Use of HMMR ligands

Abstract

The invention discloses an application of an HMMR ligand. According to the application, new application of the HMMR ligand is found, and the HMMR ligand can regulate and control ERK to inhibit a gluconeogenesis pathway, so that the HMMR ligand has potential to be used as a diabetes treatment drug; the application also finds that the FCN3 protein or the derivative thereof can be used as an HMMR ligand to be combined with a membrane receptor HMMR, regulate ERK to inhibit a gluconeogenesis pathway, reduce expression of gluconeogenesis related genes and reduce the liver gluconeogenesis level, so that the liver sugar production is reduced, and the FCN3 protein or the derivative thereof has a very remarkable long-acting hypoglycemic effect; besides, the polypeptide newly designed in the invention shows a better hypoglycemic effect, is smaller in molecule and convenient to prepare, and is a high-potential lead molecule for treating diabetes, especially type 2 diabetes.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority to the Chinese patent application filed on June 21, 2023, with application number 202310748714X and invention name “Use of HMMR Ligands”, the full text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to the use of HMMR ligands. Background Art

[0004] Diabetes is a metabolic disease characterized by severe endocrine disorders and hyperglycemia. Long-term hyperglycemia can lead to a variety of chronic complications such as retinopathy, kidney disease, hypertension, and even coronary artery disease. Survey results show that the prevalence of diabetes in adults aged 18 and above in China has reached 12.8%, and the prevalence of prediabetes has reached 35.2%. Diabetes has become one of the most important and thorny public health issues in my country. Type 2 diabetes accounts for more than 95% of the total diabetes cases in adults, and its main cause is insulin resistance. The initial treatment of diabetes is mainly oral metformin and sulfonylurea drugs. However, long-term use of these two types of drugs leads to drug resistance in the body, and the mechanism of insulin lowering blood sugar is only a temporary solution, and there are insulin complications. Therefore, this field urgently needs to develop new hypoglycemic drugs suitable for clinical treatment. Summary of the invention

[0005] The object of the present invention is to provide a use of an HMMR ligand.

[0006] Another object of the present invention is to provide a use of ficolin 3 and its derivative peptides.

[0007] Another object of the present invention is to provide an isolated polypeptide.

[0008] Another object of the present invention is to provide a method for treating diabetes.

[0009] In order to solve the above technical problems, the first aspect of the present invention provides a use of an HMMR ligand for:

[0010] (i) prevention and / or treatment of diabetes;

[0011] (ii) preparing a medicament for preventing and / or treating diabetes;

[0012] (iii) lowering blood glucose concentration;

[0013] (iv) reduce the expression of hepatic gluconeogenesis-related genes;

[0014] (v) preparing an insulin sensitizer; and / or

[0015] (vi) Reduce insulin resistance or increase insulin sensitivity.

[0016] In some preferred embodiments, the HMMR ligand is ficolin 3 or a peptide derived therefrom.

[0017] In some preferred embodiments, the liver gluconeogenesis-related gene is selected from at least one of glucose-6-phosphatase (G6PC), forkhead box protein O1 (FOXO1), and phosphoenolpyruvate kinase (PEPCK).

[0018] In some preferred embodiments, the diabetes is type 2 diabetes.

[0019] In a second aspect, the present invention provides a use of ficolin 3 or a peptide derived therefrom for:

[0020] (i) prevention and / or treatment of diabetes;

[0021] (ii) preparing a medicament for preventing and / or treating diabetes;

[0022] (iii) lowering blood glucose concentration;

[0023] (iv) reduce the expression of hepatic gluconeogenesis-related genes;

[0024] (v) preparing an insulin sensitizer; and / or

[0025] (vi) reduce insulin resistance or increase insulin sensitivity;

[0026] (vii) as a human hyaluronan-mediated motility factor receptor modulator.

[0027] In some preferred embodiments, the liver gluconeogenesis-related gene is selected from at least one of glucose-6-phosphatase (G6PC), forkhead box protein O1 (FOXO1), and phosphoenolpyruvate kinase (PEPCK).

[0028] In some preferred embodiments, the diabetes is type 2 diabetes.

[0029] In some preferred embodiments, the ficolin 3 or its derivative peptide reduces the expression of hepatic gluconeogenesis-related genes by targeting HMMR.

[0030] In some preferred embodiments, the ficolin 3 or its derivative peptide activates the ERK signaling pathway by targeting HMMR.

