Application of polypeptide hormone in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention belongs to the technical field of medicine and disease treatment, and particularly relates to application of polypeptide hormone to preparation of medicine for treating non-alcoholic fatty liver disease. The polypeptide hormone ELABELA is ELABLA-21, and the amino acid sequence of the ELABLA-21 is as shown in SEQ ID NO. 1 (sequence identifier number 1). In-vivo experiments prove that the ELABLA-21 has a positive treatment effect on the non-alcoholic fatty liver disease, specifically, the ELABLA-21 obviously improves the symptoms of the non-alcoholic fatty liver disease of a mouse, reduces the weight of the liver, improves the fatty degeneration of the liver, reduces the lipid accumulation of the liver and reduces the triglyceride level of the liver, and a candidate medicine is provided for treatment of the non-alcoholic fatty liver disease. And the traditional Chinese medicine composition is an endogenous hormone synthesized by an organism, so that compared with the existing medicine for treating the non-alcoholic fatty liver disease, the traditional Chinese medicine composition has smaller toxic and side effects, and makes remarkable progress compared with the prior art.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and disease treatment, and specifically relates to the use of a polypeptide hormone in preparing a medicine for treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease is the liver component of metabolic syndrome and the most common chronic liver disease worldwide. It is a highly prevalent dynamic disease that occurs in all age groups. Its main sign is that triglycerides accumulate more than 5% to 10% of the liver weight, and it occurs in the absence of secondary factors such as excessive drinking, viral or autoimmune hepatitis, or congenital liver disease. Nonalcoholic fatty liver disease can rapidly develop into cirrhosis and liver malignancies. Among them, the English name of nonalcoholic fatty liver disease is Nonalcoholic fatty liver disease, NAFLD.

[0003] Most existing therapeutic drugs target individual components of metabolic syndrome and therefore have limited efficacy and are often associated with side effects. As a result, not only is there no effective treatment for metabolic syndrome, but existing treatments for NAFLD are ineffective against progression to NAFLD and fibrosis, and there is currently no U.S. Food and Drug Administration-approved treatment for NAFLD.

[0004] The peptide small molecule hormone ELABELA has a homologous sequence with Apelin. As a ligand of the G protein-coupled receptor APJ, it plays an extremely important role in various pathophysiological processes in the embryonic and adult stages. A large number of studies have shown that ELABELA has the effects of promoting angiogenesis, relaxation and cardiac development, and has a significant protective effect on cardiovascular diseases such as heart failure, hypertension, atherosclerosis and vascular remodeling. At present, it is necessary to develop a new product use for the peptide hormone ELABELA in the treatment of liver diseases including non-alcoholic fatty liver disease. Among them, the full English name of APJ is Apelin Peptide Jejunum. Summary of the invention

[0005] The purpose of the present invention is to provide a new product use of the polypeptide hormone ELABELA in the treatment of liver diseases including non-alcoholic fatty liver disease. The present invention found that the application of ELABELA-21 in vivo to treat high-fat-fed mice can significantly inhibit the non-alcoholic fatty liver phenotype of mice. Therefore, the present invention provides a use of a polypeptide hormone in the preparation of a drug for the treatment of non-alcoholic fatty liver disease.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] The present invention provides a use of a polypeptide hormone in preparing a drug for treating non-alcoholic fatty liver disease, that is, the present invention provides a new drug use of the polypeptide hormone ELABELA.

[0008] Wherein, the polypeptide hormone includes the polypeptide hormone ELABELA-21, and the polypeptide hormone also includes any one or more of the enzymatically derived polypeptides ELABELA-14 and ELABELA-11 of the polypeptide hormone ELABELA-21. ELABELA-21 is referred to as ELA-21. ELABELA-14 is referred to as ELA-14. ELABELA-11 is referred to as ELA-11.

[0009] The amino acid sequence of ELA-21 is shown in SEQ ID NO.1: LRKHNCLQRRCMPLHSRVPFP.

[0010] The amino acid sequence of the enzymatically derived polypeptide ELA-14 of the polypeptide hormone ELA-21 is shown in SEQ ID NO.2: QRRCMPLHSRVPFP.

[0011] The amino acid sequence of the enzymatically derived polypeptide ELA-11 of the polypeptide hormone ELA-21 is shown in SEQ ID NO.3: CMPLHSRVPFP.

