Micropeptide MP19 regulating cholesterol metabolism, and use thereof

Abstract

The present application relates to a gene for regulating cholesterol metabolism and a micropeptide MP19 encoded thereby, and a use thereof, and also relates to a use of the micropeptide in the preparation of a drug or pharmaceutical composition for detecting, preventing, alleviating or treating diseases associated with abnormal cholesterol metabolism, said diseases comprising atherosclerosis, age-related macular degeneration, hyperlipidemia, non-alcoholic hepatitis, hepatocellular carcinoma, coronary heart disease, ischemic stroke, and obesity. By means of endogenous overexpression or exogenous synthesis of the micropeptide, diseases associated with abnormal cholesterol metabolism can be effectively prevented, alleviated, or treated.

Description

Micropeptide MP19 for regulating cholesterol metabolism and application thereof

[0001] CROSS-REFERENCE

[0002] This application claims priority to Chinese Patent Application No. 202411003823X filed on July 25, 2024 with the China National Intellectual Property Office, the content of this application is incorporated herein by reference in its entirety for all purposes. TECHNICAL FIELD

[0003] The present application belongs to the technical field of biological medicine, and specifically relates to a micropeptide MP19 for regulating cholesterol metabolism and application thereof. BACKGROUND

[0004] Atherosclerosis is a disease affecting a large number of people, is the pathological basis of cardiovascular and cerebrovascular diseases, is closely related to lipid metabolism disorder, is a chronic inflammatory disease with characteristics of autoimmune disease occurring in the intima of arterial blood vessels, has characteristics of intima wall thickening of arterial blood vessels, proliferation and increase of vascular smooth muscle cells, lipid accumulation in the vessel wall, and plaque formation, and research has found that, in particular, the increase of low-density lipoprotein cholesterol level plays a key role in the occurrence and development of atherosclerosis. The pathogenesis of atherosclerosis is still not very clear, and atherosclerosis has disadvantages of high incidence, high treatment cost, and long treatment cycle, and currently mainly used lipid-lowering drugs in clinical practice include statins, ezetimibe, and PCSK9 inhibitors. Although these drugs have good lipid-lowering effect, most patients still fail to achieve the treatment goal.

[0005] Age-related macular degeneration (AMD) is one of the main causes of blindness worldwide, a severe and progressive retinal disease that can destroy the macular area of the retina, is the main cause of irreversible vision loss in the elderly, and brings a major burden to the health care system and increases its economic burden. Research has found that dyslipidemia and changes in HDL-C levels are associated with AMD (Age-related macular degeneration. Nat Rev Dis Primers. 2021 May 6; 7(1): 31. doi: 10.1038 / s41572-021-00265-2).

[0006] First, lipids are the main component of drusen, which can account for more than 40% of their volume. The main ultrastructural component of drusen or basal linear deposits (BLinD) is a large lipoprotein secreted by retinal pigment epithelium (retinal pigment epithelial cells), which is rich in apolipoprotein B (ApoB), apolipoprotein E (ApoE), and cholesterol, which confirms that abnormal cholesterol metabolism is directly related to the occurrence and development of AMD.

[0007] Specifically, retinal pigment epithelial cells accumulate cholesterol by taking up lipoproteins in the circulatory system or phagocytosing photoreceptor extracellular segment. In a healthy state, retinal pigment epithelial cells recycle cholesterol to photoreceptor cells, or remove cholesterol by effluxing to apolipoprotein A-I (ApoA-I) to form high-density lipoprotein particles. If this reverse cholesterol transport is blocked, retinal pigment epithelial cells will secrete ApoB100 lipoprotein, the main component of low-density lipoprotein, to Bruch's membrane (BrM). In fact, the excess lipid in retinal pigment epithelial cells will accumulate after efflux, which is similar to atherosclerosis, and eventually cause Bruch's membrane to thicken. Some scholars believe that Bruch's membrane is essentially equivalent to the blood vessel wall. Studies have shown that the formation of soft drusen or basal linear deposits is due to the impaired removal function of the aging Bruch's membrane-choroidal capillary endothelium, causing metabolic products effluxed by retinal pigment epithelial cells to accumulate in the retinal pigment epithelial cell-sub-basement membrane gap.