[0031] In some preferred embodiments, the ficolin 3 or its derivative peptide reduces the expression of liver gluconeogenesis-related enzymes by activating the ERK signaling pathway.

[0032] In some preferred embodiments, the ficolin 3 or its derivative peptide reduces the expression of liver gluconeogenesis-related enzymes by inhibiting the cAMP signaling pathway.

[0033] The third aspect of the present invention provides an isolated polypeptide, wherein the polypeptide comprises an amino acid sequence selected from any one of the following:

[0034] (i) the amino acid sequence shown in SEQ ID NO: 1;

[0035] (ii) an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, most preferably at least 97% homologous to the amino acid sequence described in (i); and

[0036] (iii) an amino acid sequence complementary to the amino acid sequence described in (i) or (ii).

[0037] In some preferred embodiments, the polypeptide has an amino acid sequence selected from any one of the following:

[0038] (i) the amino acid sequence shown in SEQ ID NO: 1;

[0039] (ii) an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, most preferably at least 97% homologous to the amino acid sequence described in (i); and

[0040] (iii) an amino acid sequence complementary to the amino acid sequence described in (i) or (ii).

[0041] The fourth aspect of the present invention provides a fusion protein, ficolin 3 or a derivative peptide thereof.

[0042] In some preferred embodiments, the ficolin 3-derived peptide is the polypeptide described in the second aspect of the present invention.

[0043] In some preferred embodiments, the fusion protein comprises the polypeptide described in the second aspect of the present invention.

[0044] In some preferred embodiments, the fusion protein further includes at least one of IgG, Fc fragment, FLAG, His, GST, SUMO, TrxA, DsbA or eGFP.

[0045] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises ficolin 3 or a peptide derived therefrom, the polypeptide according to the second aspect of the present invention, and the fusion protein according to the third aspect of the present invention.

[0046] In some preferred embodiments, the pharmaceutical composition comprises the polypeptide described in the second aspect of the present invention or the fusion protein described in the third aspect of the present invention.

[0047] A sixth aspect of the present invention provides a method for preventing and / or treating diabetes, the method comprising the steps of:

[0048] A therapeutically effective amount of ficolin 3 or a peptide derived therefrom is administered to the subject.

[0049] A seventh aspect of the present invention provides a method for preventing and / or treating diabetes, the method comprising the steps of:

[0050] A therapeutically effective amount of an HMMR ligand is administered to the subject.

[0051] Compared with the prior art, the present invention has at least the following advantages:

[0052] (1) The present invention discovered a new use of HMMR ligands, which can regulate ERK to inhibit the gluconeogenesis pathway and thus have the potential to be used as a drug for the treatment of diabetes;

[0053] (2) The present invention also found that FCN3 protein or its derivatives can act as HMMR ligands, bind to the membrane receptor HMMR, regulate ERK to inhibit the gluconeogenesis pathway, reduce the expression of gluconeogenesis-related genes, reduce the level of liver gluconeogenesis, and thus reduce liver sugar production, which has a very significant long-term hypoglycemic effect;

[0054] (3) The newly designed polypeptide of the present invention has a superior hypoglycemic effect and is smaller in size and easier to prepare. It is a leading molecule with high potential for treating diabetes, especially type 2 diabetes.

[0055] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0057] Figure 1a This is a diagram showing the effect of the secretory protein FCN3 on glucose tolerance in HFD mice according to an example of the present invention.

[0058] Figure 1b This is a diagram showing the effect of the secretory protein FCN3 on insulin resistance in HFD mice according to an example of the present invention.

[0059] Figure 2 This is a diagram showing the effect of secretory protein FCN3 on reducing liver glucose metabolism level at the cellular level according to an embodiment of the present invention.

[0060] Figure 3 Schematic diagram of the long-term hypoglycemic effect of the secretory protein FCN3 according to an embodiment of the present invention.

[0061] Figure 4a This is a diagram showing the effect of secretory protein FCN3 on alleviating glucose tolerance in HFD mice via HMMR according to an embodiment of the present invention.

[0062] Figure 4b This is a diagram showing the effect of secretory protein FCN3 on alleviating insulin tolerance in HFD mice via HMMR according to an embodiment of the present invention.

[0063] Figure 5a and 5b This is a diagram showing the effect of HMMR knockdown on the secretory protein FCN3 on the transcription of genes related to liver gluconeogenesis according to an embodiment of the present invention.