[0012] Preferably, ELA-21 reduces liver weight.

[0013] Preferably, ELA-21 improves hepatic steatosis.

[0014] Preferably, ELA-21 inhibits hepatic lipid accumulation.

[0015] Preferably, ELA-21 reduces liver TG levels.

[0016] Preferably, ELA-21 inhibits steatohepatitis.

[0017] Preferably, the drug contains the polypeptide hormone ELABELA as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0018] Wherein, the polypeptide hormone ELABELA is any one of ELA-21, ELA-14 and ELA-11.

[0019] Preferably, the drug is a drug for reducing liver weight.

[0020] Preferably, the drug is a drug for improving hepatic steatosis.

[0021] Preferably, the drug is a drug that inhibits hepatic lipid accumulation.

[0022] Preferably, the drug is a drug that reduces liver TG levels.

[0023] Preferably, the drug is a drug for improving fatty liver hepatitis.

[0024] Preferably, the administration of the drug includes any one of parenteral administration and intravenous administration. Parenteral administration includes subcutaneous administration, intradermal administration, intramuscular injection and intraperitoneal injection, which is convenient for administration; intravenous administration has a rapid onset of effect.

[0025] Preferably, the medicine includes any one of tablets, capsules, granules, pills, emulsions, liquids, patches, solutions, ointments and injections.

[0026] Preferably, the drug is a tablet, and the polypeptide hormone ELABELA is mixed with an excipient to obtain a mixed powder; the mixed powder is mixed with starch slurry to obtain a soft material, which is then granulated, dried and granulated in sequence, and the obtained granules are mixed with talcum powder and then tableted to obtain the tablet.

[0027] Preferably, the drug is a capsule, and the mixed powder of the polypeptide hormone ELABELA and the excipient is sieved and then filled into a soft capsule material made of gelatin to obtain the capsule.

[0028] Preferably, the drug is a granule, and the polypeptide hormone ELABELA is mixed with an excipient to obtain a mixed powder; the mixed powder is mixed with an ethanol aqueous solution to obtain a soft material, which is then granulated, dried and granulated in sequence to obtain the granule.

[0029] Preferably, the excipients include any one or more of starch, sucrose, trehalose, mannitol and sorbitol. The excipients combine with the polypeptide hormone ELABELA to form a stable inclusion complex, increase the stability of the polypeptide hormone ELABELA, protect the polypeptide hormone ELABELA molecules from environmental factors, and improve the stability and bioavailability of the polypeptide hormone ELABLEA.

[0030] Preferably, the carrier includes any one or more of nanoparticles, liposomes, transfersomes, microspheres, and hydrogels. The above carriers all have good biocompatibility, biodegradability, and renewability, thereby improving the utilization rate, safety, and effectiveness of the drug. Among them, nanoparticles can adjust the release rate of drugs in the human body, change the biomembrane permeability and in vivo distribution of drug molecules, and can deliver drugs to targeted organs; liposomes, transfersomes, microspheres, and hydrogels can improve the ability of drugs to penetrate the skin barrier, improve the bioavailability of drugs, and reduce the frequency of drug administration.

[0031] Preferably, the drug is a solution.

[0032] The carrier used in the solution includes a solvent.

[0033] The solution is prepared by compounding the polypeptide hormone ELABELA with a solvent.

[0034] Solution drugs have a large dispersion, fast absorption, rapid action, and good bioavailability; the active ingredients are evenly dispersed and can be accurately measured and used, especially suitable for children and elderly patients; the dosage size is easy to adjust and control; it can reduce the local irritation of certain soluble drugs; and it can increase the stability and safety of certain drugs. Therefore, the dosage form of the above-mentioned drug provided by the present invention is a solution.

[0035] Preferably, the concentration of the polypeptide hormone ELABELA in the solution is 0.05 mg / mL to 0.1 mg / mL.

[0036] Preferably, the solvent is any one of DMSO, water, ethanol, glycerol, and physiological saline.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a use of a polypeptide hormone in the preparation of a drug for treating non-alcoholic fatty liver disease, and provides a new method for treating non-alcoholic fatty liver disease. The present invention proves the positive therapeutic effect of ELA-21 in non-alcoholic fatty liver disease through in vivo experiments. Specifically, ELA-21 significantly improves the symptoms of non-alcoholic fatty liver disease in mice, reduces liver weight, improves liver steatosis, reduces liver lipid accumulation, and reduces liver TG levels, providing a candidate drug for the treatment of non-alcoholic fatty liver disease. Moreover, it is an endogenous hormone synthesized by the body itself, and has less toxic and side effects than existing drugs for treating non-alcoholic fatty liver disease, and has made significant progress compared with the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the experimental process of the animal experiment in the embodiment of the present invention.