[0008] Secondly, variations in multiple lipid metabolism-related genes (such as LIPC, CETP, ABCA1 and APOE) are associated with the risk of age-related macular degeneration (AMD), which further confirms the key role of abnormal lipid metabolism in the pathogenesis of AMD.

[0009] Clinically, the treatment methods for AMD include photodynamic therapy, surgical treatment, intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs such as ranibizumab, aflibercept and conbercept, but intravitreal injection has the risk of endophthalmitis, hemorrhage, etc.

[0010] It should be noted that the methods described in this section are not necessarily the methods that have been previously conceived or used. Unless otherwise indicated, nothing in this section should be construed as a recognition that any method described in this section is an existing technology. Similarly, unless otherwise indicated, a reference in this section to a problem should not be construed as an acknowledgment that the problem is recognized in any existing technology. SUMMARY

[0011] To solve the above problems, the present application provides a polypeptide and its mutant, which can be used to regulate the cholesterol metabolism of the body, thereby having a good detection, prevention, alleviation or treatment effect on various diseases or symptoms, such as obesity, atherosclerosis, age-related macular degeneration, hyperlipidemia, non-alcoholic hepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, tumor angiogenesis, coronary heart disease, ischemic stroke, embolism caused by blood lipid plaque shedding, etc. Therefore, the polypeptide and its mutant provided by the present application have good application prospect and clinical value.

[0012] According to an embodiment of the present application, the polypeptide has an amino acid sequence of any one of (i)-(iv) below: (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) at least one of an amino acid sequence obtained by deleting, substituting or inserting at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids from the amino acid sequence set forth in SEQ ID NO: 1; (iii) at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1; and (iv) at least one of a sequence containing the amino acid sequence PVS.

[0013] According to an embodiment of the present application, a polynucleotide encoding the polypeptide described in the present application is also provided.

[0014] According to an embodiment of the present application, a recombinant vector comprising the polynucleotide described in the present application is also provided.

[0015] According to an embodiment of the present application, a pharmaceutical composition comprising the polypeptide described in the present application, the polynucleotide described in the present application, or the recombinant vector described in the present application is also provided.

[0016] According to an embodiment of the present application, the polypeptide described in the present application, the polynucleotide described in the present application, or the recombinant vector described in the present application is also provided for use in the preparation of a medicament or a pharmaceutical composition for preventing, alleviating or treating a disease or a symptom.

[0017] According to an embodiment of the present application, the polypeptide described in the present application, the polynucleotide described in the present application, or the recombinant vector described in the present application is also provided for use in the preparation of a medicament or a pharmaceutical composition for regulating cholesterol metabolism or ameliorating cholesterol metabolic abnormality.

[0018] According to an embodiment of the present application, a method for preventing, alleviating or treating a disease or a symptom is also provided, the method comprising administering the polypeptide described in the present application, the polynucleotide described in the present application, or the recombinant vector described in the present application to a subject in need.

[0019] According to an embodiment of the present application, a method for regulating cholesterol metabolism or ameliorating cholesterol metabolic abnormality is also provided, the method comprising administering the polypeptide described in the present application, the polynucleotide described in the present application, or the recombinant vector described in the present application to a subject in need.

[0020] It is to be understood that the details set forth herein do not limit the scope of the embodiments of the application. Other embodiments of the application will be readily apparent to those skilled in the art from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0021] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in

[0022] Figure 1 is a map of plvx-mCMV-zsGreen-Puro plasmid used in Example 1.

[0023] Figure 2 is the overexpression of MP19 detected by qPCR and Western blot in Example 1.