[0064] Figure 6 This is a diagram showing the effect of HMMR knockdown on the secretory protein FCN3 on glucose tolerance in HFD mice according to an embodiment of the present invention.

[0065] Figure 7a and 7b This is a schematic diagram of the secretory protein FCN3 regulating liver glucose metabolism through the cAMP-PKA pathway according to an embodiment of the present invention.

[0066] Figure 7c Schematic diagram of secretory protein FCN3 regulating liver glucose metabolism through pERK pathway according to an embodiment of the present invention.

[0067] Figure 8a This is a diagram showing the effects of the secretory protein FCN3 and its derivative hFCN3-123 on glucose tolerance in HFD mice according to an embodiment of the present invention.

[0068] Figure 8b This is a diagram showing the effects of the secretory protein FCN3 and its derivative hFCN3-123 on the insulin tolerance of HFD mice according to an embodiment of the present invention.

[0069] Figure 9a This is a diagram showing the effects of the secretory protein FCN3 and its derivative hFCN3-123 on FOXO1 gene expression according to an embodiment of the present invention.

[0070] Figure 9bThis is a diagram showing the effects of the secretory protein FCN3 and its derivative hFCN3-123 on the expression of mPCK1 and mG6PC genes according to an embodiment of the present invention.

[0071] Fig.10 Schematic diagram of the structure of FCN3 protein and its derivative hFCN3-123 according to an embodiment of the present invention. DETAILED DESCRIPTION

[0072] After extensive and in-depth research, the inventors discovered that HMMR (human hyaluronan-mediated motility factor receptor) ligands can regulate cAMP to inhibit the gluconeogenesis pathway and thus have the potential to be used as a diabetes treatment drug. On this basis, the inventors discovered a new ligand for HMMR, FCN3. FCN3 or its derivative peptides reduce the expression of gluconeogenesis-related genes and the expression of key gluconeogenesis enzymes by interacting with HMMR, thereby inhibiting the liver gluconeogenesis pathway and reducing blood glucose concentration. It has a significant therapeutic effect on diabetes, especially type 2 diabetes, and the long-term hypoglycemic effect lasts for more than 3 days. It is a potential leading small molecule for type 2 diabetes. Based on FCN3, the inventors designed and developed a derivative peptide hFCN3-123, which can inhibit the classical gluconeogenesis pathway of the liver and reduce the liver's gluconeogenesis capacity beyond the FCN3 protein, and can produce a hypoglycemic effect significantly higher than the FCN3 protein.

[0073] HMMR ligands

[0074] The present invention relates to the use of HMMR (human hyaluronan-mediated motility factor receptor) ligands for (i) preventing and / or treating diabetes; (ii) preparing drugs for preventing and / or treating diabetes; (iii) lowering blood glucose concentration; (iv) lowering the expression of genes related to liver gluconeogenesis; (v) preparing insulin sensitizers; and / or (vi) alleviating insulin resistance or increasing insulin sensitivity.

[0075] As used in the present invention, the term "receptor" refers to a cell component that binds to a drug and can transmit information to cause an effect. It is a macromolecular protein present on the cell membrane or in the cytoplasm.

[0076] As used in the present invention, the term "ligand" is a substance that can specifically bind to a receptor. Ligands can generally agonize the corresponding receptors and cause specific physiological effects. Any ligand that can bind to a receptor and activate it is called an agonist, and a ligand that can bind to a receptor and block its activity is called an antagonist. Certain cell proteins that can bind to a ligand but have no ability to trigger an effect are called binders.

[0077] In a preferred embodiment of the present invention, the HMMR ligand is FCN3 or a peptide derived therefrom.

[0078] Use of FCN3 or its derivative peptides

[0079] The present invention relates to the use of FCN3 or its derivative peptides for (i) preventing and / or treating diabetes; (ii) preparing drugs for preventing and / or treating diabetes; (iii) reducing blood glucose concentration; (iv) reducing the expression of liver gluconeogenesis-related genes; (v) preparing insulin sensitizers; (vi) alleviating insulin resistance or increasing insulin sensitivity; and / or (vii) serving as HMMR ligands.