[0040] Figure 2 The non-alcoholic fatty liver phenotype of mice in the present invention: wherein, Figure 2 Figure a shows the liver weight of mice; Figure 2 Figure b is a physical picture of the liver of mice in the Control group; Figure 2 Figure c is a physical picture of the liver of mice in the HFD group; Figure 2 Figure d is a physical picture of the liver of mice in the HFD+ELA-21 group; Figure 2 Figure e shows the HE staining results of liver tissue of mice in the Control group; Figure 2Figure f shows the HE staining results of liver tissues of mice in the HFD group; Figure 2 Figure g shows the HE staining results of liver tissues of mice in the HFD+ELA-21 group; Figure 2 Figure h shows the Oil Red O staining results of liver tissue of mice in the Control group; Figure 2 Figure i in the figure shows the results of Oil Red O staining of liver tissues of mice in the HFD group; Figure 2 Figure j shows the Oil Red O staining results of liver tissue of mice in the HFD+ELA-21 group.

[0041] Figure 3 is the serum lipid level of mice in the present invention: wherein, Figure 3 Figure a shows the serum high-density lipoprotein level in mice; Figure 3 Panel b shows the serum low-density lipoprotein level in mice; Figure 3 Figure c shows the total cholesterol level in mouse serum; Figure 3 Figure d shows the serum triglyceride level of mice.

[0042] Figure 4 is the lipid level in mouse liver in the present invention: wherein, Figure 4 Figure a shows the level of high-density lipoprotein in mouse liver; Figure 4 Panel b shows the level of low-density lipoprotein in mouse liver; Figure 4 Figure c shows the total cholesterol level in mouse liver; Figure 4 Panel d shows the triglyceride level in mouse liver. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0044] The mice used in the embodiments of the present invention are all of the C57BL / 6J strain and are raised in the Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine in a specific pathogen-free animal room. Experimental Animal Use License No.: SCXK(Gan)2023-0001.

[0045] Among them, the English name of specific pathogens is specific pathogen free, SPF.

[0046] All mice used in the experiment were 8-week-old male mice, and all mouse experiments complied with the "Regulations on the Use of Experimental Animals" formulated by the National Ethics Committee and the Jiangxi University of Traditional Chinese Medicine. The mice used in the experiment are referred to as C57BL / 6J mice below.

[0047] Example 1

[0048] 1. Fatty liver modeling, grouping, and drug administration

[0049] (1) Fatty liver model

[0050] After C57BL / 6J mice were fed a high-fat diet (45 kcal% fat, HFD) for 12 weeks, the lipid level (total cholesterol) in the serum increased, indicating that the model was successful, and the C57BL / 6J mouse model group mice were obtained.

[0051] (2) Grouping

[0052] Seventeen mice were randomly divided into three groups: control group, 5 mice; HFD group, 6 mice; HFD+ELA-21 group, 6 mice. The mice in the HFD+ELA-21 group were intraperitoneally injected with ELA-21 once a day from the 13th week, and samples were collected at the 16th week for subsequent experiments.

[0053] Among them, the control group is also called the Control group.

[0054] The experimental process of animal experiments is as follows Figure 1 shown.

[0055] The specific grouping and dosing methods are as follows:

[0056] Control group: The mice were fed with normal mouse diet and given an intraperitoneal injection of an equal volume of saline. Here, "equal volume" means that the volume of the injected saline is the same as the volume of the injected ELA-21 solution.

[0057] HFD group: fed with a high-fat diet, and given an intraperitoneal injection of an equal volume of saline. Here, "equal volume" means that the volume of the injected saline is the same as the volume of the injected ELA-21 solution.

[0058] HFD+ELA-21 group: The mice were fed with a high-fat diet and given an intraperitoneal injection of ELA-21 solution. The amount of ELA-21 injected into the mice was 1 mg / kg / day.