[0024] Figure 3 is the detection of LDL-C and HDL-C in overexpression micropeptide MP19 cells and its control cells by low density lipoprotein cholesterol (LDL-C) colorimetric test kit and high density lipoprotein cholesterol (HDL-C) content detection kit in Example 2.

[0025] Figure 4 is the detection of the membrane permeability of exogenous micropeptide MP19 by Dil staining and Hoechst 33342 staining in Example 3.

[0026] Figure 5 is the intracellular lipid droplet content of foam RAW264.7 cells treated with micropeptide MP19 detected by oil red staining in Example 4.

[0027] Figure 6 is the intracellular lipid droplet content of AML12 cells treated with micropeptide MP19 detected by oil red staining in Example 5.

[0028] Figure 7 is the intracellular lipid droplet content of AML12 cells treated with micropeptide MP19 detected by BODIPY 493 / 503 and Hoechst 33342 staining in Example 5.

[0029] Figure 8 is the intracellular ROS content level of AML12 cells treated with micropeptide MP19 analyzed by DCFH-DA staining in Example 6.

[0030] Figure 9 is the detection of the cell migration ability of HUVEC cells treated with micropeptide MP19 in Example 7.

[0031] Figure 10 is the detection result of intracellular LDL-C and HDL-C content after exogenous administration of micropeptide MP19 or its mutants MP19-3A, MP19-8A, MP19-18A, MP19-19A in the LDL-C colorimetric test kit and HDL-C content detection kit in Example 8.

[0032] Figure 11 is the detection result of intracellular LDL-C and HDL-C content after exogenous administration of truncated mutants of micropeptide MP19 (MP19-A, MP19-B, MP19-C, MP19-D, MP19-E, MP19-F) in the LDL-C colorimetric test kit and HDL-C content detection kit in Example 9.

[0033] Figure 12 is the detection result of body weight change of mice within 2 weeks of micropeptide MP19 treatment in Example 10.

[0034] Figure 13 is the detection result of body weight change of mice within 8 weeks of micropeptide MP19 treatment in Example 11.

[0035] Figure 14 is the aortic plaque accumulation of mice after 8 weeks of exogenous micropeptide MP19 treatment in Example 12.

[0036] Figure 15 is the detection result of LDL-C, HDL-C, total cholesterol (TC) and triglyceride (TG) content in serum and liver of mice after 8 weeks of micropeptide MP19 treatment in Example 13.

[0037] Figure 16 is the detection result of LDL-C, HDL-C, TC and TG content in serum and liver of mice after 8 weeks of micropeptide MP19 treatment in Example 14.

[0038] Figure 17 is the pathological detection result of heart, liver, spleen, lung, kidney, brain and other tissues of mice after 8 weeks of micropeptide MP19 treatment in Example 14. DETAILED DESCRIPTION

[0039] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratios, amounts, time, temperature, thickness, and so forth, used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. At the very least, each numerical parameter should at least be construed in light of the number of significant digits and ordinary rounding approaches at the time of the disclosure or the skilled person’s understanding.

[0040] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical range were all expressly written herein.

[0041] The terms or descriptions used herein, unless otherwise stated or contradicted by context, are to be understood independently of context and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0042] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meaning of similar expressions can be extended in this way.

[0043] As used herein, "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. Thus, polypeptides, oligopeptides, microproteins, proteins, antibodies and enzymes are included within the definition of polypeptide.

[0044] As used herein, "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0045] It should be noted that, in the context of the present application, upstream refers to the 5' end of a nucleic acid or the N-terminus of a polypeptide, and downstream refers to the 3' end of a nucleic acid or the C-terminus of a polypeptide, from upstream to downstream is from 5' end to 3' end or from N-terminus to C-terminus.