[0080] As used in the present invention, the terms "FCN3" and "Ficolin-3" are used interchangeably and refer to ficolin 3. Ficolin is a class of sugar-binding proteins that, in addition to the sugar recognition domain, also have collagen-like and fibrinogen-like domains. Three ficolin proteins have been found in the human body, namely, Ficolin-1, Ficolin-2 and Ficolin-3. Ficolin-3 was originally found in the serum of patients with systemic lupus erythematosus as an autoantigen. Subsequent studies have also found that Ficolin-3 is associated with liver disease, tumors and infectious diseases.

[0081] As used in the present invention, the term "derivative peptide" refers to a peptide obtained by mutation, extension, truncation, cyclization, modification, etc. of the original peptide. Mutation refers to the mutation of one or more natural or non-natural amino acid residues into other residues on the basis of the original peptide, extension refers to the addition of one or more natural or non-natural amino acid residues on the basis of the original peptide, truncation refers to the deletion of one or more amino acid residues on the basis of the original peptide, and modification includes C-terminal modification (amidation, sulfation, etc.), N-terminal modification (acetylation, fatty acidization, etc.), intermediate residue modification (glycosylation modification binding to Ser-, Tyr-, Asn-, Thr-; phosphorylation modification binding to Ser-, Tyr-, Thr-, and cyclization modification. In one embodiment of the present invention, the FCN3 protein is truncated to obtain the FCN3 protein derivative hFCN3-123. In a preferred embodiment of the present invention, the "derivative peptide" retains the effects of reducing the expression of gluconeogenesis-related genes, reducing the level of liver gluconeogenesis, reducing liver sugar production, and long-term hypoglycemic effect.

[0082] As used in the present invention, the term "gluconeogenesis-related genes" refers to genes encoding key enzymes in the gluconeogenesis process, as well as other genes that affect the expression of key enzymes in the gluconeogenesis process. The gluconeogenesis process refers to the process by which an organism converts a variety of non-sugar substances (such as starch, lactic acid amino acids or glycerol, etc.) into glucose or glycogen. The key enzymes in the gluconeogenesis process include glucose-6-phosphatase (G6PC), fructose-1,6-bisphosphatase (FBP), pyruvate carboxylase (PC), phosphoenolpyruvate kinase (PEPCK), etc. Genes that affect the expression of key enzymes in the gluconeogenesis process include cyclic adenosine monophosphate response element binding protein (CREB), whose activation can upregulate the expression of G6Pase and PEPCK; forkhead box protein O1 (FOXO1), whose expression can lead to upregulation of PEPCK and G6pase expression.

[0083] Isolated peptides

[0084] The present invention relates to an isolated polypeptide comprising an amino acid sequence selected from any one of the following: (i) an amino acid sequence shown in SEQ ID NO: 1; (ii) an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 97% homology with the amino acid sequence described in (i); and (iii) an amino acid sequence complementary to the amino acid sequence described in (i) or (ii).

[0085] As used in the present invention, the term "peptide" refers to a molecule comprising a sequence of amino acids bound by peptide bonds, regardless of length, post-translational modification or function. The term "polypeptide" refers to a protein that exists naturally or is produced or altered by recombination, chemically or otherwise, and is essentially a three-dimensional structure of a protein that is processed post-translationally in the same manner as the native protein. Herein, the terms "peptide", "polypeptide" and "protein" are synonymous herein.

[0086] In the present invention, the isolated polypeptide can be prepared by methods well known to those skilled in the art, such as recombinant methods and chemical synthesis methods. In the embodiments of the present invention, it is preferred to use chemical synthesis methods such as solid phase synthesis methods to link different or identical multiple amino acids in a specified order according to SEQ ID NO: 1 to form a peptide chain, and then obtain the isolated polypeptide by conventional separation operations.

[0087] Fusion Protein

[0088] The present invention also relates to a fusion protein, which is the isolated polypeptide mentioned above.

[0089] As used in the present invention, the term "fusion protein" refers to a molecule that is linked by covalent bonds to two or more proteins or fragments thereof and is contained by the main chain of each peptide. The fusion protein is preferably produced by genetic expression of a polynucleotide molecule encoding these proteins. In a preferred embodiment of the present invention, the fusion protein includes at least one fragment selected from IgG, Fc fragment, FLAG, His, GST, SUMO, TrxA, DsbA or eGFP in addition to the isolated polypeptide of the present invention as a fragment.

[0090] Pharmaceutical composition

[0091] The present invention also relates to a pharmaceutical composition, comprising at least one of ficolin 3 or its derivative peptide, the above-mentioned polypeptide, the above-mentioned fusion protein, and a pharmaceutically acceptable carrier.

