody (NOVUS, NBP1-97308) was added dropwise and incubated overnight at 4°C. The sections were washed twice with 0.01 M PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added and incubated at room temperature for 1 hour, followed by two washes with 0.01 M PBS for 5 minutes each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and then rinsed with running water for 5 minutes. After dehydration with graded alcohols, the sections were cleared with xylene, and embedded in neutral rubber. The sections were then observed under a light microscope at 200x magnification. Parkinson's disease is characterized by a loss of dopaminergic signaling in nigrostriatal neurons, which also express GLP-1R.

[35] . The results showed that the expression of GLP-1R (indicated by the arrow) in the substantia nigra of mice treated with plasminogen (Fig. 47B) was significantly higher than that of the control group treated with PBS (Fig. 47A), and the difference was statistically significant (Fig. 47C) (* indicates P<0.05). These results indicate that plasminogen can promote the expression of GLP-1R in the substantia nigra of Parkinson's disease model mice. Example 48

[0126] Example 48 relates to the effect of plasminogen on body weight and fat content in obese mice. Mouse model and group assignment Fourteen 8-week-old C57 male mice were randomly divided into two groups based on body weight: a blank control group of 4 mice and a model group of 10 mice. The blank control group mice were fed a normal maintenance diet, while the model group mice were fed a 45% fat-calorie high-fat diet (TP23000, Nantong Trophy Feed Technology Co., Ltd.) for 12 weeks to establish an obesity model.

[36] In this study, the 45% fat-calorie high-fat diet is referred to as the "high-calorie diet." After 12 weeks, the model mice were weighed and randomly divided into two groups based on their weight: a plasminogen-treated group and a PBS-treated control group, each with five mice. Human plasminogen was dissolved in PBS. Mice in the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection; the PBS-treated control group received the same volume of PBS via tail vein injection; the blank control group received no treatment. The experimental animals were treated for 28 consecutive days (the first day of treatment was counted as day 1), and on day 29, the following procedures and measurements were performed. Measurements and Results Weight measurement The experimental animals were weighed on days 1 and 29 and the weight change was calculated. The weight on day 29 minus the weight on day 1 was shown as the result. As a result, there was no significant change in body weight in the blank control group, but the body weight of the plasminogen-administered group was significantly reduced compared to the PBS-administered control group, and the difference was statistically significant (* indicates P<0.05) (Figure 48). This indicates that plasminogen can significantly reduce the body weight of obese model mice. Body mass index measurement On day 29, the mice were weighed and measured for body length, and the body weight index was calculated. Body weight index = body weight (kg) / body length 2 (m). Body mass index is a standard currently widely used internationally to measure the degree of obesity and health of the human body. Body mass index can also be used as an index of the degree of obesity in obese animal models. [37-38]As a result, the body weight index of the mice in the plasminogen-administered group was significantly lower than that of the control group administered with PBS, and the difference was statistically significant (* indicates P<0.05). (Note: * indicates P<0.01), and the body weight index of the plasminogen-treated mice was closer to that of the blank control group than that of the PBS-treated control group (Figure 49). This indicates that plasminogen can significantly reduce the body weight index of obese model mice and alleviate obesity. Measuring Lee's index The mice were weighed on the 29th day, and then their body lengths were measured to calculate Lee's index. Lee's index = 3 ￣Weight (g) / Body length (cm). Lee's index is a valid index for reflecting the degree of obesity [39-40] The results showed that the Lee's index of the plasminogen-treated mice was significantly lower than that of the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05). Furthermore, the Lee's index of the plasminogen-treated mice was closer to that of the blank control group than that of the PBS-treated control group (Figure 50). This indicates that plasminogen can significantly reduce the Lee's index of obese model mice and alleviate obesity. Detection of intra-abdominal fat mass The mice were weighed on day 29 and then sacrificed, and abdominal fat was removed and weighed. Abdominal fat index (%) = (abdominal fat weight / body weight) * 100. As a result, the abdominal fat index of the plasminogen-treated mice was significantly lower than that of the PBS-treated control group, and the difference was statistically significant (* indicates P<0.05), and was closer to that of the blank control group mice (Figure 51). This indicates that plasminogen can significantly reduce abdominal fat deposition in obese model mice. Detection of vacuolar area in abdominal subcutaneous fat On day 29, the mice were sacrificed, and abdominal fat was removed and fixed in 4% paraformaldehyde fixative for 24–48 hours. After fixation, the tissue samples were dehydrated through a graded alcohol series, permeabilized with xylene, and then embedded in paraffin. Tissue sections were 4 μm thick. The sections were deparaffinized, submerged, stained with hematoxylin and eosin (HE staining), fractionated in 1% HCl-ethanol, blued with ammonia water, dehydrated through a graded alcohol series, and mounted. The sections were then examined under a light microscope at 200x magnification. The area of ​​fat vacuoles was analyzed using Image-Pro Plus image processing software. When energy intake exceeds energy expenditure in obese individuals, a large amount of lipids accumulates in fat cells, causing adipose tissue expansion, i.e., fat cell enlargement, and the area of ​​fat vacuoles increases.

