Method and drug for promoting degradation of misfolded protein and aggregate thereof

Abstract

To provide a pharmaceutical composition that promotes the degradation of a misfolded protein and an aggregate thereof, involving the administration of a therapeutically effective amount of plasminogen activation pathway components to a subject.SOLUTION: The present invention provides a pharmaceutical composition containing plasminogen for promoting degradation of a misfolded protein and an aggregate thereof, wherein the plasminogen has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a specific sequence and still retains the proteolytic activity of plasminogen.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for promoting the degradation of misfolded proteins and their aggregates, comprising administering to a subject an effective amount of a component of the plasminogen activation pathway or a related compound, e.g., plasminogen, to promote the degradation of misfolded proteins and their aggregates and to prevent and treat diseases caused by misfolded proteins and / or their aggregates. [Background technology]

[0002] Diseases caused by protein misfolding or its polymerization include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, polyglutamine diseases, and prion-like diseases (Creutzfeldt-Jakob disease (CJD), Gerstmann syndrome (GSS), fatal sporadic or familial isomnia, and Kuru), among others. Although the etiology is different, each with a selective lesion site, and the location of the disease gene is different, they share common pathological features, namely, degeneration, death, and intraneuronal inclusions formed by aggregation of mutant proteins in specific regions of neurons, such as senile plaques and tangles in the brains of Alzheimer's disease patients, eosinophilic Lewy bodies remaining in dopaminergic neurons of Parkinson's disease patients, Bunina bodies in spinal cord and brainstem neurons of amyotrophic lateral sclerosis patients, and intranuclear inclusions in neurons involved in polyglutamine diseases patients. Current research suggests that these neuronal degeneration, death, and inclusion formation are closely related to aggregates formed by misfolded proteins.

[0003] In recent years, studies have confirmed that misfolded proteins in cells can be refolded by molecular chaperones, such as heat shock proteins, and can also be degraded by the ubiquitin-proteasome system (Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegen-eration, Nature, 2006, 443(7113):780-786). When misfolded proteins are overproduced beyond the degradation capacity or when proteasome function itself is impaired, the misfolded proteins and their aggregates are selectively mediated and ubiquitinated by polyubiquitin chains linked mainly in the K-63 manner, and the ubiquitinated proteins and their aggregates interact with histone deacetylase 6 (HDAC6) (microtubule-associated α-tubulin deacetylase), and the retrograde transport of dynein motors mediated by HDAC6 along microtubules reaches the encapsulated protein aggregates near the center of the microtubule organization, a process called the protein aggregate pathway. Studies have shown that the formation of protein aggregates may be a protective mechanism against the corresponding diseases, and its role may be to recruit potentially cytotoxic misfolded proteins and their aggregates to form protein aggregates to reduce the cytotoxic effects.

[0004] Alzheimer's disease and protein aggregates Alzheimer's disease is characterized by the formation of extensive extracellular Aβ plaques and intracellular neuronal fibrillary tangles (Braak H, Braak E. Evolution of neuronal changes in the course of Alzheimer's disease, Neural Transm Suppl, 1998, 53:127-140). Many research results indicate that there is a certain association between Alzheimer's disease and the aggregate pathway.

[0005] Parkinson's disease is a common neurodegenerative disorder characterized primarily by selective loss of dopaminergic neurons and the appearance of Lewy bodies (Spillantini MG, Schmidt ML, Lee VM, et al. Alpha-synuclein in Lewy bodies, Nature, 1997, 388(6645):839-840). The disease is associated with the formation of inclusion bodies. The main protein component of Lewy bodies in sporadic Parkinson's disease is alpha-synuclein.

[0006] Polyglutamine diseases are slow-onset, progressive neurodegenerative disorders caused by abnormal repeats of the glutamine-encoding CAG sequence, such as Huntington's disease, spinocerebellar ataxia type 3, and dentatorubral-pallidoluysian atrophy. In Huntington's disease, a polyglutamine branch in exon 1 of the Huntington protein that exceeds 37 uninterrupted glutamines causes disease.

[0007] The main pathological feature of patients with amyotrophic lateral sclerosis is the formation of Bunina bodies, Skein-like inclusions, and Lewy body-like inclusions (Mizuno Y, Fujita Y, Takatama M, et al. sclerosis, J Neurol Sci, 2011, 302(1 / 2):14-18), but the specific mechanism of their formation remains unknown.

[0008] Additionally, the following diseases have been reported in the literature to be associated with misfolded / misconformed / abnormally aggregated proteins: cystic fibrosis and cystic fibrosis transmembrane conductance regulator (CFTR), emphysema and liver damage and α1-antitrypsin, Parkinson's disease and Parkin protein, cataracts and crystallin, familial amyloidosis and transthyretin, short-chain acyl-CoA dehydrogenase (SCAD) deficiency and short-chain acyl-CoA dehydrogenase variant, familial hypercholesterolemia and low density lipoprotein receptor, huntingtin and huntingtin, amyotrophic lateral sclerosis and neurofilament protein. Amyotrophic Lateral Sclerosis and Peripherin, Motor Neuron Disease and α-Internexin, Diabetes and Islet Amyloid Polypeptide, Dialysis-Associated Amyloidosis and β2-Microglobulin, Amyloidosis and Serum Amyloid A Protein, Amyloidosis and Immunoglobulin Light Chains, Amyloidosis-Related Human Diseases and Human Lysozyme, Amyloidosis and α-Lactalbumin, Amyloidosis and Apolipoprotein E, Amyloidosis and Apolipoprotein J.

[0009] Currently, promoting the refolding or proteasomal degradation of misfolded proteins is still a suitable method for the treatment of neurodegenerative diseases, and there is a need to find related drugs that can provide effective methods for the clinical treatment of neurodegenerative diseases. Summary of the Invention

[0010] Through research, the present invention has discovered that plasminogen can promote the recovery of memory function in Alzheimer's disease patients, improve cognitive ability, significantly alleviate and alleviate various clinical symptoms and signs of Alzheimer's disease, and prevent and treat Alzheimer's disease.

[0011] Specifically, the present invention relates to the following:

[0012] In one aspect, the present application relates to a method for promoting degradation of misfolded proteins and aggregates thereof, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.

[0013] In one aspect, the present application relates to the use of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors, in the preparation of a medicament for promoting the degradation of misfolded proteins and aggregates thereof.

[0014] In one aspect, the present application relates to a drug or pharmaceutical composition that promotes the degradation of misfolded proteins and their aggregates, comprising one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0015] 2. The method, use, agent or pharmaceutical composition according to item 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA and uPA.

[0016] 3. The method, use, agent or pharmaceutical composition according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, alpha2-antiplasmin or alpha2-macroglobulin, such as an antibody.

[0017] 4. The compound is selected from the group consisting of human amyloid Aβ40, human amyloid Aβ42, α-synuclein, Tau protein, SOD-1 protein, polyglutamine, neuroserin protease inhibitor (Neuroserpin, NSP), cystic fibrosis transmembrane conductance regulator (CFTR), α1-antitrypsin, Parkin protein, crystallin, transthyretin, short-chain acyl-CoA dehydrogenase variant (SCAD variant), low density lipoprotein receptor, huntingtin, neurofilament protein, peripheral protein, α-internexin, islet amyloid polypeptide, and the like. Item 4. The method, use, agent, or pharmaceutical composition according to any one of Items 1 to 3, which promotes degradation of one or more misfolded proteins and aggregates thereof selected from the group consisting of serum albumin, serum amyloid A protein, immunoglobulin light chains, human lysozyme, α-lactalbumin, prothymosin α, apolipoprotein E, and apolipoprotein J.

[0018] In some embodiments, the compound promotes the degradation of one or more proteins selected from the group consisting of human amyloid Aβ40, human amyloid Aβ42, α-synuclein, Tau protein, SOD-1 protein, and polyglutamine in brain tissue.

[0019] 5. The method, use, agent, or pharmaceutical composition according to any one of items 1 to 3, wherein the compound promotes the cleavage of Pro-BDNF into mature BDNF or promotes the cleavage of Pro-NGF into mature NGF. In some embodiments, the compound promotes the cleavage of Pro-BDNF in brain tissue into mature BDNF or promotes the cleavage of Pro-NGF in brain tissue into mature NGF.

[0020] 6. The method, use, medicament, or pharmaceutical composition according to any one of items 1 to 5, wherein the compound is plasminogen.

[0021] 7. The method, use, agent, or pharmaceutical composition according to any one of items 1 to 6, wherein the plasminogen is human full-length plasminogen or a conservatively substituted mutant thereof.

[0022] 8. The method, use, agent or pharmaceutical composition according to any one of items 1 to 6, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and still has plasminogen lysine binding activity or proteolytic activity.

[0023] 9. The method, use, agent or pharmaceutical composition according to any one of items 1 to 6, wherein the plasminogen comprises a protein consisting of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with sequence 14 and still having plasminogen proteolytic activity.

[0024] 10. The method, use, agent, or pharmaceutical composition according to any one of items 1 to 6, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or mutants thereof that retain the proteolytic activity of plasminogen.

[0025] 11. The method, use, agent, or pharmaceutical composition according to any one of items 1 to 6, wherein the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10, or 12, or comprises a conservatively substituted variant of the amino acid sequence as shown in sequence 2, 6, 8, 10, or 12.

[0026] 12. The method, use, medicament, or pharmaceutical composition according to any one of items 1 to 11, wherein the compound is used in combination with one or more other therapeutic methods or medicaments.

[0027] 13. The method, use, medicament or pharmaceutical composition according to item 12, wherein the other treatment methods include cell therapy (including stem cell therapy), supportive therapy, and physical therapy.

[0028] 14. The method, use, agent, or pharmaceutical composition according to item 12, wherein the one or more other agents are agents for the treatment of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or Parkinson's disease.

[0029] 15. The method, use, medicament, or pharmaceutical composition according to any one of items 1 to 14, wherein the compound is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g. via the carotid artery), and intramuscular administration.

[0030] The present invention also relates to the following. 1. A method for preventing or treating a disease caused by misfolded proteins (collectively referred to as misfolded protein diseases), comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors. 2. The method of claim 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA. 3. The method according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, such as an antibody. 4. The method according to any one of items 1 to 3, wherein the disease caused by misfolded proteins (collectively referred to as misfolded protein diseases) includes Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and polyglutamine diseases, Gerstmann's syndrome, diffuse or fatal familial insomnia, kuru, cystic fibrosis, liver damage, cataracts, familial amyloidosis, short-chain acyl-CoA dehydrogenase (SCAD) deficiency, familial hypercholesterolemia, motor neuron disease, diabetes, dialysis-associated amyloidosis, and amyloidosis-related human diseases. 5. The method according to any one of items 1 to 3, wherein the polyglutamine disease includes Huntington's disease, spinocerebellar ataxia type 3, and dentate cytosis with Lewy body atrophy. 6. The method according to any one of items 1 to 5, wherein the compound is plasminogen. 7. The method according to any one of items 1 to 6, wherein the plasminogen is human full-length plasminogen or a conservatively substituted mutant thereof. 8. The method according to any one of items 1 to 6, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and still has plasminogen lysine-binding activity or proteolytic activity. 9. The method according to any one of items 1 to 6, wherein the plasminogen comprises a protein consisting of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with sequence 14 and still having plasminogen proteolytic activity. 10. The method according to any one of items 1 to 6, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or mutants thereof that retain the proteolytic activity of plasminogen. 11. The method according to any one of items 1 to 6, wherein the plasminogen comprises an amino acid sequence as shown in SEQ ID NO: 2, 6, 8, 10 or 12, or comprises a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO: 2, 6, 8, 10 or 12. 12. The method according to any one of items 1 to 11, wherein the compound is used in combination with one or more other therapeutic methods or agents. 13. The method according to item 12, wherein the other treatment methods include cell therapy (including stem cell therapy), supportive therapy, and physical therapy. 14. The method according to item 12, wherein the other drug is another drug for the treatment of Alzheimer's disease, Parkinson's disease, ALS, or Huntington's disease. 15. The method according to any one of items 1 to 14, wherein the compound is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), and intramuscular administration.

[0031] In any one of the above embodiments of the present application, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still have plasminogen activity, for example, lysine binding activity or proteolytic activity. In some embodiments, the plasminogen is a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is added, deleted, and / or substituted based on sequence 2, 6, 8, 10 or 12 and still have plasminogen activity, for example, lysine binding activity or proteolytic activity.

[0032] In some embodiments, the plasminogen is a protein that includes plasminogen fragments and still has plasminogen activity, e.g., lysine-binding activity or proteolytic activity. In some embodiments, the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen, or a variant thereof that retains plasminogen activity, e.g., lysine-binding activity or proteolytic activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity, e.g., lysine-binding activity or proteolytic activity. In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity, e.g., lysine-binding activity or proteolytic activity. In some embodiments, the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human natural plasminogen.