[0046] As used herein, "vector" refers to a DNA molecule capable of self-replication and commonly in the form of a circular double-stranded DNA molecule that transfers foreign DNA into a host organism. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. Among them, plasmid is the most common vector form, which refers to a circular double-stranded DNA capable of accepting foreign nucleic acid fragments and capable of replication in prokaryotic or eukaryotic cells.

[0047] As used herein, "expression vector" and "recombinant vector" are used interchangeably and refer to a vector containing a foreign gene, and also includes regulatory elements for expression in a designated host organism. Introduction of the expression vector into an appropriate host organism enables expression of the inserted gene of interest (e.g., a nucleotide sequence encoding a micropeptide as provided herein).

[0048] As used herein, "foreign" or "heterologous" are used interchangeably and refer to a source that is not indigenous (original) to the organism, e.g., derived from another species of organism. As used herein, "heterologous gene" or "foreign gene" refers to a gene that does not naturally occur in the host organism, which is introduced into the host organism by gene transfer.

[0049] As used herein, "alleviate" and "treat" and synonyms thereof refer to the improvement of a disease, disorder, and / or condition. "Alleviate" and "treat" can be an improvement in at least one measurable physical parameter including, but not necessarily limited to, a parameter that can be discerned by the patient. "Alleviate" and "treat" can also mean to suppress the development of a disease, disorder, and / or condition, either physically (e.g., stabilize a discernible symptom), physiologically (e.g., stabilize a physical parameter), or both. "Alleviate" and "treat" can also mean to slow down or reverse the progression of a disease, disorder, and / or condition.

[0050] As used herein, "prevent" and synonyms thereof refer to delaying the onset or reducing the risk of acquiring a particular disease, disorder, and / or condition or symptoms associated with such diseases, disorders, and / or conditions.

[0051] In order to make the above objects, features and advantages of the present application more clear, a detailed description of the specific embodiments of the present application is provided below.

[0052] Micropeptide MP19 and mutants thereof

[0053] According to an embodiment of the present application, there is provided a polypeptide having an amino acid sequence of any one of (i) to (iv) below: (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) at least one of an amino acid sequence obtained by deletion, substitution or insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (iii) at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1; and (iv) at least one of a sequence containing the amino acid sequence PVS.

[0054] In some embodiments, the site of deletion includes at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, and / or 18th amino acid of the amino acid sequence set forth in SEQ ID NO: 1. For example, the site of deletion can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 of the amino acids. In some embodiments of the present application, polypeptides obtained by deleting any 3 of the 1st-18th amino acids of the amino acid sequence set forth in SEQ ID NO: 1 have the activity of modulating cholesterol metabolism. Therefore, one skilled in the art can expect that polypeptides obtained by mutating multiple sites (e.g., multiple sites in the 1st-18th amino acids) of the amino acid sequence set forth in SEQ ID NO: 1 also have the activity of modulating cholesterol metabolism.

[0055] In some preferred embodiments, the site of deletion includes any 3 of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, or 18th amino acid of the amino acid sequence set forth in SEQ ID NO: 1.

[0056] In some preferred embodiments, the site of deletion includes any 3 adjacent of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, or 18th amino acid of the amino acid sequence set forth in SEQ ID NO: 1.

[0057] In some more preferred embodiments, the site of the deletion is selected from at least one of the following groups: (1) amino acids 1, 2, and 3 of the amino acid sequence set forth in SEQ ID NO: 1; (2) amino acids 4, 5, and 6 of the amino acid sequence set forth in SEQ ID NO: 1; (3) amino acids 7, 8, and 9 of the amino acid sequence set forth in SEQ ID NO: 1; (4) amino acids 10, 11, and 12 of the amino acid sequence set forth in SEQ ID NO: 1; (5) amino acids 13, 14, and 15 of the amino acid sequence set forth in SEQ ID NO: 1; and / or (6) amino acids 16, 17, and 18 of the amino acid sequence set forth in SEQ ID NO: 1.