[0092] In a preferred embodiment of the present invention, the polypeptide and carrier of the present invention are used as a pharmaceutical formulation (e.g., a composition, a preparation, a drug), which comprises an active compound, as described above, and one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to, a pharmaceutically acceptable carrier, an adjuvant, an excipient, a buffer, a preservative and a stabilizer. The formulation may also include other active agents. The present invention also relates to a pharmaceutical composition comprising a polypeptide or a fusion protein as described in the sixth aspect of the present invention, and a pharmaceutically acceptable formulation.

[0093] In the present invention, the term "pharmaceutically acceptable" refers to compounds, ingredients, substances, compositions, dosage forms, etc., which are suitable for contacting the tissues of the intended subjects (e.g., humans) without excessive toxicity, irritation, allergic reaction, or other problems or complications within the scope of reasonable medical judgment, and have a reasonable benefit / risk ratio. Each carrier, adjuvant, excipient, etc. must be "acceptable" in the sense of being compatible with the other formulation ingredients. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical textbooks, Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2 nd edition,1994.

[0094] Treatment

[0095] The present invention also relates to a method for treating diabetes, comprising the step of administering a therapeutically effective amount of ficolin 3 or a peptide derived therefrom to a subject.

[0096] The present invention also relates to a method for treating diabetes, comprising the steps of: reducing the expression of liver gluconeogenesis-related genes by targeting HMMR, thereby reducing blood glucose levels; preferably, in the method, a therapeutically effective amount of a drug (such as a polypeptide, compound, etc.) that targets and binds to (activates) HMMR is administered to the subject.

[0097] The present invention also relates to a method for treating diabetes, comprising the steps of: reducing the expression of liver gluconeogenesis-related enzymes by inhibiting the cAMP signaling pathway, thereby reducing blood glucose levels; preferably, in the method, a therapeutically effective amount of a drug (such as a polypeptide, compound, etc.) that activates the cAMP signaling pathway is administered to the subject.

[0098] The present invention also relates to a method for treating diabetes, comprising the steps of: lowering blood glucose levels by targeting HMMR to inhibit the cAMP signaling pathway; preferably, in the method, a therapeutically effective amount of a drug (e.g., a polypeptide, a compound, etc.) that targets (activates) HMMR is administered to the subject.

[0099] The subject to which the compounds of the present invention and / or treatment are administered is a mammal, preferably a test animal such as a rodent (rabbit, rat or mouse), dog, cat, monkey or ape, or a farm animal such as a cow, horse, sheep, pig or goat. More preferably, the subject is a human.

[0100] As used in the present invention, the term "therapeutically effective amount" refers to the amount of a drug sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a drug refers to the amount of a therapeutic agent that can provide a therapeutic effect in the treatment or management of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

[0101] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0102] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0103] Example 1

[0104] In this example, it was verified that the secreted protein FCN3 slowed down the level of glucose metabolism in the liver of DIO mice.

[0105] DIO mice were fed for 12 weeks and then subjected to a glucose tolerance test (GTT test) with a glucose dosage of 1 g / kg. The control group was injected with saline, and the experimental group was injected with 0.78 mg / kg of purified FCN3 recombinant protein. The test was performed at 0, 15, 30, 60, 90, and 120 minutes. The results are shown in the figure. Figure 1a shown. Figure 1a In the figure, the upper part is the curve of changes of glucose in the blood of the control group over time, and the lower part is the curve of changes of glucose in the blood of the experimental group over time. It can be seen that compared with the control group, the glucose tolerance level of the experimental group is improved.

[0106] DIO mice were fed for 12 weeks and then subjected to an insulin tolerance test (ITT test). The insulin dosage was 1 IU / kg. The control group was injected with normal saline, and the experimental group was injected with 0.78 mg / kg of purified FCN3 recombinant protein. The test was performed at 0, 15, 30, 60, 90, and 120 min. The results are shown in the figure. Figure 1b shown. Figure 1b In the figure, the upper part is the curve of changes of glucose in the blood of the control group over time, and the lower part is the curve of changes of glucose in the blood of the experimental group over time. It can be seen that compared with the control group, the insulin tolerance level of the experimental group is improved.

[0107] The above experimental results show that the secreted protein FCN3 slows down the level of glucose metabolism in the liver of DIO mice

[0108] Example 2

[0109] In this example, it was verified that the secretory protein FCN3 reduces the level of liver glucose metabolism at the cellular level.