[0033] In some embodiments, the subject is a human. In some embodiments, the subject is deficient or lacking in plasminogen. In some embodiments, the deficient or lacking is congenital, inherited and / or localized.

[0034] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier and plasminogen for use in the aforementioned methods. In some embodiments, the kit may be a prophylactic or therapeutic kit comprising (i) plasminogen for use in the aforementioned methods, and (ii) means for delivering the plasminogen to the subject. In some embodiments, the means is a syringe or a vial. In some embodiments, the kit further comprises a label or a protocol for instructing administration of the plasminogen to the subject to perform any of the aforementioned methods.

[0035] In some embodiments, the article of manufacture comprises a container containing a label and (i) plasminogen or a pharmaceutical composition containing plasminogen for use in the aforementioned methods, the label providing instructions for administering the plasminogen or composition to the subject to perform any of the aforementioned methods.

[0036] In some embodiments, the kit or article of manufacture further comprises one or more additional components or containers that contain other agents.

[0037] In some embodiments of the method, the plasminogen is administered systemically or locally, preferably by intravenous, intramuscular, or subcutaneous administration of plasminogen. In some embodiments of the method, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In some embodiments of the method, the plasminogen is administered at a daily dose of 0.0001-2000 mg / kg, 0.001-800 mg / kg, 0.01-600 mg / kg, 0.1-400 mg / kg, 1-200 mg / kg, 1-100 mg / kg, 10-100 mg / kg (calculated per kilogram of body weight), or 0.0001-2000 mg / cm 2 , 0.001-800mg / cm 2 , 0.01-600mg / cm 2 , 0.1-400mg / cm 2 , 1-200mg / cm 2, 1-100mg / cm 2 , 10-100mg / cm 2 (calculated per square centimeter of body surface area) and is preferably administered one or more times, preferably at least daily.

[0038] The present invention expressly covers all combinations of the technical features belonging to the embodiments of the present invention, and the technical configurations after the combinations are expressly disclosed in this application as if the above technical configurations were separately expressly disclosed. Moreover, the present invention also expressly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are expressly disclosed in this specification. [Brief description of the drawings]

[0039] [Figure 1] 1A to 1D show the effect of plasminogen on recombinant human α-synuclein in mouse brain homogenates. A shows a Tricine-SDS-PAGE electrophoretic pattern, while B, C, and D show quantitative band scan analysis results for α-synuclein, polymer a, and polymer b, respectively. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of α-synuclein in the plasminogen group was significantly lower than that in the solvent control group (*** indicates P<0.001), and the amounts of both polymers a and b were significantly lower than those in the solvent control group (** indicates P<0.01, *** indicates P<0.001), while in the brain homogenates of normal mice, the amount of α-synuclein in the plasminogen group was significantly lower than that in the solvent control group (*** indicates P<0.001), and the amounts of both polymers a and b were significantly lower than those in the solvent control group (* indicates P<0.05, ** indicates P<0.01). This indicates that plasminogen can effectively promote the degradation of recombinant human α-synuclein and its polymers in the brain homogenates of Parkinson's disease model mice and normal mice. [Diagram 2]2A-C are diagrams showing the effect of plasminogen on recombinant human α-synuclein in mouse brain homogenates. A is a Western blot, and B and C are quantitative analysis results of band scans of α-synuclein and polymers, respectively. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of α-synuclein in the plasminogen group was significantly lower than that of the solvent control group (** indicates P<0.01), and the amount of the polymers was significantly lower than that of the solvent control group (*** indicates P<0.001), while in the brain homogenates of normal mice, the amount of α-synuclein in the plasminogen group was significantly lower than that of the solvent control group (** indicates P<0.01), and the amount of the polymers was significantly lower than that of the solvent control group (*** indicates P<0.001). This indicates that plasminogen can effectively promote the degradation of recombinant human α-synuclein and its polymers in brain homogenates from Parkinson's disease model mice and normal mice. [Diagram 3] 3A-D show the immunohistochemical results of α-synuclein in the substantia nigra of Parkinson's disease model mice 14 days after plasminogen administration. A is the blank control group, B is the solvent group, C is the plasminogen administration group, and D is the result of quantitative analysis of the average optical density. As a result, the blank control group mice had only a small amount of α-synuclein in the substantia nigra, the solvent group mice had a significantly higher amount of α-synuclein than the blank control group mice (* indicates P<0.05), the plasminogen administration group mice had a significantly lower amount of α-synuclein than the solvent group mice, and the difference was statistically significant (* indicates P<0.05), and the plasminogen administration group mice had a similar amount of α-synuclein to the blank control group mice. This indicates that plasminogen can reduce the expression of α-synuclein in the substantia nigra of Parkinson's disease model mice and ameliorate neuronal damage and degeneration. [Figure 4]4A-B are diagrams showing the results of the effect of plasminogen on human amyloid Aβ40 in a PBS buffer system. A is a Tricine-SDS-PAGE electrophoresis diagram, and B is the result of quantitative scanning analysis of Aβ40 dissolution in vitro. As a result, the amount of Aβ40 in the solvent control group was defined as 100%, and there was no change, and in the plasminogen administration group, Aβ40 was partially degraded when only plasminogen was added, and in the plasminogen + tPA group, i.e., when plasminogen and tPA were added simultaneously, the in vitro degradation of Aβ40 was obvious, and there was a significant difference compared with the solvent control group (** represents P<0.01). This indicates that plasminogen can promote the degradation of human amyloid Aβ40 in a PBS buffer system. [Diagram 5] Figure 5A-B shows the results of the effect of plasminogen on human amyloid Aβ40 in rabbit cerebrospinal fluid. A is a diagram of Tricine-SDS-PAGE electrophoresis, and B is the result of quantitative scan analysis of Aβ40 dissolution. As a result, the amount of Aβ40 in the solvent control group was defined as 100%, and there was no change, while in the plasminogen administration group, Aβ40 was partially degraded when only plasminogen was added, and it was degraded to 74.81%. This indicates that plasminogen can promote the degradation of human amyloid Aβ40 in rabbit cerebrospinal fluid. [Figure 6]6A-B show the results of the effect of plasminogen on human amyloid Aβ40 in Alzheimer's disease model and normal mouse brain homogenate. A is a diagram of Tricine-SDS-PAGE electrophoresis, and B is the result of quantitative scanning analysis of Aβ40 dissolution in vitro. As a result, in the brain homogenate of Alzheimer's disease model mice, the amount of amyloid Aβ40 in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001); in the normal mouse brain homogenate, the amount of human amyloid Aβ40 in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (P=0.001). This indicates that plasminogen can effectively promote the degradation of human amyloid Aβ40 in Alzheimer's disease model and normal mouse brain homogenate. [Figure 7] 7A-B show the effect of plasminogen on human amyloid Aβ42 in Alzheimer's disease model and normal mouse brain homogenates, in which A shows Tricine-SDS-PAGE and B shows the results of quantitative scanning analysis of the dissolution of Aβ42 and its polymers in vitro. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of human amyloid Aβ42 in the plasminogen administration group was significantly lower than that in the solvent control group, and the amounts of its polymers a, b, and c were all significantly lower than those in the solvent group, and the difference was highly significant (* indicates P<0.05; *** indicates P<0.001); in the brain homogenates of normal mice, the amount of amyloid Aβ42 in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001), and the amounts of its polymers a, b, and c were all lower than those in the solvent group, and the difference was highly significant (*** indicates P<0.001). This indicates that plasminogen can effectively promote the degradation of human amyloid Aβ42 and its polymers in the brain homogenates of Alzheimer's disease model and normal mice. [Figure 8]8A-B are diagrams showing the results of the effect of plasminogen on human amyloid Aβ42 in Alzheimer's disease model and normal mouse brain homogenates. A is a Western blot diagram, and B is the result of a quantitative scan analysis of the dissolution of Aβ42 and its polymers in vitro. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of human amyloid Aβ42 in the plasminogen administration group was significantly lower than that of the solvent control group, and the amount of the polymers was both significantly lower than that of the solvent group, and the difference was extremely significant (* represents P<0.05); in the normal mouse brain homogenates, the amount of amyloid Aβ42 in the plasminogen administration group was significantly lower than that of the solvent control group, and the difference was extremely significant (*** represents P<0.001), and the amount of the polymers was both lower than that of the solvent group, and the difference was extremely significant (*** represents P<0.001). This indicates that plasminogen can effectively promote the degradation of human amyloid Aβ42 and its polymers in Alzheimer's disease models and normal mouse brain homogenates. [Figure 9] 9A-B are diagrams showing the effect of plasminogen on Tau protein in normal mouse brain homogenates. A is a Western blot image, and B is the quantitative analysis result of the optical density of the Tau protein band. As a result, in normal mouse brain homogenates, the amount of Tau protein in the plasminogen group was significantly lower than that in the solvent control group, and the difference was significant (* represents P<005, ** represents P<0.01, and *** represents P<0.001). This suggests that plasminogen can promote the degradation of Tau protein in normal mouse brain homogenates. [Figure 10]10A-B are diagrams showing the effect of plasminogen on Tau protein in brain homogenates of Alzheimer's disease mice. A is a Western blot image, and B is a quantitative analysis result of the optical density of the Tau protein band. As a result, in the brain homogenates of Alzheimer's disease mice, the amount of Tau protein in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was statistically significant (* represents P<005, ** represents P<0.01). This suggests that plasminogen can promote the degradation of Tau protein in the brain homogenates of Alzheimer's disease mice. [Figure 11] Figure 11 shows the Western blot detection results of Tau proteins of different molecular weights in the brain tissue of Alzheimer's disease mice administered plasminogen for 28 days. The results showed that there were certain levels of Tau proteins of different molecular weights in the brain homogenates of mice in the blank control group, and the levels of Tau proteins of each molecular weight and total Tau protein in the brain tissue of mice in the administration group were significantly lower than those of mice in the solvent group, and the statistical analysis P values ​​of 35kd, 35-40kd, 40kd, and 54kd molecular weight Tau protein levels and total Tau protein levels of the two groups were 0.174, 0.0406, 0.052, 0.067, and 0.055. This indicates that plasminogen can promote the degradation of Tau protein in the brain tissue of Alzheimer's model mice. [Figure 12] 12A-B show the effect of plasminogen on recombinant human Pro-BDNF in brain homogenates of normal mice. A is an image of SDS-PAGE, and B is the quantitative analysis result of the bands in SDS-PAGE. As a result, in the brain homogenates of normal mice, the amount of Pro-BDNF in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This suggests that plasminogen can promote the cleavage of Pro-BDNF in brain homogenates of normal mice. [Figure 13]Figures 13A-B show the effect of plasminogen on recombinant human Pro-BDNF in brain homogenates of normal mice, where A is an image of a Western blot and B is an analysis result of the optical density (OD) value of the Pro-BDNF band in the Western blot. As a result, in the brain homogenates of normal mice, the amount of Pro-BDNF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (** indicates P<0.01). This suggests that plasminogen can promote the cleavage of recombinant human Pro-BDNF in brain homogenates of normal mice. [Figure 14] 14A-B are diagrams showing the effect of plasminogen on recombinant human Pro-BDNF in brain homogenates of Parkinson's disease model mice. A is an image of SDS-PAGE, and B is the result of quantitative analysis of SDS-PAGE bands. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of Pro-BDNF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This suggests that plasminogen can promote the cleavage of recombinant human Pro-BDNF in brain homogenates of Parkinson's disease model mice. [Figure 15]15A-C are diagrams showing the effect of plasminogen on recombinant human Pro-BDNF in brain homogenates of Parkinson's disease model mice. A is an image of a Western blot, B is an analysis result of the optical density (OD) value of the Pro-BDNF band in the Western blot, and C is an analysis result of the optical density (OD) value of the BDNF band in the Western blot. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of Pro-BDNF in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was significant (* indicates P<0.05, *** indicates P<0.001), and the amount of BDNF in the plasminogen administration group was significantly higher than that in the solvent control group, and the difference was highly significant. This suggests that plasminogen can promote the cleavage of recombinant human Pro-BDNF and the formation of mature BDNF in the brain homogenates of Parkinson's disease model mice. [Figure 16] 16A-B are diagrams showing the effect of plasminogen on recombinant human Pro-BDNF in brain homogenates of Alzheimer's disease model mice. A is an image of SDS-PAGE, and B is the quantitative analysis result of the Pro-BDNF band in SDS-PAGE. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of Pro-BDNF in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This suggests that plasminogen can promote the cleavage of recombinant human Pro-BDNF in brain homogenates of Alzheimer's disease model mice. [Figure 17]17A-C are figures showing the effect of plasminogen on recombinant human Pro-BDNF in brain homogenates of Alzheimer's disease model mice, where A is an image of a Western blot, B is an analysis result of the optical density (OD) value of the Pro-BDNF band in the Western blot, and C is an analysis result of the optical density (OD) value of the BDNF band in the Western blot. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of Pro-BDNF in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (** indicates P<0.01, *** indicates P<0.001); the amount of BDNF in the plasminogen administration group was significantly higher than that in the solvent control group, and the difference was highly significant. This suggests that plasminogen can promote the cleavage of recombinant human Pro-BDNF and the formation of mature BDNF in the brain homogenates of Alzheimer's disease model mice. [Figure 18] Figure 18A-D shows the results of immunohistochemical staining of BDNF in the hippocampus of schizophrenia model mice administered plasminogen for 35 days. A is the blank control group, B is the solvent PBS control group, C is the plasminogen-administered group, and D is the quantitative analysis result of the average optical density. The results showed that the hippocampus of the mice in the blank control group expressed a certain level of BDNF (marked with an arrow), the level of BDNF in the hippocampus of the mice in the solvent group was improved, and the expression level of BDNF in the hippocampus of the mice in the plasminogen-administered group was significantly higher than that of the solvent group, and the statistical difference was close to significance (P = 0.095). This suggests that plasminogen can improve the expression level of BDNF in the hippocampus of schizophrenia model mice. [Figure 19]Figure 19A-D shows the results of immunohistochemical staining of BDNF in the hippocampus of Alzheimer's disease model mice administered plasminogen for 28 days. A is the blank control group, B is the solvent group, C is the treatment group, and D is the quantitative analysis result of the average optical density. The results showed that the hippocampus of the mice in the blank control group expressed a certain level of BDNF (marked with arrows), the expression of BDNF in the hippocampus of the mice in the solvent group was significantly lower than that of the blank control group, and the expression of BDNF in the hippocampus of the mice in the treatment group was significantly higher than that of the solvent group, and the statistical difference was significant (* represents P<0.05). This indicates that plasminogen can promote the expression of BDNF in the hippocampus of Alzheimer's disease model mice. [Figure 20] 20A-B show the representative brain tissue Western blot detection results of SMA mice after plasminogen administration and the quantitative analysis of the optical density (OD) ratio of NGF / Pro-NGF. The results showed that the brain tissue of the mice in the blank control group had a certain NGF / ProNGF ratio, and the NGF / ProNGF ratio of the brain tissue of the mice in the administration group was significantly higher than that of the mice in the vehicle group, and the statistical difference was highly significant (*** represents P<0.001). This suggests that plasminogen can promote the conversion of ProNGF to NGF in the brain tissue of SMA model mice and promote the formation of mature NGF. [Figure 21]21A-C are diagrams showing the effect of plasminogen on recombinant human Pro-NGF in brain homogenates of normal mice. A is an image of a Western blot, B is an analysis result of the optical density (OD) value of the Pro-NGF band in the Western blot, and C is an analysis result of the optical density (OD) value of the NGF band in the Western blot. As a result, in the brain homogenates of normal mice, the amount of Pro-NGF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (* represents P<0.05, *** represents P<0.001); the amount of NGF in the plasminogen-administered group was significantly higher than that in the solvent control group, and the difference was significant. This suggests that plasminogen can promote the cleavage of recombinant human Pro-NGF and the formation of mature NGF in brain homogenates of normal mice. [Figure 22] 22A-C are diagrams showing the effect of plasminogen on recombinant human Pro-NGF in brain homogenates of Alzheimer's disease model mice. A is an image of a Western blot, B is an analysis result of the optical density (OD) value of the Pro-NGF band in the Western blot, and C is an analysis result of the optical density (OD) value of the NGF band in the Western blot. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of Pro-NGF in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001); the amount of NGF in the plasminogen administration group was significantly higher than that in the solvent control group, and the difference was significant. This suggests that plasminogen can promote the cleavage of recombinant human Pro-NGF and the formation of mature NGF in the brain homogenates of Alzheimer's disease model mice. [Figure 23]23A-B show the effect of plasminogen on recombinant SOD-1 protein in brain homogenates of normal mice. A is an SDS-PAGE electrophoretogram, and B is the result of quantitative scanning analysis of SOD-1 protein dissolution in vitro. As a result, in brain homogenates of normal mice, the amount of SOD-1 in the plasminogen-administered group was lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in brain homogenates of normal mice. [Figure 24] 24A-B show the effect of plasminogen on recombinant SOD-1 protein in brain homogenates of SOD1-G93A transgenic mice. A is an SDS-PAGE electrophoretogram, and B is the result of quantitative scanning analysis of SOD-1 protein dissolution in vitro. As a result, in the brain homogenates of SOD1-G93A transgenic mice, the amount of SOD-1 protein in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in brain homogenates of ALS model mice. [Diagram 25] 25A-B show the effect of plasminogen on recombinant SOD-1 protein in brain homogenates of normal mice. A is an image of a Western blot, and B is an analysis result of the optical density (OD) value of the SOD-1 protein band in the Western blot. As a result, in the brain homogenates of normal mice, the amount of SOD-1 protein in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in brain homogenates of normal mice. [Figure 26]26A-B are diagrams showing the effect of plasminogen on recombinant SOD-1 protein in brain homogenates of SOD1-G93A transgenic mice. As a result, in the brain homogenates of SOD1-G93A transgenic mice, the amount of SOD-1 protein in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in brain homogenates of SOD1-G93A transgenic mice. Detailed Description of the Invention