[0058] In some more preferred embodiments, the site of the substitution is selected from at least one, at least two, or at least three of the following: (1) amino acid 3 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from G to A; (2) amino acid 8 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; (3) amino acid 18 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; and / or (4) amino acid 19 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from L to A.

[0059] In some more preferred embodiments, the site of the substitution is selected from at least one, at least two, or at least three of the following: (1) amino acid 3 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from G to A; (2) amino acid 8 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; (3) amino acid 18 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; and / or (4) amino acid 19 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from L to A.

[0060] In some more preferred embodiments, the site of the substitution is selected from at least one, at least two, or at least three of the following: (1) amino acid 3 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from G to A; (2) amino acid 8 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; (3) amino acid 18 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; and / or (4) amino acid 19 of the amino acid sequence set forth in SEQ ID NO: 1 is mutated from L to A.

[0061] In some more preferred embodiments, the polypeptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 1-11. In some more preferred embodiments, the polypeptide comprises the amino acid sequence PVS.

[0062] Polynucleotide, recombinant vector

[0063] According to an embodiment of the present application, there is further provided a polynucleotide encoding the polypeptide of the present application.

[0064] According to an embodiment of the present application, there is further provided a recombinant vector comprising the polynucleotide of the present application.

[0065] In some embodiments, the polynucleotide or the recombinant vector further comprises a promoter. The promoter can be any promoter sequence suitable, i.e. a nucleic acid sequence that is recognized by the host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the polypeptide. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be derived from genes operating under the control of either a constitutive or inducible promoter.

[0066] In some embodiments, the polynucleotide or the recombinant vector further comprises a transcription termination sequence. The transcription termination sequence, i.e. a sequence recognized by the host cell to terminate transcription. In some embodiments, the transcription termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that is functional in the host cell of choice can be used in the present application.

[0067] Pharmaceutical composition

[0068] According to an embodiment of the present application, there is further provided a pharmaceutical composition comprising the polypeptide of the present application, the polynucleotide of the present application, or the recombinant vector of the present application.

[0069] In some embodiments, the pharmaceutical composition further comprises a glucagon-like peptide-1 receptor agonist. In some preferred embodiments, the glucagon-like peptide-1 receptor agonist is Semaglutide.

[0070] In some embodiments, the pharmaceutical composition further comprises an HMG-CoA reductase inhibitor. In some preferred embodiments, the HMG-CoA reductase inhibitor is Atorvastatin.

[0071] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable carrier. The carrier can be any compatible, physiologically acceptable, non-toxic substance suitable for delivering the polypeptide, polynucleotide or recombinant vector provided herein into the body of a mammal, e.g. a human.

[0072] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the excipient comprises at least one of a solubilizing agent, a disintegrant, a wetting agent, a stabilizer, a thickening agent, a diluent, a buffer, a flavoring agent.

[0073] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the excipient comprises at least one of a solubilizing agent, a disintegrant, a wetting agent, a stabilizer, a thickening agent, a diluent, a buffer, a flavoring agent.

[0074] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the excipient comprises at least one of a solubilizing agent, a disintegrant, a wetting agent, a stabilizer, a thickening agent, a diluent, a buffer, a flavoring agent.

[0075] In some non-limiting embodiments, the carrier and / or excipient for the pharmaceutical composition used in the present application can comprise, for example, a liquid, gel or solid carrier, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffering agent, an antioxidant, a suspending agent, a dispersing agent, a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, other components known in the art or various combinations thereof.

[0076] Uses and methods

[0077] According to an embodiment of the present application, there is also provided a use of the polypeptide described herein, the polynucleotide described herein, the recombinant vector described herein, or the pharmaceutical composition described herein in the manufacture of a medicament or a pharmaceutical composition for preventing, alleviating or treating a disease or a symptom.