[0110] In the primary liver cell experiment, the blank group was given normal saline, the control group was given DEX (dexamethasone) + FSK (forskolin), and the experimental group was given DEX (dexamethasone) + FSK (forskolin) and FCN3 protein 0.4ug / ml.

[0111] After administration of FCN3 protein at 0.4ug / ml, the cell supernatant was collected 36h later, and the glucose level in the supernatant was detected using a liquid sample glucose GLU content assay kit (oxidase method). The results are shown in Figure 2 . Figure 2 In each group of bar graphs, the left side is the blank group, the middle is the DEX+FSK group, and the right side is the DEX+FSK+FCN3 group. The experimental results show that the secretory protein FCN3 can reduce sugar production.

[0112] Example 3

[0113] In this example, it was verified that the secretory protein FCN3 has a long-lasting hypoglycemic effect.

[0114] After HFD mice were fed for 12 weeks, the control group was injected with normal saline, and the experimental group was injected with 0.78 mg / kg of purified FCN3 recombinant protein. The results were shown in Table 1. Figure 3 . Figure 3 In the figure, the upper part is the curve of the change of glucose in the blood of the control group over time, and the lower part is the curve of the change of glucose in the blood of the experimental group over time. It can be seen that compared with the control group, the purified hFCN3 recombinant protein in the experimental group has a long-term effect in lowering blood sugar.

[0115] Example 4

[0116] In this example, it was verified that the secretory protein FCN3 alleviated the level of glucose metabolism in the liver of HFD mice through HMMR.

[0117] AAV-mediated receptor knockout mice were fed a high-fat diet for 12 weeks and then subjected to a glucose tolerance test (GTT test) with a glucose dosage of 1 g / kg. AAV-shLacz was the AAV-mediated receptor knockout control group, and AAV-HR was the AAV-mediated HMMR receptor knockout group. The two groups were injected with saline and purified FCN3 recombinant protein 0.78 mg / kg, respectively, and tested at 0, 15, 30, 60, 90, and 120 minutes. The results are shown in Figure 2. Figure 4a shown. Figure 4a In the experiment, the receptor knockout control group verified that the purified FCN3 protein improved glucose tolerance in DIO mice. After AAV-dependent HMMR knockout, shLacz-hFCN3 is the curve of glucose changes in the blood of the receptor knockout control group over time, and shHR-hFCN3 is the curve of glucose changes in the blood of the AAV-mediated HMMR receptor knockout group over time. It can be seen that the improvement effect of purified FCN3 protein on glucose tolerance in mice was restored, verifying that the secretory protein FCN3 regulates the level of glucose tolerance by binding to the receptor HMMR.

[0118] AAV-mediated receptor knockout mice were fed a high-fat diet for 12 weeks and then subjected to an insulin tolerance test (ITT test). The insulin dosage was 1 IU / kg. AAV-shLacz was the AAV-mediated receptor knockout control group, and AAV-HR was the AAV-mediated HMMR receptor knockout group. The two groups were injected with saline and purified FCN3 recombinant protein 0.78 mg / kg, respectively, and tested at 0, 15, 30, 60, 90, and 120 min. The results are shown in Figure 2. Figure 4b shown. Figure 4bIn the experiment, the receptor knockout control group verified that the purified FCN3 protein improved the insulin tolerance level of DIO mice. After AAV-dependent HMMR knockout, shLacz-hFCN3 is the curve of glucose changes in the blood of the receptor knockout control group over time, and shHR-hFCN3 is the curve of glucose changes in the blood of the AAV-mediated HMMR receptor knockout group over time. It can be seen that the improvement effect of purified FCN3 protein on insulin tolerance in mice was restored, verifying that the secretory protein FCN3 regulates the insulin tolerance level by binding to the receptor HMMR.

[0119] The above experimental results indicate that the secreted protein FCN3 regulates hepatic glucose metabolism by binding to the receptor HMMR.

[0120] Example 5

[0121] In this example, qPCR primary liver cell experiments verified that the secretory protein FCN3 binds to HMMR and regulates liver glucose metabolism.

[0122] The experimental group was given purified FCN3 protein and qPCR experiment was performed 48 hours later. The results are shown in Figure 6 . Figure 6 In the middle, the left side is the verification of HMMR knockdown efficiency, followed by BLANK, DEX+FSK, DEX+FSK+FCN3 and DEX+FSK+FCN3+siHMMR on the transcriptional effects of sugar-producing genes FOXO1, G6PC, CREB, GP and PEPCK.