[0040] "Protein folding" is the process by which a polypeptide chain coils into a specific functional three-dimensional structure or conformation, either simultaneously with or after synthesis on the ribosome, according to thermodynamic and kinetic principles, or with the assistance of molecular chaperones. Correctly folded proteins function normally, whereas misfolded proteins can cause disease.

[0041] The fibrinolytic system, also called the fibrinolytic system, is a system of a series of chemicals involved in the process of fibrinolysis (fibrinolysis), mainly including plasminogen (PLG), plasmin, plasminogen activators, and fibrinolysis inhibitors. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease and is synthesized by vascular endothelial cells. t-PA activates plasminogen, a process that mainly occurs in fibrin. Urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without the need for fibrin as a cofactor. Plasminogen (PLG) is synthesized in the liver. When blood clots, PLG is adsorbed in large quantities to the fibrin net and activated to plasmin by the action of t-PA or u-PA to promote fibrinolysis. Plasminase (PL) is a serine protease that degrades fibrin and fibrinogen, hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc., converts plasminogen to plasmin, hydrolyzes complement, etc. Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and α2-antithyplasmin (α2-AP). There are two main forms of PAI, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio and inactivate t-PA while activating PLG. α2-AP is synthesized in the liver and binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby weakening the susceptibility of fibrin to PL. Substances that inhibit the activity of the fibrinolysis system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0042] As used herein, the term "a component of the plasminogen activation pathway" refers to a 1. Plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen, their variants or analogs; 2. Plasmin and its variants or analogues; and 3. Plasminogen activators, including, for example, tPA and uPA, as well as tPA or uPA variants and analogs that contain one or more domains of tPA or uPA, such as one or more of the kringle domain and the proteolytic domain.

[0043] The above "variants" of plasminogen, plasmin, tPA and uPA include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as proteins having, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid added, deleted and / or substituted and still having plasminogen activity, plasmin activity, tPA or uPA activity. For example, a "variant" of plasminogen, plasmin, tPA or uPA includes, for example, mutants of these proteins resulting from substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acids.

[0044] The "plasminogen variant" of the present invention covers a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still having plasminogen activity, e.g., lysine binding activity or proteolytic activity. For example, the "plasminogen variant" of the present invention can be a protein having 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid added, deleted and / or substituted based on sequence 2, 6, 8, 10 or 12 and still having plasminogen activity, e.g., lysine binding activity or proteolytic activity. In particular, the plasminogen variants of the present invention include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as mutants of these proteins obtained by conservative substitution of, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids.

[0045] The plasminogen of the present invention may be an orthologue of human plasminogen derived from a primate or rodent, or a variant which still retains plasminogen activity, e.g., lysine-binding activity or proteolytic activity, such as the plasminogen shown in sequences 2, 6, 8, 10 or 12, e.g., human native plasminogen shown in sequence 2.

[0046] The above-mentioned "analogs" of plasminogen, plasmin, tPA and uPA include compounds that provide substantially the same effect as plasminogen, plasmin, tPA or uPA, respectively.

[0047] The above "variants" and "analogs" of plasminogen, plasmin, tPA and uPA cover "variants" and "analogs" of plasminogen, plasmin, tPA and uPA that contain one or more domains (e.g., one or more kringle domains and a proteolytic domain). For example, "variants" and "analogs" of plasminogen cover plasminogen variants and analogs, such as mini-plasminogen, that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain). "Variants" and "analogs" of plasmin cover plasmin "variants" and "analogs" such as mini-plasmin and delta-plasmin that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain).

[0048] Whether the above-mentioned "variants" or "analogs" of plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA, respectively, or whether they exert substantially the same effect as plasminogen, plasmin, tPA or uPA, respectively, can be measured by the level of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting). For example, the measurement can be performed with reference to the methods described in the following documents:Ny,A.,Leonardsson,G.,Hagglund,A.C,Hagglof,P.,Ploplis,V.A.,Carmeliet,P. and Ny,T. (1999). Ovulation inplasminogen-deficient mice. Endocrinology 140,5030-5035;Silverstein RL, Leung LL, Harpel PC, Nachman RL (November 1984). “Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator”. J. Clin. Invest. 74 (5): 1625-33;Gravanis I, Tsirka SE (February 2008). “Tissue-type plasminogen activator as a therapeutic target in stroke”. Expert Opinion on Therapeutic Targets. 12 (2): 159-70;Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). “Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo”. Blood. 74 (2): 722-8。

[0049] In some embodiments of the present invention, the "component of the plasminogen activation pathway" of the present invention is plasminogen. In some embodiments, the plasminogen is human full-length plasminogen or a conservative mutant thereof that retains its plasminogen activity (e.g., its lysine-binding activity or proteolytic activity). In some embodiments, the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen or a variant thereof that retains their plasminogen activity (e.g., their lysine-binding activity or proteolytic activity). In some embodiments, the plasminogen is natural or synthetic human plasminogen or a conservative mutant or fragment thereof that still retains plasminogen activity (e.g., its lysine-binding activity or proteolytic activity). In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a conservative mutant or fragment thereof that still retains plasminogen activity. In some embodiments, the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen comprises a conservative substitution sequence of the amino acid sequence shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the amino acids of the plasminogen are shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a conservative substitution variant of the plasminogen shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human native plasminogen or a conservative variant thereof. In some embodiments, the plasminogen is human native plasminogen shown in sequence 2 or a conservative variant thereof.

[0050] "A compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, including, for example, tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0051] The "antagonist of fibrinolysis inhibitors" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolysis inhibitors, such as PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. Said antagonists are antibodies to PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or for example antisense RNA or miniRNA that blocks or downregulates the expression of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but do not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that block the binding and / or activity domains of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0052] Plasmin is an important component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to become active matrix metalloproteinases (MMPs). Thus, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Plasminogen is present at relatively high relative levels in plasma and other body fluids, and traditionally, it has been believed that the regulation of the PA system is mainly achieved by the synthesis and activity levels of PA. The synthesis of the components of the PA system is tightly regulated by different factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is simultaneously inhibited by plasminogen activator inhibitor-1 (PAI-1) of uPA and tPA, and is regulated by plasminogen activator inhibitor-2 (PAI-2), which mainly inhibits uPA. Some cells have a uPA-specific cell surface receptor (uPAR) with direct hydrolytic activity.

[0053] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. It is synthesized mainly in the liver and is present in large amounts in extracellular fluids. The plasminogen content in plasma is approximately 2 μM. Thus, plasminogen is a large potential source of proteolytic activity in tissues and body fluids. There are two molecular forms of plasminogen: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and undegraded form of plasminogen has one amino-terminal (N-terminal) glutamic acid and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of the two forms of plasminogen is cleaved by uPA or tPA, leading to the formation of the disulfide-linked double-chain protease plasmin. The amino-terminal part of plasminogen contains five homologous tricycles, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain a lysine-binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. The most recently discovered plasminogen is a 38 kDa fragment, containing kringlel-4, which is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is generated from several protease-hydrolyzed plasminogens.

[0054] The main substrate of plasmin is fibrin, and fibrin lysis is a key point to prevent the formation of pathological thrombi. Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycan, and gelatin, indicating that plasmin plays an important role in ECM reconstruction. Indirectly, plasmin also degrades other components of the ECM by converting some protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Therefore, it may be proposed that plasmin is an important upstream regulator of extracellular protein hydrolysis. Plasmin also has the ability to activate some latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system to release chemotactic complement fragments.

[0055] "Plasmin" is a very important enzyme present in blood, which hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0056] "Plasminogen" is the proenzyme form of plasmin, which, based on the sequence in swiss prot, is calculated as the amino acid sequence of natural human plasminogen including the signal peptide (Sequence 4), and consists of 810 amino acids, has a molecular weight of about 90 kD, is synthesized mainly in the liver, and is a glycoprotein that can circulate in the blood, and the cDNA sequence encoding said amino acid sequence is as shown in Sequence 3. Full-sized 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). With reference to the sequence in swiss prot, the signal peptide comprises residues Met1-Gly19, PAp comprises residues Glu20-Val98, Kringle1 comprises residues Cys103-Cys181, Kringle2 comprises residues Glu184-Cys262, Kringle3 comprises residues Cys275-Cys352, Kringle4 comprises residues Cys377-Cys454, and Kringle5 comprises residues Cys481-Cys560. According to the NCBI data, the serine protease domain comprises residues Val581-Arg804.