[0078] According to an embodiment of the present application, there is also provided a method of preventing, alleviating or treating a disease or a symptom, the method comprising administering to a subject in need thereof the polypeptide described herein, the polynucleotide described herein, the recombinant vector described herein, or the pharmaceutical composition described herein.

[0079] In some embodiments, the disease or condition comprises a disease associated with abnormal cholesterol metabolism. A variety of diseases or conditions are currently known to be associated with abnormal cholesterol metabolism, such as obesity, atherosclerosis, hyperlipidemia, age-related macular degeneration, non-alcoholic hepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, tumor angiogenesis, coronary heart disease, ischemic stroke, embolism caused by blood lipid plaque shedding, etc. (Cholesterol Regulation in Age-Related Macular Degeneration: A Framework for Mathematical Modelling of Drusen Biogenesis, J Bull Math Biol. 2020, 82(10): 135. Doi: 10.1007 / s11538-020-00812-0; Metabolic-associated fatty liver disease and lipoprotein metabolism. J Mol Metab. 2021, 50: 101238. Doi: 10.1016 / j.molmet.2021.101238; Cholesterol Metabolism: A Double-Edged Sword in Hepatocellular Carcinoma, J Front Cell Dev Biol. 2021, 9: 762828. Doi: 10.3389 / fcell.2021.762828; Soybean oil lowers circulating cholesterol levels and coronary heart disease risk, and has no effect on markers of inflammation and oxidation, J Nutrition. 2021, 89: 111343 Doi: 10.1016 / j.nut.2021.111343; Low-density lipoprotein cholesterol lowering for the prevention of cardiovascular outcomes in patients with ischemic stroke, J Int J Stroke. 2019, 14(5): 476-482. Doi: 10.1177 / 1747493019851283), the disclosures of which are incorporated herein by reference in their entirety.In some embodiments of the present application, the polypeptides, polynucleotides, and recombinant vectors described in the present application all show good activity in regulating cholesterol metabolism, and thus a person skilled in the art can reasonably predict, based on the disclosure of the present application, that the polypeptides, polynucleotides, and recombinant vectors described in the present application have therapeutic effects on various diseases related to cholesterol metabolism. In some preferred embodiments, the diseases or symptoms include obesity, atherosclerosis, age-related macular degeneration, hyperlipidemia, non-alcoholic hepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, tumor angiogenesis, coronary heart disease, ischemic stroke, and embolism caused by blood lipid plaque shedding.

[0080] In some embodiments, the prevention, alleviation, or treatment of diseases or symptoms includes at least one of the following groups: (1) reducing the content of triglycerides and total cholesterol in cells, serum, or tissues; (2) reducing the content of low density lipoprotein cholesterol (LDL-C) in cells, serum, or tissues; (3) increasing the content of high density lipoprotein cholesterol (HDL-C) in cells, serum, or tissues; (4) reducing lipid accumulation in cells; (5) reducing the level of oxidative damage in cells; (6) inhibiting the migration of vascular endothelial cells; (7) reducing body weight; and (8) reducing the accumulation of arterial plaques.

[0081] According to an embodiment of the present application, a method for regulating cholesterol metabolism or improving cholesterol metabolism disorders is also provided, which comprises administering the polypeptides described in the present application, the polynucleotides described in the present application, the recombinant vectors described in the present application, or the pharmaceutical compositions described in the present application to a subject in need thereof.

[0082] According to an embodiment of the present application, the use of the polypeptides described in the present application, the polynucleotides described in the present application, the recombinant vectors described in the present application, or the pharmaceutical compositions described in the present application in the preparation of a medicament or a pharmaceutical composition for regulating cholesterol metabolism or improving cholesterol metabolism disorders is also provided.