[41] . The results showed that the fat vacuole area in the plasminogen-treated group (Figure 52C) was significantly smaller than that in the PBS-treated control group (Figure 52B), and the difference was statistically significant (** indicates P<0.01) (Figure 52D). Moreover, the fat vacuole area in the plasminogen-treated group was closer to that in the blank control group compared with that in the PBS-treated control group (Figure 52A). This indicates that plasminogen can significantly reduce adipocyte size and abdominal fat deposition in obese model mice. Example 49

[0127] Example 49 is a study on the effect of plasminogen on reducing lipid deposition in the liver. Ten male db / db mice aged 24–25 weeks were weighed on the day of the experiment (day 0) and randomly divided into two groups based on weight: a PBS-treated control group and a plasminogen-treated group, each with five mice. Plasminogen or PBS administration began on day 1. Mice in the plasminogen-treated group received plasminogen at 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection for 35 days. On day 36, mice were sacrificed, and liver tissue was collected and fixed in 4% paraformaldehyde fixative for 24–48 hours. The tissue was then submerged in 15% or 30% sucrose at 4°C overnight, embedded in OCT, and frozen. The sections were 8 μm thick, stained with Oil Red O for 15 minutes, separated in 75% alcohol for 5 seconds, and stained for nuclei with hematoxylin for 30 seconds. The sections were then mounted in glycerinated gelatin and examined under a light microscope at 200x magnification. Oil Red O staining can represent lipid deposition and reflect the degree of lipid deposition.

[42] . The staining results showed that the area of ​​fat deposits in the livers of mice in the plasminogen-treated group (Fig. 53B) was significantly smaller than that in the PBS-treated control group (Fig. 53A), and the difference was statistically significant (P = 0.02) (Fig. 53C), indicating that plasminogen can reduce fat deposition in the livers of diabetic mice. Example 50

[0128] Example 50 is a second study on plasminogen reducing lipid deposition in the liver. Thirteen 6-week-old male ApoE mice were fed a high-fat, high-cholesterol diet (Nantong Trophy, TP2031) for 16 weeks to induce atherosclerosis. [31-32]After modeling, the mice continued to be fed a high-fat, high-cholesterol diet. Three days before dosing, 50 μL of blood was collected from each mouse and the total cholesterol (T-CHO) content was measured. The model mice were randomly divided into two groups based on their T-CHO content: a PBS-treated control group (seven mice) and a plasminogen-treated group (six mice). Starting on day 1, the mice in the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. These treatments were continued for 30 days. The mice continued to be fed the model diet throughout the treatment period. On day 31, the mice were sacrificed, and liver tissue was collected and fixed in 4% paraformaldehyde fixative for 24–48 hours. The sections were submerged overnight in 15% or 30% sucrose at 4°C, embedded in OCT, and then cut into 8-μm-thick frozen sections, stained with Oil Red O for 15 minutes, fractionated in 75% alcohol for 5 seconds, and stained for nuclei with hematoxylin for 30 seconds. The sections were then mounted in glycerinated gelatin and examined under a light microscope at 200x magnification. The staining results showed that the fat deposition in the livers of mice in the plasminogen-treated group (Fig. 54B) was significantly less than that in the PBS-treated control group (Fig. 54A), and the difference in quantitative analysis was statistically significant (P = 0.02) (Fig. 54C). This indicates that plasminogen can reduce fat deposition in the livers of atherosclerosis model mice. Example 51