[0057] Glu-plasminogen is a natural full-size human plasminogen, and consists of 791 amino acids (not including a signal peptide consisting of 19 amino acids). The cDNA sequence encoding this sequence is as shown in sequence 1, and its amino acid sequence is as shown in sequence 2. In vivo, Lys-plasminogen is formed by hydrolysis at the 76th-77th amino acid positions of Glu-plasminogen, and is, for example, as shown in sequence 6, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 5. Delta-plasminogen (δ-plasminogen) is a fragment of full-size plasminogen lacking the Kringle2-Kringle5 structure, and contains only Kringle1 and a serine protease domain (also called the protease domain (PD)). There is a literature that reports the amino acid sequence of δ-plasminogen (sequence 8), and the cDNA sequence encoding this amino acid sequence is, for example, sequence 7. Mini-plasminogen consists of Kringle5 and serine protease domains, and the literature reports residues Val443-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence without the signal peptide), and its amino acid sequence is as shown in sequence 10, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 9. However, literature reports that microplasminogen only contains a serine protease domain, and its amino acid sequence is residues Ala543-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and patent document CN102154253A discloses that it contains residues Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and in this patent application, the sequence of microplasminogen can be referred to patent document CN102154253A, and its amino acid sequence is as shown in sequence 12, and the cDNA sequence encoding the amino acid sequence is as shown in sequence 11.

[0058] The structure of full-length plasminogen is also described in a paper by Aisina et al. (Aisina RB, Mukhametova L I. Structure and function of plasminogen / plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605). According to the above mentioned article by Aisina et al., plasminogen contains Kringle 1, 2, 3, 4, 5 domains and a serine protease domain (also called protease domain (PD)), Kringles are responsible for binding plasminogen to low and high molecular weight ligands (i.e., lysine binding activity), which results in the conversion of plasminogen to a more open configuration and makes it more accessible to activation, and the protease domain (PD) is residues Val562-Asn791, and tPA and UPA specifically cleave the Arg561-Val562 activating bond of plasminogen, thereby allowing plasminogen to form plasmin. Thus, the protease domain (PD) is the region that confers the proteolytic activity of plasminogen.

[0059] 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.

[0060] In this application, the term "insufficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being lower than that of a normal person, and being sufficiently low to affect the subject's normal physiological function. The term "deficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being significantly lower than that of a normal person, the activity or expression being extremely low, and the normal physiological function can only be maintained by external supply.

[0061] Those skilled in the art can understand as follows. All technical configurations of plasminogen in the present invention can be applied to plasmin, so the technical configurations described in the present invention cover plasminogen and plasmin. In the circulation process, plasminogen takes a closed inactive conformation, but when it binds to a thrombus or cell surface, it becomes active plasmin with an open conformation under the mediation of plasminogen activator (PA). The active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. Among them, the PAp domain of plasminogen contains an important epitope that maintains plasminogen in an inactive closed conformation, but the KR domain can bind to lysine residues on receptors and substrates. Several types of enzymes as plasminogen activators have already been known, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hagemann factor), etc.

[0062] "Plasminogen active fragment" refers to an active fragment that binds to lysine in the target sequence of a substrate (lysine binding activity), or an active fragment that exerts a proteolytic function (proteolytic activity), or a fragment that has both proteolytic activity and lysine binding activity. The plasminogen technical configuration of the present invention encompasses technical configurations that replace plasminogen with a plasminogen active fragment. In some embodiments, the plasminogen active fragment of the present invention comprises the serine protease domain of plasminogen or consists of the serine protease domain of plasminogen. In some embodiments, the plasminogen active fragment of the present invention comprises sequence 14, or comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity to sequence 14, or consists of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity to sequence 14. In some embodiments, the plasminogen active fragments of the present invention comprise one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or conservative substitution variants thereof, or consist of one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or conservative substitution variants thereof. In some embodiments, the plasminogen of the present invention comprises a protein comprising an active fragment of plasminogen as described above.

[0063] At present, the methods for measuring plasminogen and its activity in blood include tissue plasminogen activator activity (t-PAA), plasma tissue plasminogen activator antigen (t-PAAg), plasma tissue plasminogen activity (plgA), plasma tissue plasminogen antigen (plgAg), plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibitor antigen, and plasma plasmin-antiplasmin complex (PAP). The most common method is the chromogenic substrate method: streptokinase (SK) and luminescent substrate are added to the plasma of the subject, and the PLG in the subject's plasma becomes PLM under the action of SK, which acts on the luminescent substrate, and then measured by a spectrophotometer, and the increase in absorbance is directly proportional to the activity of plasminogen. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, etc. can also be used to measure plasminogen activity in blood.

[0064] "Ortholog" refers to a homologue between different species, including both protein and DNA homologues, also called a direct lineage gene. It specifically refers to a protein or gene that is evolved from the same ancestral gene between different species. The plasminogen of the present invention includes human native plasminogen, and further includes orthologs or orthologs of plasminogens with plasminogen activity derived from different species.

[0065] A "conservative substitution variant" is a variant in which a given amino acid residue is modified but does not change the overall conformation and function of a protein or enzyme, including but not limited to those that substitute an amino acid in the amino acid sequence of a parent protein with an amino acid of similar properties (e.g., acidic, alkaline, hydrophobic, etc.). Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic alkaline 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 of two proteins or amino acid sequences with similar functions may vary. For example, they have a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" further include polypeptides or enzymes with an amino acid identity of 60% or more based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and most preferably 90% or more, and have the same or essentially similar properties or functions compared to the natural or parent protein or enzyme.

[0066] "Isolated" plasminogen is 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% (by weight), as determined by 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 includes plasminogen produced from recombinant cells by bioengineering techniques and separated by at least one further purification step.

[0067] 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 having modified peptide backbones. The terms also include 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.

[0068] The definition of "percentage of amino acid sequence identity (%)" of a reference peptide sequence is the percentage of amino acid residues in a candidate sequence that are the same as those in a reference polypeptide sequence, after introducing gaps as necessary to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by several methods within the skill of the art, for example by publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm that achieves maximum comparison requirements for the full size of the sequences to be compared. However, for the purposes of the present invention, the percentage of amino acid sequence identity is obtained by the sequence comparison computer software ALIGN-2.

[0069] 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 (or alternatively, a given amino acid sequence A that has or contains a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0070] 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 Y is the total number of amino acid residues in B. It should be understood as follows: if the length of amino acid sequence A and the length of amino acid sequence B are not equal, then the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used in this text are as described in the previous paragraph and are obtained by the ALIGN-2 computer program.

[0071] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be completely or partially preventing the onset or development of a disease or its symptoms, or partially or completely reducing the disease and / or its symptoms, and / or partially or completely curing the disease and / or its symptoms. It also includes: (a) preventing a disease from occurring in a subject, where the subject has the causative agent for the disease but has not been diagnosed with the disease; (b) inhibiting the disease or inhibiting its formation; and (c) attenuating a disease and / or its symptoms, i.e., causing a decrease or disappearance of the disease and / or its symptoms.

[0072] 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, cows, sheep, pigs, goats), and the like.

[0073] A "therapeutically effective amount" or "effective amount" is an amount of a component of the plasminogen activation pathway or its related compound (e.g., plasminogen) that is capable of achieving said prevention and / or treatment of a disease when administered to a mammal or other subject and used to treat the disease. A "therapeutically effective amount" will vary according to the component of the plasminogen activation pathway or its related compound (e.g., plasminogen) used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0074] Preparation of the Plasminogen of the Invention For therapeutic use, plasminogen may be isolated and purified from nature or may be synthesized by standard chemical peptide synthesis techniques. When polypeptides are synthesized by chemical methods, the synthesis may be performed in liquid phase 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 in order, is suitable for the chemical synthesis of plasminogen. Various forms of SPPS, e.g., Fmoc and Boc, can be used to synthesize plasminogen. The techniques used for solid-phase synthesis are described in: Barany and Solid-Phase Peptide Synthesis; pages 3-284, The Peptides: Analysis, Synthesis, Biology. Volume 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, small insoluble porous beads are treated with functional units on which the peptide chain is built. After repeated coupling / deprotection cycles, the free N-terminal amine of the attached solid phase is coupled with a single N-protected amino acid unit, which is then deprotected to expose a new N-terminal amine for linking with another amino acid. The peptide remains immobilized on the solid phase and is then excised.

[0075] 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. Control of expression can be a eukaryotic promoter system in a vector, which is transformed or transfected into a eukaryotic host cell (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 collection and purification of the plasminogen.

[0076] Suitable expression vectors usually replicate in the host cell either free or as an integrated part of the host cell chromosomal DNA. Usually, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) which is useful for detecting those cells which have been transformed in vitro with the desired DNA sequence.

[0077] Escherichia coli is an example of a prokaryotic host cell in which a polynucleotide encoding a target antibody can be cloned. Other suitable microbial hosts include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can be generated, which usually contain expression control sequences (e.g., an origin of replication) that are compatible with the host cell. There are also many known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the phage lambda-derived promoter system. The promoter generally controls expression and, if necessary, may further include a ribosome binding site sequence, for initiating transcription and translation, in the operator gene sequence.

[0078] 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 necessary. Exemplary promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0079] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce the anti-Tau antibodies of the present invention (e.g., polynucleotides encoding such anti-Tau antibodies of interest). 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 can include 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 ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0080] Once synthesized (chemically or recombinantly), the plasminogen according to the 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 is essentially pure, e.g., at least about 80% to 85% pure, at least about 85%-90% pure, at least about 90%-95% pure, or 98%-99% pure or even more pure, e.g., free of contaminants, such as cellular debris, large molecules other than such antibodies, etc.

[0081] Drug combinations A component of the plasminogen activation pathway or related compound (e.g., plasminogen) of the desired purity is mixed with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) to form a lyophilized formulation or aqueous solution to obtain a therapeutic combination. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects at the required dosages and concentrations, and further include buffers such as, for example, phosphates, citrates, and other organic acids. Antioxidants include ascorbyl methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoic acid esters, such as methyl or propyl parahydroxybenzoic acid ester; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (those having less than about 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 include 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 (such as zinc-protein complexes); and / or non-ionic surfactants, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). A preferred lyophilized anti-VEGF antibody formulation is described in WO97 / 04801, incorporated herein by reference.

[0082] The combinations of the present invention may contain one or more active compounds as required for the particular condition requiring treatment, preferably those having complementary activities and no adverse effects on each other.

[0083] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, can be incorporated, for example, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or can be incorporated into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in crude emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0084] The plasminogen activation pathway components or related compounds (e.g., plasminogen) of the present invention to be administered to the body must be sterile, which can be readily achieved by filtration through sterile filtration membranes, prior to or after lyophilization and recombination.

[0085] The plasminogen activation pathway components or related compounds (e.g., plasminogen) of the present invention can be prepared in sustained release formulations. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers having a defined shape and containing the glycoprotein, such as membranes or microcapsules. Examples of sustained release 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. Pat. No. 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., same source as above), or degradable lactic acid-hydroxyacetic acid copolymers, e.g., Lupron. 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, but some aqueous gels release proteins for a shorter period of time. Rational strategies can be designed to stabilize proteins depending on the mechanism involved. For example, if the aggregation mechanism is the exchange of sulfur disulfide bonds to form intermolecular SS 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.

[0086] Dosage and dosage Administration of the pharmaceutical compositions of the present invention can be achieved by different modes, such as nasal inhalation, aerosol inhalation, nasal or eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, and intrarectal administration.

[0087] Preparations for use in 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 solutions, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolytic replenishers, and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.

[0088] A medical practitioner may determine a dosage regimen based on various clinical factors. For example, as is well known in the medical arts, the dosage regimen for any given patient is determined by several factors, including the patient's body type, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other drugs being administered at the same time. The dosage range of the plasminogen pharmaceutical composition of the present invention may be, for example, about 0.0001-2000 mg / kg of subject body weight daily, or about 0.001-500 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, the dosage may 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 illustrative range are also covered, particularly when considering the above factors. Intermediate doses within the above range are also within the scope of the present invention. Subjects may receive such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dose schedule is 0.01-100 mg / kg for several consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.