[0083] In some embodiments, wherein the modulating or ameliorating cholesterol metabolism comprises at least one of the following group: (1) reducing the level of triglyceride and total cholesterol in cells, serum or tissues; (2) reducing the level of low density lipoprotein cholesterol (LDL-C) in cells, serum or tissues; (3) increasing the level of high density lipoprotein cholesterol (HDL-C) in cells, serum or tissues; (4) reducing lipid accumulation in cells; (5) reducing the level of oxidative damage in cells; (6) inhibiting vascular endothelial cell migration; (7) reducing body weight; (8) reducing arterial plaque accumulation.

[0084] In the methods described herein, the dosage of the polypeptide, polynucleotide, recombinant vector or composition provided herein can depend on several factors, including the severity and responsiveness of the symptoms, the route of administration, the time of administration (days to months to years), and the time period for improvement of symptoms. The dosage regimen can be adjusted to provide a therapeutic response according to the specific situation of the patient by a person skilled in the art. For example, a single dose can be administered, several separate doses can be administered over a predetermined period of time, or the dose can be reduced or increased as indicated by the therapeutic situation. The size of the dose is determined by the specific therapeutic effect to be achieved. The dose value can also vary depending on the type and severity of the condition to be alleviated. For any particular subject, the specific dose regimen can be adjusted over time according to the individual need and the professional judgment of the treating clinician.

[0085] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by the combination are all considered part of the disclosure of the present application.

[0086] Exemplary embodiments of the present application will be described below with reference to the accompanying drawings, which include various details of the embodiments of the present application to assist in understanding. It should be understood that these are considered only exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined only by the claims. Therefore, those of ordinary skill in the art should recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Also, descriptions of well-known functions and structures are omitted in the following description for clarity and conciseness.

[0087] Embodiments

[0088] In the present embodiment, unless a specific technique or condition is specified, the technique or condition described in the literature in the art or according to the product manual is used. Unless the manufacturer is specified, all reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0089] Example 1: Construction of MP19 overexpression cell line

[0090] Firstly, the plvx-mCMV-zsGreen-Puro plasmid (constructed by General Biotech) was constructed, and the element map of the plasmid is shown in Figure 1, wherein the nucleotide sequence of the element "MP19 gene sequence" is shown in SEQ ID NO: 12. HEK-293T cells (Cell Resource Center of Shanghai Institutes for Biological Sciences, CAS) were selected for lentivirus packaging, and the target cells AML12 (Shanghai Chenying Biotechnology Co., Ltd.) were infected to construct the MP19 overexpression cell line. The experimental process is briefly described as follows:

[0091] HEK-293T was passaged into a 100mm culture dish, and the density reached 60-70% for lentivirus packaging. Two 1.5mL EP tubes were prepared, 500μL DMEM basic medium, 10μg expression vector, 7.5μg psPAX2 (purchased from General Biotech (Anhui) Co., Ltd.) and 2.5μg pMD2.G (purchased from General Biotech (Anhui) Co., Ltd.) were added into tube No. 1, 500μL DMEM basic medium and 72μL EZ Trans transfection reagent (AC04L091, Shanghai Liji Biological Technology Co., Ltd.) were added into tube No. 2, the contents of tube No. 2 were added into tube No. 1, mixed well by blowing and sucking, incubated for 10min, added the transfection complex along the wall of the tube, gently shaken, replaced with fresh culture medium after 12h, collected the culture supernatant at 48h and 72h after transfection, centrifuged the virus supernatant at 1000rpm for 5min to remove cell debris, the supernatant was filtered through a 0.45μm filter to an ultracentrifuge tube, added virus concentrate (AC04L441, Shanghai Liji Biological Technology Co., Ltd.) according to virus liquid: virus concentrate = 4:1, incubated at 4°C overnight, centrifuged the virus liquid at 4000g for 15min at 4°C, discarded the supernatant to obtain the virus.