[0129] Example 51 is the third study on plasminogen reducing lipid deposition in the liver. A model of hyperlipidemia was induced in 11 6-week-old male C57 mice by feeding them a high-fat, high-cholesterol diet (Nantong Trophy, TP2031) for 16 weeks. [29-30]This model was used as a 16-week-old hyperlipidemia model. After modeling, mice were subsequently fed a high-fat, high-cholesterol diet. Three days before dosing, 50 μL of blood was collected from each mouse to measure the total cholesterol (T-CHO) content. The model mice were randomly divided into two groups based on their T-CHO content: a PBS-treated control group (six mice) and a plasminogen-treated group (five mice). Starting on day 1, the plasminogen-treated group received 1 mg / 0.1 mL / mouse / day of human plasminogen via tail vein injection, while the PBS-treated control group received the same volume of PBS via tail vein injection. The mice continued to receive the model diet throughout the treatment period. After 30 days of treatment, the mice were sacrificed on day 31, and their livers were removed and fixed in 4% paraformaldehyde fixative for 24–48 hours. The sections were submerged overnight in 15% or 30% sucrose at 4°C, embedded in OCT, and then cut into 8-μm-thick frozen sections, stained with Oil Red O for 15 minutes, fractionated in 75% alcohol for 5 seconds, and stained for nuclei with hematoxylin for 30 seconds. The sections were then mounted in glycerinated gelatin and examined under a light microscope at 200x magnification. As a result, the fat deposition in the liver of mice in the plasminogen-treated group (Fig. 55B) was significantly less than that in the PBS-treated control group (Fig. 55A), and the difference in quantitative analysis was statistically significant (* indicates P<0.05) (Fig. 55C). This indicates that plasminogen can reduce fat deposition in the liver of hyperlipidemia model mice. Example 52

[0130] Example 52 relates to the effect of plasminogen on the regeneration of corpus callosum myelin in a mouse model of cuprizone-induced demyelination. Twenty 8-week-old C57 male mice were randomly divided into two groups: a blank control group of 6 mice and a model group of 14 mice. The blank control group mice were fed a normal maintenance diet, while the model group mice were fed a 0.2% cuprizone model diet (Nantong Trophy Feed Technology Co., Ltd.) for 6 weeks to induce the mouse myelin loss model.

[43] After 6 weeks, the model mice were further randomly divided into two groups based on body weight: a plasminogen-treated group and a PBS-treated control group, each consisting of seven mice. Plasminogen was administered to the plasminogen-treated group at 1 mg / 0.1 mL / mouse per day via tail vein injection. The PBS-treated control group received the same volume of PBS via tail vein injection, while the blank control group received no injection. The administration continued for 14 consecutive days. All mice were fed a normal maintenance diet during the administration period. The first day of administration was designated day 1. On day 15, mice were dissected and their brains were removed. They were fixed in 4% paraformaldehyde fixative, dehydrated, and embedded. After fixation, the tissue samples were dehydrated through graded alcohols, permeabilized in xylene, and embedded in paraffin. Coronal sections of the brain tissue were 3 μm thick. After deparaffinization and immersion, the sections were stained with LFB myelin stain. The sections were dehydrated in graded alcohols, cleared with xylene, and then encapsulated in neutral rubber. They were then observed under an optical microscope and photographed. LFB (luxol fast blue) staining is an effective method for staining myelin using luxol fast blue staining, and for studying the positioning of the corticospinal tract and the morphological observation of myelin lesions, damage, and regenerative repair. [44-45] . The results showed that the morphology of corpus callosum myelin in the blank control group (Fig. 56A) was essentially normal, whereas the positive staining (indicated by the arrows) in the corpus callosum myelin in the plasminogen-treated group (Fig. 56C) was significantly greater than that in the PBS-treated control group (Fig. 56B), and the difference was statistically significant (Fig. 56D) (* indicates P<0.05). This indicates that plasminogen can promote the regeneration of corpus callosum myelin in the cuprizone-induced demyelination model mice. Example 53

[0131] Example 53 relates to the promotion of neurofilament protein expression in injured nerves by plasminogen. Twenty 8-week-old C57 male mice were randomly divided into two groups: a blank control group of 6 mice and a model group of 14 mice. The blank control group mice were fed a normal maintenance diet, while the model group mice were fed a 0.2% cuprizone model diet (Nantong Trophy Feed Technology Co., Ltd.) for 6 weeks to induce the mouse myelin loss model.