[0059] Among them, ELA-21 was synthesized by Nanjing GenScript Biotechnology Co., Ltd. with a purity of >98% and stored at -20°C. Before use, it was dissolved in physiological saline and sterilized with a 0.22μm filter to obtain an ELA-21 solution with a concentration of 0.07mg / mL.

[0060] 2. Sample collection and processing

[0061] The mice in each group obtained at the end of the experiment were fasted for 12 hours before sampling, but not water deprivation, and weighed, and their eyeballs were removed to collect blood. The liver tissue was removed, weighed, and the body was photographed with a macrophotographer.

[0062] The liver weight of mice was Figure 2 As shown in Figure a; The liver of mice in the Control group is as follows Figure 2 As shown in Figure b; the liver of mice in the HFD group Figure 2 As shown in Figure c; the liver of mice in the HFD+ELA-21 group Figure 2 As shown in Figure d. Under high-fat diet conditions, the liver of mice turned yellow, became diffusely enlarged, and the liver weight increased, while ELA-21 treatment could significantly reduce the liver weight of mice and alleviate liver enlargement.

[0063] The 2 cm × 1.5 cm × 0.3 cm liver tissues of the mice in the control group, HFD group, and HFD+ELA-21 group were fixed in 4% by mass paraformaldehyde, and the remaining liver tissues were quickly frozen in liquid nitrogen for 30 min and then stored at -80°C for later use.

[0064] The mouse blood collected from each group was placed in 1.5 mL centrifuge tubes, left to stand at room temperature for 1 hour, centrifuged at 4°C and 3500 r / min for 10 minutes, and the supernatant was collected to obtain serum samples, which were stored at -80°C for later use.

[0065] 3. Liver HE staining and Oil Red O staining

[0066] After the liver tissue in step 2 is fixed in a 4% by volume paraformaldehyde solution at 4°C for 24 hours, the liver tissue is removed and rinsed with running water, and then automatically dehydrated and wax-immersed before being embedded in paraffin. The slices are sliced ​​using a paraffin slicer with a thickness of 4 μm, and baked in an oven at 65°C for 2 hours, and stored at room temperature for later use to obtain paraffin sections.

[0067] Paraffin sections were dewaxed to water by an automatic stainer, and then stained with hematoxylin for 5 minutes, washed with distilled water, differentiated with differentiation solution, washed with distilled water, and blued with blueing solution until the cytoplasm was dark blue and the nucleus was not stained, rinsed with running water, and the sections were sequentially dehydrated with 85% ethanol and 95% ethanol for 5 minutes each, and stained with eosin solution for 5 minutes. The sections were then sequentially placed in 100% ethanol I for 3 minutes, 100% ethanol II for 3 minutes, 100% ethanol III for 3 minutes, xylene I for 3 minutes, and xylene II for 3 minutes; the sections were sealed with neutral gum and photographed under a microscope. The results are shown in the figure. Figure 2 As shown in Figure e.

[0068] After the liver tissue in step 2 was fixed in a 4% by volume paraformaldehyde solution at 4°C for 24 hours, the tissue was removed and rinsed with running water overnight, precipitated with a 30% sucrose solution, embedded in OCT, sliced ​​with a frozen slicer, the slice thickness was 8 μm, incubated with 100% by volume isopropanol for 5 minutes, incubated with 0.5% by volume Oil Red O solution at 60°C for 7 minutes, washed with 85% by volume isopropanol solution for 3 minutes, washed with distilled water, stained with hematoxylin for 1 minute, washed with distilled water, and sealed with a sealing agent. The lipids were red or orange, and the cell nuclei were light blue. The slides were photographed under a microscope. The results are as follows Figure 2 As shown in Figure f.

[0069] Depend on Figure 2 As shown in Figures e and f, mice can develop fatty liver under high-fat diet conditions, with pathological manifestations of elevated liver steatosis and excessive fat accumulation. However, after ELA-21 treatment, the symptoms were significantly improved, and the level of liver steatosis and fat accumulation were significantly reduced. Figure 2 The result of Figure e is HE staining. Figure 2 The results in Figure f are Oil Red O staining.

[0070] Depend on Figure 2 It can be seen that ELA-21 treatment can significantly improve non-alcoholic fatty liver disease induced by a high-fat diet.

[0071] 4. Detection of blood lipid levels

[0072] The serum samples prepared above were taken and the levels of LDL-C, HDL-C, TC and TG in mouse serum were detected using LDL-C kit, HDL-C kit, TC kit and TG kit respectively. The results are as follows: Figure 3 shown.