[0123] According to Figure 5, the purified FCN3 protein reduced the transcriptional levels of liver gluconeogenesis-related genes FOXO1, PEPCK, and G6PC, verifying that the purified FCN3 protein reduced liver gluconeogenesis levels. After administration of HMMR siRNA, the inhibitory effect of the purified FCN3 protein on the transcriptional levels of liver gluconeogenesis-related genes FOXO1, PEPCK, and G6PC was restored, verifying that the secretory protein FCN3 regulates liver glucose metabolism by binding to the receptor HMMR.

[0124] Example 6

[0125] In this example, it was verified that the secretory protein FCN3 alleviated the level of glucose metabolism in the liver of HFD mice through HMMR.

[0126] The HMMR receptor liver-specific knockout mice were fed a high-fat diet for 12 weeks and then a glucose tolerance test (GTT test) was performed. The glucose dosage was 1 g / kg. fl / fl were the recipient liver-specific knockout mice of the same littermate. LKOThe HMMR receptor liver-specific knockout mice were injected with saline and purified FCN3 recombinant protein 0.78 mg / kg respectively, and the HMMR receptor liver-specific knockout mice were injected with purified FCN3 recombinant protein 0.78 mg / kg. The results were tested at 0, 15, 30, 60, 90 and 120 minutes. Figure 6 shown. Figure 6 In the experiment, the receptor knockout control group verified that the purified FCN3 protein improved glucose tolerance in DIO mice. After liver-specific knockout of the HMMR receptor, the improvement of glucose tolerance in mice by the purified FCN3 protein was restored, verifying that the secretory protein FCN3 regulates glucose tolerance levels by binding to the receptor HMMR.

[0127] The above experimental results indicate that the secreted protein FCN3 regulates hepatic glucose metabolism by binding to the receptor HMMR.

[0128] Example 7

[0129] In this example, WB experiments of primary liver cells and ELISA verified that the secretory protein FCN3 regulates liver glucose metabolism through the cAMP-PKA pathway.

[0130] WB experiment was performed 30 minutes after administration of 0.4ug / ml of FCN3 purified protein. The results are shown in Figure 7a , pCREB level decreased. ELISA experiment was performed 15 minutes after administration of FCN3 purified protein 0.4mg / ml, and cAMP level decreased. The results are shown in Figure 7b . It is proved that FCN3 protein reduces the protein level of key enzymes of gluconeogenesis by inhibiting the cAMP signaling pathway, thereby inhibiting the gluconeogenesis pathway in the liver and lowering blood sugar levels. In each group of bar graphs, from left to right are BLANK, DEX+FSK, DEX+FSK+FCN3.

[0131] At the same time, WB liver primary experiments verified that the secreted protein FCN3 can also regulate liver glucose metabolism through the ERK pathway.

[0132] WB experiment was performed 4 hours after administration of 0.4ug / ml of FCN3 purified protein. The results are shown in Figure 7c .according to Figure 7c , the purified protein FCN3 activated the protein level of pERK, and its downstream pCREB and PEPCK levels decreased, proving that FCN3 protein reduces the protein level of key enzymes of gluconeogenesis by activating the ERK signaling pathway, thereby inhibiting the gluconeogenesis pathway in the liver and reducing blood sugar levels. In each group of bar graphs, from left to right are BLANK, DEX+FSK, DEX+FSK+FCN3.

[0133] Example 8

[0134] In this example, a new FCN3 derivative hFCN3-123 was designed based on the FCN3 protein, and it was verified that the secreted protein hFCN3-123 slowed down the level of glucose metabolism in the liver of DIO mice.

[0135] DIO mice were fed for 12 weeks and then subjected to a glucose tolerance test (GTT test) with a glucose dosage of 1 g / kg. The control group was injected with normal saline, and the experimental group was injected with 0.15 mg / kg of purified hFCN3-123 recombinant protein. The test was performed at 0, 15, 30, 60, 90, and 120 minutes. The results are shown in the figure. Figure 8a shown. Figure 8a In the figure, the upper part is the curve of changes of glucose in the blood of the control group over time, and the lower part is the curve of changes of glucose in the blood of the experimental group over time. It can be seen that compared with the control group, the glucose tolerance level of the experimental group is improved.