[0089] Product or drug kit One embodiment of the present invention is directed to an article of manufacture or drug kit comprising a component of the plasminogen activation pathway or its associated compound (e.g., plasminogen). The article of manufacture preferably comprises a container, a label or a protocol. Suitable containers are bottles, vials, syringes, etc. The containers can be made of a variety of materials, such as glass or plastic. The container contains a composition, the composition is effective to treat a disease or condition of the present invention, and has a sterile access (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 a component of the plasminogen activation pathway or its associated compound (e.g., plasminogen). A label on or attached to the container describes the composition as being used to treat a disease or condition of the present invention. The article of manufacture further comprises a second container containing a pharmaceutical buffer, the pharmaceutical buffer including, for example, phosphate buffered saline, Ringer's solution, and glucose solution. It may further comprise other materials as may be required from a commercial and user perspective, i.e., other buffers, diluents, filters, needles, and syringes. The article of manufacture also includes a protocol with instructions for use, including, for example, instructing a user of the composition to administer to a patient a composition of a component of the plasminogen activation pathway or its related compound (e.g., plasminogen) and other medications associated with the treatment of a disease. EXAMPLES

[0090] The human plasminogen used in the following examples was derived from the plasma of a human donor and has been described in the following publications: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Based on the method described in Apr;235:1005-10, the process was optimized and purified from human donor plasma, where the plasminogen alone was greater than 98%. Example 1

[0091] Example 1 relates to the promotion of degradation of recombinant human α-synuclein in brain homogenates from Parkinson's disease model mice. Eight C57BL / 6J male mice, aged 11-12 weeks and weighing 18-25g, were taken, and the weight of all mice was measured one day before modeling. They were randomly divided into two groups according to their weight, four in the blank control group and four in the model group. The modeling time was set at 9 am every day, and the blank control group was administered 200 μl of saline by intraperitoneal injection, and the model group was administered 5 mg / ml MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) solution at 35 mg / kg / mouse by intraperitoneal injection for 5 consecutive days to construct the Parkinson's disease model. [1] Preparation of MPTP solution: 45 mg MPTP (Sigma, M0896) was dissolved in 9 ml of saline solution to prepare a final concentration of 5 mg / ml. After modeling was completed, i.e., on the 6th day after modeling, an open field test was performed on all mice to confirm whether modeling was successful. All mice were killed and whole brain tissue was collected and weighed, then 1×PBS (Thermo Fisher, pH 7.4; 10010-031) was added at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3-4 times), and after homogenization, it was centrifuged at 4°C (12000 rpm, 20 min), and the supernatant (brain tissue homogenate) was taken and transferred to an EP tube. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank control group, (2) solvent control group, and (3) plasminogen group. The blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL α-synuclein solution (ChinaPeptides Co., Ltd., custom-expressed human α-synuclein, UniProtKB-P37840, 1.0 mg / mL), 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the plasminogen group was added with 21.5 mL α-synuclein solution (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate. After adding the sample to each group, the mixture was incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken and loaded. The electrophoresis conditions were run at 30 V for 1.5 h, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and placed in 1‰ Coomassie Brilliant Blue staining solution (1 g of Coomassie Brilliant Blue R250 was dissolved in 1000 ml of a mixture of ethanol:glacial acetic acid:purified water in a volume ratio of 5:2:13) for 30 min, and then destained using a destaining solution (purified water:glacial acetic acid:absolute ethanol = 17:2:1 in volume). The gels were photographed and quantitatively scanned with a biomolecular imager. Parkinson's disease is currently believed to be caused by the loss of dopaminergic neurons in the substantia nigra of the midbrain and the appearance of Lewy bodies. α-synuclein is a neuronal protein consisting of 140 amino acid residues that may cause neuronal damage and participate in the process of neurodegeneration in the central nervous system. Studies have revealed that Lewy bodies in neurons and aggregated α-synuclein in neuronal synapses are characteristic of brain lesions in Parkinson's disease. [2] . As a result, in the brain homogenates of Parkinson's disease model mice, the amount of α-synuclein in the plasminogen group was significantly lower than that in the solvent control group (*** indicates P<0.001), and the amounts of both polymers a and b were significantly lower than those in the solvent control group (** indicates P<0.01, *** indicates P<0.001), while in the brain homogenates of normal mice, the amount of α-synuclein in the plasminogen group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001), and the amounts of both polymers a and b were significantly lower than those in the solvent control group, and the difference was significant (* indicates P<0.05, ** indicates P<0.01) (Figure 1). This indicates that plasminogen can effectively degrade human α-synuclein and its polymers in the brain homogenates of Parkinson's disease model mice and normal mice. Example 2

[0092] Example 2 relates to the fact that plasminogen promotes the degradation of α-synuclein in brain homogenates from Parkinson's disease model mice. Eight C57BL / 6J male mice, aged 11-12 weeks and weighing 18-25 g, were taken, and the weights of all mice were measured one day before modeling. They were randomly divided into two groups according to their weights, with four mice in the blank control group and four mice in the model group. The Parkinson's disease model was constructed as described in Example 1. [5] , brain tissue homogenates were prepared and transferred to EP tubes. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the vehicle control group was added with 21.5 μL α-synuclein solution (ChinaPeptides Co., Ltd., custom-expressed human α-synuclein, UniProtKB-P37840, 1.0 mg / mL), 4.6 μL vehicle solution, and 23.9 μL mouse brain homogenate; the plasminogen-treated group was added with 21.5 μL saline, 4.6 μL vehicle solution, and 23.9 μL mouse brain homogenate. α-synuclein solution (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate were added. After adding the samples to each group, the samples were incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was mixed uniformly with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL was taken for loading. The electrophoresis conditions were run at 30 V for 1.5 h, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to a PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human α-synuclein antibody (Proteintech, 10842-1-AP) and incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of α-synuclein in the plasminogen group was significantly lower than that in the solvent control group (** indicates P<0.01), and the amount of its polymers was both significantly lower than that in the solvent control group (*** indicates P<0.001), while in the brain homogenates of normal mice, the amount of α-synuclein in the plasminogen group was significantly lower than that in the solvent control group, and the difference was highly significant (** indicates P<0.01), and the amount of its polymers was significantly lower than that in the solvent control group, and the difference was significant (*** indicates P<0.001) (Figure 2). This indicates that plasminogen can effectively degrade human α-synuclein and its polymers in the brain homogenates of Parkinson's disease model mice and normal mice. Example 3

[0093] Example 3 relates to the ability of plasminogen to reduce the expression of α-synuclein in the substantia nigra of Parkinson's disease model mice. Twenty-eight C57BL / 6J male mice aged 10-12 weeks were taken, and all mice were weighed one day before modeling. They were randomly divided into two groups according to their weight, with 8 mice in the blank control group and 20 mice in the model group. The Parkinson's disease model was constructed as described in Example 1. [1] After modeling was completed, i.e., on the 6th day after modeling, the mice in the model group were randomly divided into two groups according to body weight, with 10 mice in each of the solvent group and the treatment group, and treatment was started, which was recorded as day 1. Plasminogen solution was administered to the treatment group mice at 1 mg / 100 μl / mouse via tail vein injection, and the solvent group was injected with solvent solution at 100 μl / mouse for 14 consecutive days, and on the 15th day of treatment, the mice were sacrificed, and the substantia nigra of the mice was collected and fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed substantia nigra (brain tissue) was dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. The substantia nigra of the section was positioned, the thickness of the section was 3 μm, and the section was deparaffinized, rehydrated, and washed once with water. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 min, washed twice with 0.01M PBS, 5 min each time. Blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 min, discarded the goat serum when the time was up, dropped rabbit anti-mouse α-synuclein antibody (α-synuclein) (Proteintech, 10842-1-AP) and incubated overnight at 4°C, washed twice with 0.01M PBS, 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 h, washed twice with 0.01M PBS, 5 min each time. Coloring was performed with a DAB kit (Vector laboratories, Inc., USA), and counterstained with hematoxylin for 30 s after washing three times with water, and rinsed with running water for 5 min. The sections were dehydrated in a graded alcohol solution, cleared in xylene, and embedded in neutral rubber, and the sections were observed under a light microscope at 400x magnification. As a result, there was only a small amount of α-synuclein in the substantia nigra of the mice in the blank control group (Figure 3A), the amount of α-synuclein in the substantia nigra of the mice in the vehicle group (Figure 3B) was significantly higher than that of the mice in the blank control group (* indicates P<0.05), and the amount of α-synuclein in the substantia nigra of the mice in the plasminogen administration group (Figure 3C) was significantly lower than that of the vehicle group and close to that of the blank control group, and the difference was statistically significant (* indicates P<0.05) (Figure 3D). This indicates that plasminogen can reduce the expression of α-synuclein in the substantia nigra of Parkinson's disease model mice and improve nerve damage degeneration. Example 4

[0094] Example 4 relates to the ability of plasminogen to promote the degradation of human amyloid (Aβ40) in a PBS buffer system. Eppendorf (EP) tubes were taken and the following groups were set up: (1) blank control group, (2) solvent control group, (3) plasminogen group, and (4) plasminogen + tPA group, with four tubes in each group. The blank control group was added with 43.3 μL saline, 16 μL plasminogen solution (0.575 mg / mL), 10 μL ultrapure water, and 30.7 μL PBS buffer (10 mM, pH 7.4, Thermo Fisher, 10010-031); the solvent control group was added with 43.3 μL Aβ40 (ChinaPeptides Co., Ltd., 04010011521, 1.0 mg / mL), 16 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), 10 μL ultrapure water, and 30.7 μL PBS buffer; the plasminogen group was added with 43.3 μL Aβ40 (1.0 mg / mL), 16 μL plasminogen solution (0.575 mg / mL), 10 μL ultrapure water, and 30.7 μL PBS buffer were added; for the plasminogen + tPA group, 43.3 μL Aβ40 (1.0 mg / mL), 8 μL plasminogen solution (1.15 mg / mL), 8 μL tPA solution (1.0 mg / mL), 10 μL lysine solution (0.1 mM), and 30.7 μL PBS buffer were added. After the addition of each sample, the reaction was stopped by adding 100 μL of 0.1% trifluoroacetic acid solution after incubation at 37 °C for 3 hours. A 20% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 1 min, and then 20 μL of sample was taken and loaded. The electrophoresis conditions were run at 30 V for 1 h, followed by running at 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and placed in 1‰ Coomassie Brilliant Blue staining solution (1 g of Coomassie Brilliant Blue R250 was dissolved in 1000 ml of a mixture of ethanol:glacial acetic acid:purified water in a volume ratio of 5:2:13) for 30 min, and then destained using a destaining solution (purified water:glacial acetic acid:absolute ethanol = 17:2:1 in volume). The gels were photographed with a biomolecular imager and quantitatively scanned. Accumulation of amyloid (Aβ) is an important factor in the formation of Alzheimer's disease. Aβ40 and Aβ42, which contain 40 and 42 residues, are the main forms that constitute senile plaques. In other words, deposition of Aβ in the hippocampus and striatum of the brain to form senile plaques is the main cause of Alzheimer's disease. [3] Detection of Aβ40 and Aβ42 contents in cerebrospinal fluid is gradually becoming a physiological indicator of clinical Alzheimer's disease. As a result, the amount of Aβ40 in the solvent control group was defined as 100% and remained unchanged, in the plasminogen group, Aβ40 was partially degraded when only plasminogen was added, and in the plasminogen + tPA group, when plasminogen and tPA were added, the in vitro degradation of Aβ40 was obvious, with a significant difference compared with the solvent control group (** represents P < 0.01) (Figure 4). This indicates that plasminogen can promote the degradation of human amyloid Aβ40 in the PBS buffer system. Example 5

[0095] Example 5 relates to the ability of plasminogen to promote the degradation of human amyloid (Aβ40) in rabbit cerebrospinal fluid. Eppendorf (EP) tubes were taken and designated as (1) blank control group, (2) solvent control group, (3) plasminogen group, and (4) plasminogen + tPA group, respectively. The blank control group was added with 43.3 μL saline, 16 μL plasminogen solution (0.575 mg / mL), and 40.7 μL rabbit (purchased from the market) cerebrospinal fluid; the solvent control group was added with 43.3 μL Aβ40 (ChinaPeptides Co., Ltd., 04010011521, 1.0 mg / mL), 16 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 40.7 μL rabbit cerebrospinal fluid; the plasminogen group was added with 43.3 μL Aβ40 (1.0 mg / mL), 16 μL plasminogen solution (0.575 mg / mL), and 40.7 μL rabbit cerebrospinal fluid. To the plasminogen + tPA group, 43.3 μL Aβ40 (1.0 mg / mL), 8 μL plasminogen solution (1.15 mg / mL), 8 μL tPA solution (1.0 mg / mL), and 40.7 μL PBS buffer were added. After addition, each sample was incubated at 37 °C for 3 hours, and then 100 μL of 0.1% trifluoroacetic acid solution was added to each sample to stop the reaction. A 20% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained and quantitatively scanned. As a result, the amount of Aβ40 in the solvent control group was defined as 100% and remained unchanged, while in the plasminogen group, Aβ40 was partially degraded when only plasminogen was added, being degraded to 74.81% (Figure 5). This indicates that plasminogen can promote the degradation of human amyloid Aβ40 in the cerebrospinal fluid of rabbits. Example 6