[43] After 6 weeks, the model mice were further randomly divided into two groups based on body weight: a plasminogen-treated group and a PBS-treated control group, each consisting of seven mice. Plasminogen was administered to mice in the plasminogen-treated group at 1 mg / 0.1 mL / mouse / day via tail vein injection. The PBS-treated control group received the same volume of PBS via tail vein injection. The blank control group received no injection. The treatment was continued for 14 consecutive days. All mice were fed a normal maintenance diet during the treatment period. The first day of treatment was designated Day 1. On Day 15, mice were dissected and their brains were removed. They were fixed in 4% paraformaldehyde fixative, dehydrated, and embedded. Fixed tissue samples were dehydrated through graded alcohols, permeabilized in xylene, and then embedded in paraffin. Coronal sections of the brain tissue were 3 μm thick. The sections were deparaffinized, immersed, and washed once in water. Repaired with citric acid for 30 minutes, cooled at room temperature for 10 minutes, then gently rinsed with water. The sections were then incubated in 10% hydrogen peroxide for 15 minutes, and the tissue was circled with a PAP marker. They were then blocked with 10% sheep serum (Vector Laboratories, Inc., USA) for 1 hour; after the time had elapsed, the sheep serum was discarded, and rabbit anti-NFP antibody (Abcam, ab207176) was added dropwise and incubated overnight at 4°C, followed by two 5-minute washes with PBS. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and then washed twice with PBS for 5 minutes. The sections were developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, blued in running water for 5 minutes, and washed once with PBS. After stepwise dehydration, clearing, and mounting, the sections were examined under a light microscope at 200x magnification. Neurofilament proteins (NFPs) are proteins that form the intermediate filaments of nerve cell axons. Their function is to provide elasticity to nerve fibers, making them easier to stretch and preventing breakage, and they are crucial for maintaining the cytoskeleton, stabilizing cell morphology, and axonal transport.

[46] . The results showed that the expression of NFP in the corpus callosum (indicated by the arrow) in the plasminogen-treated group (Fig. 57C) was significantly higher than that in the PBS-treated control group (Fig. 57B), and the difference was statistically significant (* indicates P<0.05). Furthermore, the expression of NFP in the corpus callosum in the plasminogen-treated group was closer to that in the blank control group (Fig. 57A) than in the PBS-treated control group. This indicates that plasminogen can promote NFP expression, thereby promoting nerve fiber regeneration. Example 54

[0132] Example 54 relates to the promotion of cutaneous nerve regeneration by plasminogen. Thirty female db / db mice were used. Before the experiment, non-fasting blood glucose (blood glucose above 15 mM) was measured and their weights were measured. The mice were divided into two groups based on blood glucose and weight: a control group administered PBS and a plasminogen-treated group, each with 15 mice. All mice were anesthetized by intraperitoneal injection of 50 mg / kg body weight of sodium pentobarbital. After anesthetizing the mice, a portion of the hair on their backs was removed. A copper block was heated to 95-100°C in boiling water, and immediately after removal, it was lightly touched perpendicularly to the bald area of ​​the mouse for 6 seconds. Avoiding excessive pressure during contact, the skin burn model was constructed.