[0136] DIO mice were fed for 12 weeks and then subjected to an insulin tolerance test (ITT test). The insulin dosage was 1 IU / kg. The control group was injected with normal saline, and the experimental group was injected with 0.15 mg / kg of purified hFCN3-123 recombinant protein. The test was performed at 0, 15, 30, 60, 90, and 120 min. The results are shown in the figure. Figure 8b shown. Figure 8b In the figure, the upper part is the curve of changes of glucose in the blood of the control group over time, and the lower part is the curve of changes of glucose in the blood of the experimental group over time. It can be seen that compared with the control group, the insulin tolerance level of the experimental group is improved.

[0137] The above experimental results show that the secreted protein hFCN3-123 slows down the level of glucose metabolism in the liver of DIO mice.

[0138] Example 9

[0139] In this example, a new FCN3 derivative hFCN3-123 was designed based on the FCN3 protein, and the qPCR primary liver cell experiment verified that the FCN3 derivative hFCN3-123 also had a hypoglycemic effect and regulated liver glucose metabolism.

[0140] hFCN3-123 amino acid sequence SEQ ID NO: 1

[0141] MDLLWILPSLWLLLLGGPACLKTQEHPSCPGPRELEASKVV hFCN3-123 base sequence SEQ ID NO: 2

[0142] atgg atctactgtg gatcctgccc tccctgtggc ttctcctgct tggggggcctgcctgcctga agacccagga

[0143] acaccccagc tgcccaggac ccagggaact ggaagccagc aaagttgtc

[0144] FCN3 protein and purified protein derivative hFCN3-123 0.32ug / ml ( Fig.10 ) 36h after the qPCR experiment, the results are shown in Figure 9a and 9b In the figure, they are BLANK group, DEX+FSK group, DEX+FSK+FCN3 group, and DEX+FSK+FCN3-123 group.

[0145] according to Figure 9a and 9b The purified FCN3 protein derivative hFCN3-123 reduced the transcription levels of liver gluconeogenesis-related genes FOXO1, PEPCK, and G6PC. The results confirmed that compared with the full-length FCN3 purified protein, the FCN3 derivative hFCN3-123 could more significantly reduce the transcription levels of liver gluconeogenesis-related genes FOXO1, PEPCK, and G6PC.

[0146] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. Use of HMMR ligands: (i) prevention and / or treatment of diabetes; (ii) preparing a medicament for preventing and / or treating diabetes; (iii) lowering blood glucose concentration; (iv) reduce the expression of hepatic gluconeogenesis-related genes; (v) preparing an insulin sensitizer; and / or (vi) Reduce insulin resistance or increase insulin sensitivity.

2. The use according to claim 1, characterized in that The HMMR ligand is ficolin 3 or a peptide derived therefrom.

3. Use of ficolin 3 or its derivative peptides for: (i) prevention and / or treatment of diabetes; (ii) preparing a medicament for preventing and / or treating diabetes; (iii) lowering blood glucose concentration; (iv) reduce the expression of hepatic gluconeogenesis-related genes; (v) preparing insulin sensitizers; (vi) reducing insulin resistance or increasing insulin sensitivity; and / or (vii) As HMMR ligand.

4. The use according to claim 3, characterized in that The diabetes is type 2 diabetes.

5. The use according to claim 3, characterized in that: The liver gluconeogenesis-related gene is selected from at least one of glucose-6-phosphatase (G6PC), forkhead box protein O1 (FOXO1), and phosphoenolpyruvate kinase (PEPCK).

6. An isolated polypeptide, characterized in that The polypeptide comprises an amino acid sequence selected from any one of the following: (i) the amino acid sequence shown in SEQ ID NO: 1; (ii) an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 97% homologous to the amino acid sequence described in (i); and (iii) an amino acid sequence complementary to the amino acid sequence described in (i) or (ii).

7. A fusion protein, characterized in that The fusion protein comprises ficolin 3 or a peptide derived therefrom.

8. The fusion protein according to claim 6, characterized in that The fusion protein comprises the isolated polypeptide according to claim 6.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: ficolin 3 or a peptide derived therefrom; The isolated polypeptide of claim 6; or The fusion protein according to claim 7 or 8.

10. A method for preventing and / or treating diabetes, characterized in that: The method comprises the steps of: A therapeutically effective amount of a HMMR ligand is administered to the subject, and the HMMR ligand is preferably ficolin 3 or a peptide derived therefrom.

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