[0096] Example 6 relates to the fact that plasminogen promotes the degradation of human amyloid Aβ40 in brain homogenates of Alzheimer's disease models and normal mice. Four 11-week-old B6SJLTg(APPSwFlLon, PSEN1*M146L*L286V)6799Vas / Mmjax(FAD) (Stock number: 034840) (abbreviated as FAD) and four C57BL / 6 (normal) mice were selected and sacrificed to collect whole brain tissue, and the supernatant brain homogenate was transferred to an EP tube as in Example 1. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank control group, (2) solvent control group, and (3) plasminogen group. The blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Aβ40 (ChinaPeptides Co., Ltd., 04010011521, 1.0 mg / mL), 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the plasminogen group was added with 21.5 mL Aβ40 (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate. After adding the sample to each group, the mixture was incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 20% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained and quantitatively scanned. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of human amyloid Aβ40 in the plasminogen group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001); in the brain homogenates of normal mice, the amount of amyloid Aβ40 in the plasminogen group was significantly lower than that in the solvent control group, and the difference was highly significant (P=0.001) (Figure 6). This indicates that plasminogen can effectively promote the degradation of human amyloid Aβ40 in the brain homogenates of Alzheimer's disease model and normal mice. Example 7

[0097] Example 7 relates to the promotion of degradation of human amyloid Aβ42 in Alzheimer's disease models and normal mouse brain homogenates. Four 11-week-old B6SJLTg(APPSwFlLon, PSEN1*M146L*L286V)6799Vas / Mmjax(FAD) (Stock number: 034840) (abbreviated as FAD) and four C57BL / 6 (normal) mice were selected and sacrificed to collect whole brain tissue. The supernatant brain homogenate was transferred to an EP tube as in Example 1. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank control group, (2) solvent control group, and (3) plasminogen group. The blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Aβ42 (ChinaPeptides Co., Ltd., 04010011526, 1.0 mg / mL), 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the plasminogen group was added with 21.5 mL Aβ42 (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate. After adding the sample to each group, the mixture was incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 20% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained and quantitatively scanned. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of amyloid Aβ42 in the plasminogen group was lower than that in the solvent control group, and the amounts of polymers a, b, and c were all significantly lower than those in the solvent group, and the difference was highly significant (* represents P<0.05, *** represents P<0.001); in the brain homogenates of normal mice, the amount of amyloid Aβ42 in the plasminogen group was significantly lower than that in the solvent control group, and the difference was highly significant (*** represents <0.001), and the amounts of polymers a, b, and c were all lower than those in the solvent group, and the difference was highly significant (*** represents <0.001 (Figure 7). This indicates that plasminogen can effectively promote the degradation of human amyloid Aβ42 and its polymers in the brain homogenates of Alzheimer's disease model mice and normal mice. Example 8

[0098] Example 8 relates to the fact that plasminogen promotes the degradation of human amyloid Aβ42 in brain homogenates of Alzheimer's disease models and normal mice. Four 11-week-old B6SJLTg(APPSwFlLon, PSEN1*M146L*L286V)6799Vas / Mmjax(FAD) (Stock number: 034840) (abbreviated as FAD) and four C57BL / 6 (normal) mice were selected and sacrificed to collect whole brain tissue. The supernatant brain homogenate was transferred to an EP tube as in Example 1. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank control group, (2) solvent control group, and (3) plasminogen group. The blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Aβ42 (ChinaPeptides Co., Ltd., 04010011526, 1.0 mg / mL), 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the plasminogen group was added with 21.5 mL Aβ42 (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate. After adding the sample to each group, the mixture was incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 20% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320). Each group of samples was mixed uniformly with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL was taken for loading. The electrophoresis conditions were run at 30 V for 1.5 h, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to a PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added mouse anti-human Aβ42 antibody (Nanjing GenScript Biotechnology Co., Ltd., 10842-1-AP) and incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-mouse IgG (HRP) antibody (Abcam, ab6789) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of human amyloid Aβ42 in the plasminogen-administered group was significantly lower than that in the solvent control group, and the amount of its polymers was also significantly lower than that in the solvent group, and the difference was highly significant (* indicates P<0.5); in the brain homogenates of normal mice, the amount of amyloid Aβ42 in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (***P<0.001), and the amount of its polymers was also lower than that in the solvent group, and the difference was highly significant (***P<0.001) (Figure 8). This indicates that plasminogen can effectively promote the degradation of human amyloid Aβ42 and its polymers in the brain homogenates of Alzheimer's disease model and normal mice. Example 9

[0099] Example 9 relates to the fact that plasminogen promotes the degradation of Tau protein in brain homogenates of normal mice. Four C57BL / 6J male mice aged 11–12 weeks and weighing 18–25 g were selected and sacrificed to collect whole brain tissue, which was then weighed and added to 1× PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3–4 times), and centrifuged at 4°C (12000 rpm, 20 min) after homogenization, and the supernatant brain homogenate was transferred to an EP tube. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (0.5 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Tau (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human Tau To the plasminogen group, 21.5 μL of Tau (1.0 mg / mL), 4.6 μL of plasminogen solution (0.5 mg / mL), and 23.9 μL of mouse brain homogenate were added; to the plasminogen group, 21.5 μL of Tau (1.0 mg / mL), 4.6 μL of plasminogen solution (0.5 mg / mL), and 23.9 μL of mouse brain homogenate were added. After adding the samples to each group, the samples were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 10% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 45 min, followed by 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit Tau protein antibody (Abcam, ab151559) and incubated at room temperature for 2 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. Tau protein is the most abundant microtubule-associated protein. Tau protein is a phosphate-containing protein, and Tau protein molecules in normal mature brains contain 2-3 phosphate groups. Tau protein in the brains of patients with Alzheimer's disease (senile dementia) is abnormally hyperphosphorylated, and each molecule of Tau protein may contain 5-9 phosphate groups, which may cause loss of normal biological function. [4] . As a result, in the brain homogenates of normal mice, the amount of Tau protein in the plasminogen group was significantly lower than that in the solvent control group, and the difference was significant (* represents P<005, ** represents P<0.01, *** represents P<0.001) (Figure 9). This suggests that plasminogen can promote the degradation of Tau protein in the brain homogenates of normal mice. Example 10

[0100] Example 10 relates to the promotion of Tau protein degradation in brain homogenates from Alzheimer's disease mice. Four 11-week-old B6SJLTg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax(FAD)(Stock number: 034840)(abbreviated as FAD) mice were selected and sacrificed to collect whole brain tissue, which was weighed and then added to 1x PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3-4 times), centrifuged at 4°C (12000 rpm, 20 min), and the supernatant brain homogenate was placed in an EP tube. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (0.5 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Tau (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human Tau To the plasminogen group, 21.5 μL of Tau (1.0 mg / mL), 4.6 μL of plasminogen solution (0.5 mg / mL), and 23.9 μL of mouse brain homogenate were added; to the plasminogen group, 21.5 μL of Tau (1.0 mg / mL), 4.6 μL of plasminogen solution (0.5 mg / mL), and 23.9 μL of mouse brain homogenate were added. After adding the samples to each group, the samples were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 10% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 45 min, followed by 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit Tau protein antibody (Abcam, ab151559) and incubated at room temperature for 2 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. As a result, in the brain homogenates of Alzheimer's disease mice, the amount of Tau protein in the plasminogen group was significantly lower than that in the solvent control group, and the difference was statistically significant (* indicates P<005, ** indicates P<0.01) (Figure 10). This suggests that plasminogen can promote the degradation of Tau protein in the brain homogenates of Alzheimer's disease mice. Example 11

[0101] Example 11 relates to the effect of plasminogen in reducing Tau protein levels in brain tissue of Alzheimer's disease model mice. B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax mice (catalog number: 034840, purchased from Jackson Laboratory) and C57BL / 6J mice were backcrossed three times to breed offspring (referred to as B6-F3-FAD). Eighteen female B6-F3-FAD mice aged 20-25 weeks and nine female C57BL / 6J mice aged 9 weeks were taken. The B6-F3-FAD mice were randomly divided into two groups, a vehicle group and a treatment group, with nine mice in each group, according to their body weight and the results of the Y-maze test. Nine C57BL / 6J mice were used as a blank control group. After grouping was completed, the mice in the blank control group and the mice in the vehicle group were administered the vehicle by tail vein injection, and the injection volume for each mouse was 5 ml / kg. Plasminogen was administered to the mice in the treatment group via tail vein injection, with each mouse receiving plasminogen at a dose of 50 mg / kg for 28 consecutive days. Seven days after administration, mice were randomly selected from each group and sacrificed, and their brain tissues were collected and homogenized at 4°C. The homogenate supernatant was used to detect total protein concentration by BCA method and Western blot detection. A 10% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed uniformly with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 1.5 h, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to a PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-mouse Tau antibody (Abcam, ab151559) and incubated at room temperature for 2 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. The results showed that there were certain levels of Tau proteins of different molecular weights in the brain homogenates of mice in the blank control group, and the levels of Tau proteins of different molecular weights and total Tau proteins in the brain tissues of mice in the treatment group were significantly lower than those of mice in the vehicle group, and the statistical analysis P values ​​of 35kd, 35-40kd, 40kd, and 54kd molecular weight Tau protein levels and total Tau protein levels of the two groups were 0.174, 0.0406, 0.052, 0.067, and 0.055, respectively (Figure 11). This indicates that plasminogen can promote the degradation of Tau protein in the brain tissues of Alzheimer's model mice. Example 12

[0102] Example 12 relates to the fact that plasminogen promotes the cleavage of Pro-BDNF in brain homogenates of normal mice. Four C57BL / 6J male mice, aged 11–12 weeks and weighing 18–25 g, were taken and sacrificed to collect whole brain tissue, which was then weighed and added to 1× PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3–4 times), and after homogenization, centrifuged at 4°C (12000 rpm, 20 min), and the supernatant brain homogenate was placed in a new EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human BDNF-α (Na- ... Pro-BDNF, UniProtKB-P23560, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21 μL of Pro-BDNF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained, and quantitatively scanned. Brain-derived neurotrophic factor (BDNF) is a basic protein with a molecular weight of 12.3 kD, consisting of 119 amino acid residues and containing three pairs of disulfide bonds. It exists in a dimerized form in vivo and is synthesized in the form of a BDNF precursor, which is cleaved by enzymatic hydrolysis to form mature BDNF. It has been reported in the literature that Pro-BDNF has the opposite effect to the mature BDNF it is cleaved to form. Pro-BDNF promotes neuronal apoptosis and reduces synaptic plasticity. [5] Mature BDNF and its receptors are widely distributed in the central nervous system and play an important role in the survival, differentiation, growth and development of neurons during the development of the central nervous system. They can prevent neurons from being damaged and dying, improve the pathological condition of neurons, and promote biological effects such as the regeneration and differentiation of damaged neurons, and are necessary for the survival and normal physiological functions of neurons in the mature central nervous system and peripheral nervous system. [6] . As a result, in the brain homogenates of normal mice, the amount of Pro-BDNF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (Figure 12). This suggests that plasminogen can promote the cleavage of Pro-BDNF in the brain homogenates of normal mice. Example 13

[0103] Example 13 relates to the fact that plasminogen promotes the cleavage of Pro-BDNF and the formation of mature BDNF in brain homogenates from normal mice. Four C57BL / 6J male mice, aged 11–12 weeks and weighing 18–25 g, were taken and sacrificed to collect whole brain tissue, which was then weighed and added to 1× PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3–4 times), and after homogenization, centrifuged at 4°C (12000 rpm, 20 min), and the supernatant brain homogenate was placed in a new EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human BDNF-α (Na- ... Pro-BDNF, UniProtKB-P23560, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21 μL of Pro-BDNF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 45 min, followed by 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human BDNF antibody (Boster Biological Technology, PB9075), incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. As a result, in the brain homogenates of normal mice, the amount of Pro-BDNF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (** indicates P<0.01) (Figure 13). This suggests that plasminogen can promote the cleavage of Pro-BDNF in the brain homogenates of normal mice. Example 14

[0104] Example 14 relates to the fact that plasminogen promotes the degradation of Pro-BDNF in brain homogenates of Parkinson's disease model mice. Four C57BL / 6J male mice, aged 11-12 weeks and weighing 18-25g, were taken. The modeling time was set at 9 am every day. The blank control group was administered 200 μl of saline by intraperitoneal injection, and the model group was administered 5 mg / ml MPTP solution by intraperitoneal injection at 35 mg / kg / mouse for 5 consecutive days to establish the Parkinson's disease model. [6] Preparation of MPTP solution: 45 mg MPTP (Sigma, M0896) was dissolved in 9 ml of saline solution to prepare a final concentration of 5 mg / ml. After modeling was completed, i.e., on the 6th day after modeling, an open field test was performed on all mice to confirm whether modeling was successful. All mice were killed and whole brain tissue was collected, weighed, and then added with 1×PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3-4 times), and after homogenization, centrifuged at 4°C (12000 rpm, 20 min), and the supernatant, i.e., brain homogenate, was taken and transferred to a new EP tube. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human Pro-BDNF, UniProtKB-P23560, 1.0 mg / mL), 4.6 μL solvent solution, and 23.9 μL mouse brain homogenate; the plasminogen group was added with 21 μL Pro-BDNF (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate were added. After adding the samples to each group, the samples were incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained, and quantitatively scanned. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of Pro-BDNF was significantly lower in the plasminogen-administered group than in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (Figure 14). This suggests that plasminogen can promote the cleavage of Pro-BDNF in the brain homogenates of Parkinson's disease model mice. Example 15