[47] Five minutes after the model was established, mice in the plasminogen-treated group received plasminogen at 2 mg / 0.2 mL / mouse / day via tail vein injection, while the control group received the same volume of PBS via tail vein injection. The first day of treatment was designated Day 1. On Days 4 and 8, five mice from each group were sacrificed and their burned skin was removed. On Day 15, the remaining mice were sacrificed and their burned skin was removed. The skin was fixed in 4% paraformaldehyde fixative for 24–48 hours and embedded in paraffin. Sections were 3 μm thick. The sections were deparaffinized, immersed in water, and washed once with water. They were then fixed in citric acid for 30 minutes, cooled at room temperature for 10 minutes, and gently rinsed with water. They were then incubated in 3% hydrogen peroxide for 15 minutes, and the tissue was circled with a PAP marker. The sections were blocked with 10% sheep serum (Vector Laboratories, Inc., USA) for 1 hour; after the time had elapsed, the sheep serum was discarded, and rabbit anti-PGP9.5 antibody (Abcam, ab10404) was added dropwise and incubated overnight at 4°C. The sections were then washed twice with PBS for 5 minutes each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added at room temperature for 1 hour, followed by two 5-minute washes with PBS. The sections were then developed with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, blued in running water for 5 minutes, and washed once with PBS. The sections were dehydrated stepwise, cleared, and mounted, and the sections were observed under a light microscope at 200x magnification. Protein gene product 9.5 PGP9.5 is a specific ubiquitin hydroxyhydrolase in nerve fibers. As a marker of nerve axons, anti-PGP9.5 antibodies can bind to any nerve fiber, whether unmyelinated or myelinated. [48-49] . The results showed that the positive expression of PGP9.5 in the burned skin of mice in the plasminogen-treated group was higher than that in the control group treated with PBS. Furthermore, the difference in PGP9.5 expression between the two groups of mice approached statistical significance on day 8 and reached statistical significance on day 15 (* indicates P<0.05) (Figure 58). This indicates that plasminogen can promote nerve regeneration in diabetic burn skin. A is a representative photograph of PGP9.5 staining; a-c are representative photographs of the control group treated with PBS on days 4, 8, and 15, respectively; d-f are representative photographs of the plasminogen-treated group on days 4, 8, and 15. B is the quantitative analysis result of immunostaining on days 4 and 8 of treatment; C is the quantitative analysis result on day 15 of treatment. References: [1] Habener JF, Stanojevic V. cells come of age. Trends Endocrinol Metab, 2013, 24: 153-163. [2]Tura A, Muscelli E, Gastaldelli A et al. Altered pattern of the incretin effect as assessed by modeling in individuals with glucose tolerance ranging from normal to diabetic. Diabetologia, 2014, 57: 1199-1203. [3]Kang ZF, Deng Y, Zhou Y et al. Pharmacological reduction of NEFA restores the Efficacy of incretin-based therapies through GLP-1 receptor signaling in the beta cell in mouse models of diabetes. Diabetologia, 2013, 56: 423-433. [4]Wilding J. Managing patients with type 2 diabetes and obesity. Practitioner, 2015, 259: 25-28. [5]Perry TA, Greig NH. A new Alzheimer’s disease interventive strategy: GLP-1 [J]. Curr Drug Targets,2004,5(6):565-571. [6]During MJ, Cao L, Zuzga DS et al. Glucagon-like peptide-1 receptor is involved in learning and neuroprotection[J].Nat Med,2003,9(9):1173-1179. [7]Gault VA, Holshcer C. GLP-1 agonists facilitate hippocampal LTP and reverse the impairment of LTP induced by beta-amyloid [J]. Eur J Pharmacol, 2008, 587:112-117. [8]Wang XH, Li L, Holsher C et al. Val8-glucagon-like peptide-1 protects against ab1-40-induced impairment of hippocamapllate-phase long-term potentiation and spatial learning in rats [J]. Neuroscience, 2010, 170:1239-1248. [9]Holscher C. Incretin anlogues that have been developed to treat type 2 diabetes hold promise as a novel treatment strategy for Alzheimer's disease [J]. Recent Pat CNS Drug Discov, 2010,5:109-117.

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Claims

1. A method for treating a disease by regulating GLP-1 / GLP-1R, comprising administering to a subject an effective amount of plasminogen.

2. The method according to claim 1, wherein the disease is a disease associated with a carbohydrate metabolism disorder, a disease associated with a fat metabolism disorder, or a GLP-1 / GLP-1R-associated nervous system disease.

3. 3. The method of claim 2, wherein the disease is one or more diseases selected from the group consisting of diabetes, diabetic nephropathy, diabetic neuralgia, diabetic retinopathy, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral column sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

4. A method for regulating the function of GLP-1 / GLP-1R, comprising administering to a subject an effective amount of plasminogen.

5. The method of claim 4, wherein the plasminogen promotes expression of GLP-1 and / or GLP-1R.

6. A method for treating a GLP-1 / GLP-1R-related disorder, comprising administering to a subject an effective amount of plasminogen.

7. 7. The method according to claim 6, wherein the GLP-1 / GLP-1R-associated disease comprises one or more selected from the group consisting of elevated blood glucose levels, impaired glucose tolerance, elevated blood lipids, obesity, fatty liver, and cognitive impairment.

8. The method of claim 6, wherein the GLP-1 / GLP-1R-related disease comprises one or more selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

9. The method of any one of claims 1 to 8, wherein the plasminogen is a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12.

10. The method according to any one of claims 1 to 9, wherein the plasminogen is a protein that contains plasminogen active fragments and still has plasminogen activity.