[0073] Among them, the Chinese name of LDL-C is low-density lipoprotein cholesterol; the Chinese name of HDL-C is high-density lipoprotein cholesterol; the Chinese name of TC is serum total cholesterol; and the Chinese name of TG is triglyceride.

[0074] LDL-C kit, HDL-C kit, TC kit, and TG kit were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0075] The catalog number of the LDL-C kit is A113-1-1.

[0076] The catalog number of the HDL-C kit is A112-1-1.

[0077] The catalog number of the TC kit is A111-1-1.

[0078] The catalog number of the TG kit is A110-1-1.

[0079] Depend on Figure 3 It can be seen that a high-fat diet significantly increased the levels of serum LDL-C, HDL-C and TC, and decreased the level of TG. Although ELA-21 treatment did not affect the levels of serum LDL-C, HDL-C and TC, it slightly increased the level of serum TG.

[0080] Depend on Figure 3 It can be seen that ELA-21 has no significant effect on blood lipid levels.

[0081] 5. Detection of liver lipid levels

[0082] The liver tissues retained above were taken, and the weight of the liver tissues was accurately weighed. The tissues were homogenized with anhydrous ethanol, and the levels of LDL-C, HDL-C, TC and TG in the extracts were detected using LDL-C kit, HDL-C kit, TC kit and TG kit, respectively. The results were as follows: Figure 4 shown.

[0083] Among them, the Chinese name of LDL-C is low-density lipoprotein cholesterol; the Chinese name of HDL-C is high-density lipoprotein cholesterol; the Chinese name of TC is serum total cholesterol; and the Chinese name of TG is triglyceride.

[0084] LDL-C kit, HDL-C kit, TC kit, and TG kit were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0085] The catalog number of the LDL-C kit is A113-1-1.

[0086] The catalog number of the HDL-C kit is A112-1-1.

[0087] The catalog number of the TC kit is A111-1-1.

[0088] The catalog number of the TG kit is A110-1-1.

[0089] Depend on Figure 4 It can be seen that a high-fat diet increases the levels of HDL, LDL, TC and TG in liver tissue, and ELA-21 treatment significantly reduces the level of TG in liver tissue, but has no effect on the levels of HDL, LDL and TC in liver tissue.

[0090] Depend on Figure 4 It can be seen that ELA-21 treatment significantly inhibited the accumulation of TG in the liver.

[0091] From the above experimental results, it can be seen that in non-alcoholic fatty liver disease caused by a high-fat diet, ELA-21 significantly reduces liver weight, improves liver fatty degeneration, reduces liver lipid accumulation, and reduces liver TG levels, which can effectively alleviate the progression of this type of fatty liver disease.

[0092] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0093] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of a polypeptide hormone in the preparation of a drug for treating non-alcoholic fatty liver disease, characterized in that: The polypeptide hormones include polypeptide hormone ELABELA-21; The amino acid sequence of the polypeptide hormone ELABELA-21 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The polypeptide hormones also include enzyme-cut derivative polypeptides ELABELA-14 and ELABELA-11 of the polypeptide hormone ELABELA-21; the amino acid sequence of ELABELA-14 is shown in SEQ ID NO.2; the amino acid sequence of ELABELA-11 is shown in SEQ ID NO.

3.

3. The use according to claim 1, characterized in that The medicine uses the polypeptide hormone ELABELA as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

4. The use according to claim 3, characterized in that The medicine includes any one of tablets, capsules, granules, pills, emulsions, liquids, patches, solutions, ointments and injections.

5. The use according to claim 3, characterized in that: The excipients include any one or more of starch, sucrose, trehalose, mannitol and sorbitol.

6. The use according to claim 3, characterized in that: The carrier includes any one or more of nanoparticles, liposomes, transfersomes, microspheres, and hydrogels.

7. The use according to claim 3, characterized in that: The drug is a solution; The carrier aided by the solution includes a solvent; The solution is prepared by compounding the polypeptide hormone ELABELA with a solvent.

8. The use according to claim 7, characterized in that The concentration of the polypeptide hormone ELABELA in the solution is 0.05 mg / mL to 0.1 mg / mL.

9. The use according to claim 7, characterized in that: The solvent is any one or more of DMSO, water, ethanol, glycerol, and physiological saline.

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