[0105] Example 15 relates to the fact that plasminogen promotes the cleavage of Pro-BDNF and the formation of mature BDNF in brain homogenates of Parkinson's disease model mice. Eight C57BL / 6J male mice, aged 11-12 weeks and weighing 18-25g, were taken, and all mice were weighed one day before modeling, and randomly divided into two groups according to their weight, 4 mice in the blank control group and 4 mice in the model group. The modeling time was set at 9 am every day, and the blank control group was administered 200 μl of saline by intraperitoneal injection, and the model group was administered 5 mg / ml MPTP solution by intraperitoneal injection at 35 mg / kg / mouse for 5 consecutive days to construct the Parkinson's disease model. [6] Preparation of MPTP solution: 45 mg MPTP (Sigma, M0896) was dissolved in 9 ml of saline solution to prepare a final concentration of 5 mg / ml. After modeling was completed, i.e., on the 6th day after modeling, an open field test was performed on all mice to confirm whether modeling was successful. All mice were killed and whole brain tissue was collected, weighed, and then added with 1×PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min, 3-4 times), and after homogenization, centrifuged at 4°C (12000 rpm, 20 min), and the supernatant, i.e., brain homogenate, was taken and transferred to a new EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human Pro-BDNF, UniProtKB-P23560, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21 μL of Pro-BDNF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 45 min, followed by 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human BDNF antibody (Boster Biological Technology, PB9075), incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed with Image J. As a result, in the brain homogenates of Parkinson's disease model mice, the amount of Pro-BDNF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was significant (* indicates P<0.05, *** indicates P<0.001), and the amount of BDNF in the plasminogen-administered group was significantly higher than that in the solvent control group, and the difference was highly significant (Figure 15). This suggests that plasminogen can promote the cleavage of Pro-BDNF and the formation of mature BDNF in the brain homogenates of Parkinson's disease model mice. Example 16

[0106] Example 16 relates to the fact that plasminogen promotes the cleavage of Pro-BDNF in brain homogenates of Alzheimer's disease model mice. Four 11-week-old B6SJLTg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax(FAD) (Stock number: 034840) (abbreviated as FAD) mice were selected and sacrificed to collect whole brain tissue. A brain homogenate of the supernatant was prepared as in Example 1 and placed in an EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human BDNF-α (Na- ... Pro-BDNF, UniProtKB-P23560, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21.5 mL of Pro-BDNF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained, and quantitatively scanned. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of Pro-BDNF in the plasminogen group was significantly lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (Figure 16). This suggests that plasminogen can promote the cleavage of Pro-BDNF in the brain homogenates of Alzheimer's disease model mice. Example 17

[0107] Example 17 relates to the fact that plasminogen promotes the cleavage of Pro-BDNF and the formation of mature BDNF in brain homogenates of Alzheimer's disease model mice. Four 11-week-old B6SJLTg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax(FAD) (Stock number: 034840) (abbreviated as FAD) mice were selected and sacrificed to collect whole brain tissue. A brain homogenate of the supernatant was prepared as in Example 1 and placed in an EP tube. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) treatment group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-BDNF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human BDNF-α (NaBDNF-α) ... Pro-BDNF, UniProtKB-P23560, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21.5 mL of Pro-BDNF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 45 min, followed by 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to a PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human BDNF antibody (Boster Biological Technology, PB9075), incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed the band optical density with Image J. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of Pro-BDNF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was highly significant (** indicates P<0.01, *** indicates P<0.001); the amount of BDNF in the plasminogen-administered group was significantly higher than that in the solvent control group, and the difference was highly significant (Figure 17). This suggests that plasminogen can promote the cleavage of Pro-BDNF and the formation of mature BDNF in the brain homogenates of Alzheimer's disease model mice. Example 18

[0108] Example 18 relates to the fact that plasminogen promotes an increase in BDNF levels in the hippocampus of schizophrenia model mice. Thirty C57 female mice were taken and weighed before modeling. After excluding abnormal mice based on body weight, all mice were randomly divided into two groups, 8 mice in the blank control group and 22 mice in the model group. After grouping, the blank control group was fed with maintenance feed, and the model group was fed with modeling feed containing 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S30349) for 42 consecutive days to induce the schizophrenia model. 7,8After modeling, an open field test was performed on all mice, and according to the results of the test, the model group mice were divided into two groups, 11 mice in the solvent group and 11 mice in the treatment group. After grouping, all mice were administered the treatment, which was the first day. The mice in the blank control group were administered the solvent (4% arginine + 2% glycine solution) at 0.1 ml / mouse / day by tail vein injection, the mice in the solvent group were administered the solvent at 0.1 ml / mouse / day by tail vein injection, and the mice in the treatment group were administered plasminogen at 1 mg / 0.1 ml / mouse / day by tail vein injection, for 35 consecutive days. All mice were normally fed a maintenance diet during the treatment period. On the 36th day, the mice were sacrificed, and brain tissue was collected and fixed in 10% formaldehyde, dehydrated, and embedded. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. Brain tissue coronal sections were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. They were fixed with citric acid for 30 min, cooled at room temperature for 10 min, and then rinsed with water. They were incubated with 3% hydrogen peroxide for 15 min, and the tissue was circled with a PAP marker. They were blocked with 10% goat serum (Vector Laboratories, Inc., USA) for 1 h, and when the time was up, the goat serum was discarded, and rabbit anti-BDNF antibody (BosterBio, PB9075) was added and incubated overnight at 4 °C, and washed twice with PBS, for 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 h, and washed twice with PBS, for 5 min 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 washed once with PBS. The sections were dehydrated with a gradient, cleared, and mounted. The sections were observed and photographed under a light microscope at 400x magnification, and the photographs were positively stained and analyzed for optical density using Imaging-Pro software. Mature BDNF and its receptors are widely distributed in the central nervous system and play an important role in the survival, differentiation, growth and development of neurons during the development of the central nervous system. They can prevent neurons from being damaged and dying, improve the pathological condition of neurons, and promote biological effects such as the regeneration and differentiation of damaged neurons, and are necessary for the survival and normal physiological functions of neurons in the mature central nervous system and peripheral nervous system. [5] . The results showed that the hippocampus of the mice in the blank control group (Figure 18A) had a certain level of BDNF (marked with an arrow), the level of BDNF in the hippocampus of the mice in the vehicle group (Figure 18B) was improved, and the level of BDNF in the hippocampus of the mice in the treatment group (Figure 18C) was significantly higher than that of the vehicle group, with the statistical difference approaching significance (P=0.095) (Figure 18D). This suggests that plasminogen can promote the increase of the level of BDNF in the hippocampus of schizophrenia model mice. Example 19

[0109] Example 19 relates to the fact that plasminogen promotes the expression of BDNF in the hippocampus of Alzheimer's disease model mice. Twenty-three 24-week-old C57 male mice were taken, and their weights were measured before modeling. After excluding abnormal mice according to their weights, all mice were randomly divided into two groups, seven in the blank control group and 16 in the model group. All mice were anesthetized and located in the granulocyte layer of the hippocampus according to the mouse stereotaxic atlas (located according to the coordinates of the bregma point: AP-2.0mm, ML±1.5mm, DV2.0mm), and slowly microinjected into both sides of each mouse, injecting Aβ1-42 oligomer solution into the mice in the model group and the same amount of PBS solution into the mice in the blank control group, with an injection speed of 0.4μL / min and an injection volume of 2μL. After injection, the syringe was stopped for 5 minutes and slowly withdrawn, and the Alzheimer's model was injected. [3]was constructed. Preparation of Aβ1-42 oligomer solution (10 μM): Take β-amyloid (1-42) (catalog number: D2650, manufacturer: Sigma), add cold hexafluoroisopropanol to make the concentration 1 mg / ml, leave at room temperature for 3 days, then dispense into 45 μL / tube, i.e., 10 nmol / mL, leave in a fume hood overnight, dry in a 25 °C dry oven for 1 hour, and store at -80 °C. When used, add 10 μl of dimethyl sulfoxide solution to each tube to dissolve, and when injected, add 990 μL of sterile PBS solution and leave at 4 °C for 24 hours before use. 28 days after brain localization injection, all mice were weighed and subjected to Y-maze test, and abnormal mice in the blank control group and model group were eliminated according to the results of the test, and the model group mice were randomly divided into two groups, 6 mice in the solvent group, 7 mice in the treatment group, and 6 mice in the blank control group. Administration was started to mice in the vehicle group and the treatment group, which was designated as the first day. Plasminogen was administered to the mice in the treatment group at 1 mg / 0.1 ml / mouse / day by tail vein injection, and the mice in the vehicle group were administered the solvent (4% arginine + 2% glycine solution) at 0.1 ml / mouse / day by tail vein injection for 28 consecutive days, and the mice in the blank control group were not administered. On the 29th day, the mice were sacrificed, and the brains were collected and fixed in 10% formaldehyde for 24 to 48 hours. The fixed brain tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. The substantia nigra was located, the thickness of the sections was 4 μm, and the sections were deparaffinized, rehydrated, 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 goat serum (Vector Laboratories, Inc., USA) for 30 minutes, and the goat serum was discarded when the time was up. Rabbit anti-mouse BDNF antibody (BosterBio, PB9075) was added and incubated overnight at 4°C, washed twice with 0.01M PBS, each time for 5 minutes. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour, washed twice with 0.01M PBS, each time for 5 minutes.The sections were 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. They were dehydrated in a graded alcohol stream, cleared with xylene, and embedded in neutral rubber. Sections were observed under an optical microscope at 200x magnification. The results showed that the hippocampus of the mice in the blank control group (Figure 19A) expressed a certain level of BDNF (marked with an arrow), the expression of BDNF in the hippocampus of the mice in the vehicle group (Figure 19B) was significantly lower than that of the blank control group, and the expression of BDNF in the hippocampus of the mice in the treatment group (Figure 19C) was significantly higher than that of the vehicle group, with statistical differences being significant (* represents P<0.05) (Figure 19D). This indicates that plasminogen can promote the expression of BDNF in the hippocampus of Alzheimer's disease model mice. Example 20

[0110] Example 20 relates to the fact that plasminogen promotes the formation of mature NGF in brain tissue of spinal muscular atrophy (SMA) model mice. FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1MsdTg(SMN2*delta7)4299Ahmb / J mutant mice (hereafter referred to as SMNΔ7 SMA mice) have a homozygous mutation in the SMN1 gene and express the human SMN2 gene, and the clinical and pathological symptoms of these mice resemble those of human SMA. Breeding mice were purchased from The Jackson Laboratory, USA (Pedigree Number: 005025). Seven 3-day-old SMNΔ7 SMA mice were taken, four mice in the vehicle group were given 6 μl of bovine serum albumin solution (5 mg / ml) by intraperitoneal injection once every morning and afternoon for the first 9 days, and 6 μl of bovine serum albumin solution (10 mg / ml) by intraperitoneal injection once every day thereafter, and three mice in the treatment group were given 30 μg / 6 μl of plasminogen by intraperitoneal injection once every morning and afternoon for the first 9 days, and 60 μg / 6 μl of plasminogen by intraperitoneal injection once every day thereafter. Four wild-type mice served as blank control groups, which were administered 6 μl of bovine serum albumin solution (5 mg / ml) by intraperitoneal injection once daily in the morning and afternoon, respectively, during the first 9 days, and 6 μl of bovine serum albumin solution (10 mg / ml) was administered by intraperitoneal injection once daily from the 10th day. On the 12th day, the mice were sacrificed to collect hindlimb muscle tissues and prepare tissue homogenates for Western blot detection of NGF protein. 12% gels were prepared according to the instructions of the SDS-PAGE gel preparation kit. Samples from each group were mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of samples were taken for loading. The electrophoresis conditions were run at 30 V for 45 min, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h.The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-mouse NGF antibody (Abcam, ab52918) and incubated at room temperature for 2 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed the optical density value of each band with Image J. Nerve growth factor (NGF) is an important member of the neurotrophic factor family. It is synthesized in vivo in the form of a precursor that contains a signal peptide, a leader peptide, and a mature peptide. Research has reported that the precursor of nerve growth factor NGF (Pro-NGF) plays an opposing role to the NGF that is formed by cleavage. Pro-NGF can promote apoptosis of neurons. Mature NGF is involved in the regulation of processes such as neuronal growth, development, differentiation, survival, and repair after injury, and also plays an important role in regulating the functional expression of central and peripheral neurons. [7] The ratio of NGF / Pro-NGF=NGF optical density (OD) / Pro-NGF optical density (OD) value. The results showed that the brain tissue of the blank control mice had a certain NGF / ProNGF ratio, and the NGF / ProNGF ratio of the brain tissue of the treated mice was significantly higher than that of the vehicle mice, and the statistical difference was highly significant (*** indicates P<0.001) (Figure 20). This suggests that plasminogen can promote the conversion of ProNGF to NGF in SMA model mice and promote the formation of mature NGF. Example 21