11. The method according to claim 10, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or a mutant thereof that retains plasminogen activity.

12. The method of any one of claims 1 to 11, wherein the plasminogen is used in combination with one or more other drugs or therapeutic methods.

13. The plasminogen is used in a drug or method for treating diabetes, a drug or method for treating atherosclerosis, a drug or method for treating cardiovascular disease, or a method for treating cerebrovascular disease.

13. The method of claim 12, which can be used in combination with one or more drugs or therapeutic methods selected from the group consisting of drugs or therapeutic methods for treating thrombosis, drugs or therapeutic methods for treating high blood pressure, drugs or therapeutic methods for lowering blood lipids, drugs or therapeutic methods for treating fatty liver, drugs or therapeutic methods for treating Parkinson's disease, drugs or therapeutic methods for treating Alzheimer's disease, and anti-infective drugs or therapeutic methods.

14. A drug for treating a GLP-1 / GLP-1R-associated disease, comprising an effective amount of plasminogen.

15. A product or kit for treating a GLP-1 / GLP-1R-related disorder, comprising a container containing an effective amount of plasminogen and a protocol explaining the use of plasminogen to treat a GLP-1 / GLP-1R-related disorder.

16. The drug, product, or kit according to claim 14 or 15, wherein the GLP-1 / GLP-1R-related disease comprises one or more selected from the group consisting of elevated blood glucose levels, impaired glucose tolerance, elevated blood lipids, obesity, fatty liver, and cognitive impairment.

17. The drug, product, or kit according to claim 14 or 15, wherein the GLP-1 / GLP-1R-related disease comprises one or more selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

18. Use of plasminogen in the manufacture of a medicament for treating a disease by regulating GLP-1 / GLP-1R.

19. The use according to claim 18, wherein the disease is a disease associated with a carbohydrate metabolism disorder, a disease associated with a fat metabolism disorder, or a GLP-1 / GLP-1R-related nervous system disease.

20. 20. The use of claim 19, wherein the disease is one or more diseases selected from the group consisting of diabetes, diabetic nephropathy, diabetic neuralgia, diabetic retinopathy, hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, liver cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral trabecular sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcer.

21. Use of plasminogen in the manufacture of a medicament for regulating the function of GLP-1 / GLP-1R.

22. The use according to claim 21, wherein the plasminogen promotes the expression of GLP-1 / GLP-1R.

23. Use of plasminogen in the manufacture of a medicament for treating a GLP-1 / GLP-1R related disease.

24. The use according to claim 23, wherein the GLP-1 / GLP-1R-associated disease comprises one or more selected from the group consisting of elevated blood glucose levels, impaired glucose tolerance, elevated blood lipids, obesity, fatty liver, and cognitive impairment.

25. The GLP-1 / GLP-1R-related diseases include diabetes, diabetic complications, hyperlipidemia, and atherosclerosis. The use of claim 23, comprising one or more selected from the group consisting of rheumatoid arteriosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, obesity, fatty liver, cirrhosis, osteoporosis, cognitive impairment, Parkinson's syndrome, lateral column sclerosis, Alzheimer's disease, inflammatory bowel disease, dyspepsia, and gastrointestinal ulcers.

26. 26. The use according to any one of claims 18 to 25, wherein the plasminogen is a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12.

27. The use according to any one of claims 18 to 26, wherein the plasminogen is a protein that comprises plasminogen active fragments and still has plasminogen activity.

28. The use according to claim 27, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or a mutant thereof that retains plasminogen activity.

29. The use according to any one of claims 18 to 28, wherein the plasminogen can be used in combination with one or more other drugs or therapeutic methods.

30. The use of claim 29, wherein the plasminogen can be used in combination with one or more drugs or therapeutic methods selected from the group consisting of drugs or therapeutic methods for treating diabetes, drugs or therapeutic methods for treating atherosclerosis, drugs or therapeutic methods for treating cardiocerebrovascular disease, drugs or therapeutic methods for treating thrombosis, drugs or therapeutic methods for treating hypertension, drugs or therapeutic methods for lowering blood lipids, drugs or therapeutic methods for treating fatty liver, drugs or therapeutic methods for treating Parkinson's disease, drugs or therapeutic methods for treating Alzheimer's disease, and anti-infective drugs or therapeutic methods.

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