[0111] Example 21 relates to the fact that plasminogen promotes the cleavage of Pro-NGF and the formation of mature NGF in brain homogenates from normal mice. Four C57BL / 6J male mice aged 11–12 weeks and weighing 18–25 g were selected and sacrificed to collect whole brain tissue, which was then weighed and added with 1× PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS, homogenized at 4°C (1 min / time, 3–4 times), and centrifuged at 4°C (12000 rpm, 20 min) after homogenization, and the supernatant brain homogenate was transferred to a new EP tube. Eppendorf (EP) tubes were taken and five parallels were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-NGF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed Pro-NGF, sequence derived from UniProtKB-P01138, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21 μL of Pro-NGF solution (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 15% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken for loading. The electrophoresis conditions were run at 30 V for 30 min, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to a PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human NGF antibody (Abcam, ab52918) and incubated at room temperature for 2 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed the band optical density with Image J. NGF is an important member of the neurotrophic factor family. It is synthesized in vivo in the form of a precursor that contains a signal peptide, a leader peptide, and a mature peptide. Studies have reported that the precursor of nerve growth factor (NGF) (Pro-NGF) plays an opposing role to the NGF that is formed by cleavage. Pro-NGF can promote apoptosis of neurons. [8] Mature NGF is involved in the regulation of processes such as neuronal growth, development, differentiation, survival, and repair after injury, and also plays an important role in regulating the functional expression of central and peripheral neurons. [9] . As a result, in the brain homogenates of normal mice, the amount of Pro-NGF was significantly lower in the plasminogen-administered group than in the solvent control group, and the difference was highly significant (* indicates P<0.05, *** indicates P<0.001); the amount of NGF in the plasminogen group was significantly higher than that in the solvent control group, and the difference was significant (Figure 21). This suggests that plasminogen can promote the cleavage of Pro-NGF and the formation of mature NGF in the brain homogenates of normal mice. Example 22

[0112] Example 22 relates to the fact that plasminogen promotes the cleavage of NGF and the formation of mature NGF in brain homogenates of Alzheimer's disease model mice. Four 11-week-old B6SJLTg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax(FAD) (Stock number: 034840) (abbreviated as FAD) mice were selected and sacrificed to collect whole brain tissue. A brain homogenate of the supernatant was prepared as in Example 1 and placed in an EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, (4) plasminogen group. The blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL Pro-NGF (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed Pro-NGF, UniProtKB-P01138, 1.0 mg / mL), 4.6 μL solvent solution, and 23.9 μL mouse brain homogenate; the plasminogen group was added with 21. μL Pro-NGF (1.0 mg / mL), 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate. After adding the sample to each group, the mixture was incubated at 37° C. for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 15% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed with 4× loading buffer (TaKaRa, e2139) in a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL of sample was taken and loaded. The electrophoresis conditions were run at 30 V for 30 min, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to a PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human NGF antibody (Abcam, ab52918) and incubated at room temperature for 2 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed the band optical density with Image J. As a result, in the brain homogenates of Alzheimer's disease model mice, the amount of Pro-NGF was significantly lower in the plasminogen group than in the vehicle control group, and the difference was highly significant (*** indicates P<0.001); the amount of NGF in the plasminogen administration group was significantly higher than in the vehicle control group, and the difference was significant (Figure 22). This suggests that plasminogen can promote the cleavage of Pro-NGF and the formation of mature NGF in the brain homogenates of Alzheimer's disease model mice. Example 23

[0113] Example 23 relates to the fact that plasminogen promotes the degradation of SOD-1 protein in brain homogenates of normal mice. Four 11-week-old C57BL / 6 (normal) mice were selected and sacrificed to collect whole brain tissue. A brain homogenate was prepared as the supernatant in the same manner as in Example 1 and transferred to an EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL SOD-1 protein (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human SOD-1 protein). SOD-1, UniProtKB-P00441, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21.5 μL of SOD-1 (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, the samples were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained, and quantitatively scanned. Superoxide dismutase-1 (SOD-1) is a metalloprotein that binds copper and zinc ions and plays an important role in converting the highly reactive reactive oxygen species superoxide into molecular oxygen and hydrogen peroxide. It is known that the SOD-1 gene is mutated in some patients with amyotrophic lateral sclerosis (ALS), an incurable neurological disease, and misfolded SOD-1 with an abnormal three-dimensional structure accumulates in motor neurons in the spinal cord. Furthermore, highly toxic misfolded SOD-1 has been detected in the cerebrospinal fluid of sporadic ALS patients of unknown etiology who do not have mutations in the gene encoding SOD-1. This means that structural abnormalities in SOD-1 may be related to the pathogenesis of ALS.

[10] . As a result, in the brain homogenates of normal mice, the amount of SOD-1 in the plasminogen group was lower than that in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (Figure 23), indicating that plasminogen can effectively promote the degradation of SOD-1 protein in the brain homogenates of normal mice. Example 24

[0114] Example 24 relates to the fact that plasminogen promotes the degradation of SOD-1 protein in brain homogenates of amyotrophic lateral sclerosis (ALS) model mice. Eight-week-old B6.Cg-Tg(SOD1-G93A)1Gur / J transgenic male mice (abbreviated as SOD1-G93A transgenic mice) were selected and sacrificed to collect whole brain tissue. The supernatant brain homogenate was prepared as in Example 6 and transferred to an EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) treatment group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL SOD-1 protein (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human plasminogen solution). SOD-1, UniProtKB-P00441, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen-treated group; 21.5 μL of SOD-1 (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen-treated group. After adding the samples to each group, the samples were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 12% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was electrophoresed as in Example 1, stained with Coomassie Brilliant Blue staining solution, destained, and quantitatively scanned. The SOD1-G93A transgenic mouse is a mouse model that has been widely used in research on amyotrophic lateral sclerosis (ALS).

[11] . As a result, the amount of SOD-1 protein in the brain homogenates of SOD1-G93A transgenic mice was significantly lower in the plasminogen-treated group than in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (Figure 24). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in the brain homogenates of ALS model mice. Example 25

[0115] Example 25 relates to the fact that plasminogen promotes the degradation of SOD-1 protein in brain homogenates of normal mice. Four 11-week-old C57BL / 6 (normal) mice were selected and sacrificed to collect whole brain tissue. A brain homogenate was prepared as the supernatant in the same manner as in Example 1 and transferred to an EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL SOD-1 protein (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human SOD-1 protein). SOD-1, UniProtKB-P00441, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21.5 μL of SOD-1 (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, the samples were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 15% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed uniformly with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL was taken for loading. The electrophoresis conditions were run at 30 V for 30 min, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human SOD-1 antibody (BOSTER Biological Technology, PB0453), incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed the band optical density with Image J. As a result, the amount of SOD-1 protein in the brain homogenates of normal mice was significantly lower in the plasminogen-administered group than in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (FIG. 25). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in the brain homogenates of normal mice. Example 26

[0116] Example 26 relates to the fact that plasminogen promotes the degradation of SOD-1 protein in brain homogenates of amyotrophic lateral sclerosis (ALS) model mice. Four 8-week-old B6.Cg-Tg(SOD1-G93A)1Gur / J transgenic male mice (abbreviated as SOD1-G93A transgenic mice) were selected and sacrificed to collect whole brain tissue. A brain homogenate of the supernatant was prepared as in Example 1 and transferred to an EP tube. Take Eppendorf (EP) tubes and set up five parallels for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL saline, 4.6 μL solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL mouse brain homogenate; the blank control group was added with 21.5 μL saline, 4.6 μL plasminogen solution (2 mg / mL), and 23.9 μL mouse brain homogenate; the solvent control group was added with 21.5 μL SOD-1 protein (Nanjing GenScript Biotechnology Co., Ltd., custom-expressed human SOD-1 protein). SOD-1, UniProtKB-P00441, 1.0 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate were added to the plasminogen group; 21 μL of SOD-1 (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to the plasminogen group. After adding the samples to each group, they were incubated at 37 °C for 6 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction. A 15% gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. Each group of samples was mixed uniformly with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 min, cooled and centrifuged for 2 min, and then 20 μL was taken for loading. The electrophoresis conditions were run at 30 V for 30 min, followed by running to the bottom of the gel at 100 V. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 h. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed four times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), added rabbit anti-human SOD-1 antibody (BOSTER Biological Technology, PB0453), incubated at room temperature for 3 hours, washed four times with TBST, added goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody, incubated at room temperature for 1 hour, washed four times with TBST, placed on a clean imaging plate, added Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographed with a biomolecular imager, and quantitatively analyzed the band optical density with Image J. As a result, in the brain homogenates of SOD1-G93A transgenic mice, the amount of SOD-1 protein was significantly lower in the plasminogen group than in the solvent control group, and the difference was highly significant (*** indicates P<0.001) (FIG. 26). This indicates that plasminogen can effectively promote the degradation of SOD-1 protein in the brain homogenates of SOD1-G93A transgenic mice. References: [1]Won-Seok Choi, et al. Conditional deletion of Ndufs4 in dopaminergic neurons promotes Parkinson’s disease-like nonmotor symptoms without loss of dopamine neurons. Scientific Reports. [2] KALIA L V,KALIA S K. Alpha-Synuclein and Lewy pathology in Parkinson’s disease[J]. Curr Opin Neurol,2015,28(4):375-381. [3]Selkoe D J. Alzheimer's disease: genes, proteins, and therapy[J]. Physiol. Rev, 2001, 81 (2): 741-766. [4]Naseri NN, Wang H, Guo J, Sharma M, Luo W. The complexity of tau in Alzheimer's disease. Neurosci Lett. 2019 Jul 13;705:183-194. [5]Gray K, Ellis V . Activation of pro-BDNF by the pericellular serine protease plasmin [J]. Febs Letters, 2008, 582(6):907-910. [6]Kowianski, Przemys?aw, Lietzau G , Czuba E , et al. BDNF: A Key Factor with Multipotent Impact on Brain Signaling and Synaptic Plasticity[J]. Cellular & Molecular Neurobiology, 2017 [7]Aloe L, Rocco M L , Bianchi P , et al. Nerve growth factor: from the early discoveries to the potential clinical use[J]. Journal of Translational Medicine, 2012, 10(1). [8] Aloe L , Rocco M L , Bianchi P , et al. Nerve growth factor: from the early discoveries to the potential clinical use[J]. Journal of Translational Medicine, 2012, 10(1). [9] Lewin G R , Lechner S G , Smith E S J . Nerve Growth Factor and Nociception: From Experimental Embryology to New Analgesic Therapy[J]. 2014.

[10] Anzai I, Tokuda E, Handa S, Misawa H, Akiyama S, Furukawa Y. Oxidative misfolding of Cu / Zn-superoxide dismutase triggered by non-canonical intramolecular disulfide formation. Free Radic Biol Med. 2020 Feb 1;147:187-199.

[11] Zhao J, Boyd AW, Bartlett PF. The identification of a novel isoform of EphA4 and ITS expression in SOD1G93A mice. Neuroscience. 2017 Apr 7;347:11-21.

Claims

1. A pharmaceutical composition comprising plasminogen for promoting degradation of misfolded proteins and aggregates thereof, said plasminogen having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2 and still having the proteolytic activity of plasminogen.

2. 2. The pharmaceutical composition of claim 1, wherein the plasminogen promotes degradation of one or more misfolded proteins and aggregates thereof selected from human amyloid Aβ40, human amyloid Aβ42, α-synuclein, Tau protein, SOD-1 protein, polyglutamine, neuroserin protease inhibitor (NSP), cystic fibrosis transmembrane conductance regulator, α1-antitrypsin, parkin protein, crystallin, transthyretin, short chain acyl-CoA dehydrogenase mutant, low density lipoprotein receptor, huntingtin, neurofilament protein, peripheral protein, α-internexin, islet amyloid polypeptide, β2-microglobulin, serum amyloid A protein, immunoglobulin light chain, human lysozyme, α-lactalbumin, prothymosin α, apolipoprotein E, and apolipoprotein J.

3. The pharmaceutical composition according to claim 1 or 2, wherein the plasminogen promotes the cleavage of Pro-BDNF into mature BDNF or promotes the cleavage of Pro-NGF into mature NGF.

Images