[0092] Resuspend with 1 mL medium, add 3 μL polybrene (TA003, General Biosystems (Anhui) Co., Ltd.), and infect AML12 cells after mixing. Replace the fresh DMEM / F12 complete medium after 9 hours of cell infection. Start drug screening of the resistance gene after 72 hours of infection to construct a stable cell line overexpressing the micropeptide MP19. Take photos under a fluorescence microscope and detect the positive rate of overexpressing cells by flow cytometry. Verify the overexpression efficiency by qPCR and Western Blot. The antibodies used in Western Blot are Anti-GAPDH Rabbit pAb (AB-P-R001, Hangzhou Xianzhi Biological Technology Co., Ltd.) and Anti-MP19 Mouse mAb (Aibima Medical Technology (Shanghai) Co., Ltd.). The primer information used in qPCR is shown in Table 1.

[0093] The results are shown in FIG. 2. The qPCR and Western blot results show that the expression amount of the micropeptide MP19 in the overexpression cell plvx-MP19 (the coordinate axis and lane in FIG. 2 are marked as “MP19”) is significantly higher than that in the control cell (the coordinate axis and lane in FIG. 2 are marked as “plvx”), indicating that the overexpression MP19 cell line is successfully constructed.

[0094] Table 1 Primer sequences used in qPCR

[0095] Example 2: Overexpression of MP19 increases the content of HDL-C and reduces the content of LDL-C in cells

[0096] The stable cell line overexpressing the micropeptide MP19 and its control cell line obtained in Example 2 are used to determine the content of LDL-C in the cells according to the Elabscience Low Density Lipoprotein Cholesterol (LDL-C) Colorimetric Assay Kit (Double Reagent Direct Method), and the content of HDL-C in the cells is determined according to the Solabio High Density Lipoprotein Cholesterol (HDL-C) Content Assay Kit.

[0097] The results are shown in FIG. 3. The content of LDL-C in the cell overexpressing the micropeptide MP19 (the coordinate axis in FIG. 3 is marked as “MP19”) is significantly lower than that in the control cell (the coordinate axis in FIG. 3 is marked as “plvx”), and the content of HDL-C in the cell overexpressing the micropeptide MP19 is higher than that in the control cell. The results show that the micropeptide MP19 can reduce LDL-C, and the increase of LDL-C level is an inducing factor of diseases related to abnormal cholesterol metabolism such as atherosclerosis. Therefore, the micropeptide may have a potential function of preventing or treating diseases related to abnormal cholesterol metabolism.

[0098] In summary, the results in Example 2-3 show that overexpression of MP19 micropeptide in cells can have a significant impact on cholesterol metabolism, including increasing the content of HDL-C in cells and reducing the content of LDL-C in cells, and thus micropeptide MP19 can have a good therapeutic effect on various cholesterol metabolism-related diseases or symptoms, including but not limited to atherosclerosis, hyperlipidemia, non-alcoholic hepatitis, coronary heart disease, ischemic stroke, and embolism caused by blood lipid plaque shedding.

[0099] Example 3: Transmembrane test of exogenous synthetic micropeptide MP19

[0100] 5000 AML12 cells in the growth phase were plated in a 96-well plate, given DMEM / F12 complete medium, and adherently cultured for 24 h, then replaced with DMEM / F12 complete medium containing 1 μM FITC-MP19 (synthesized by Changzhou Kanglong Biotechnology Co., Ltd., using solid-phase synthesis method, directly connecting FITC at the end of the polypeptide, FITC purchased from Nanjing Mingcheng Aikang Pharmaceutical Biotechnology Co., Ltd., CAS No. 3326-32-7), and cultured for 24 h.

[0101] Then, whether the exogenous synthetic micropeptide MP19 can pass through the cell membrane was observed by Dil staining and Hoechst 33342 staining, as follows: washed twice with PBS, fixed with 4% paraformaldehyde for 30 min; washed twice with PBS, stained with Dil staining solution for 20 min; washed twice with PBS, stained with Hoechst 33342 staining solution for 5 min; washed twice with PBS, and observed under a fluorescence microscope.

[0102] The results are shown in FIG. 4. In the immunofluorescence imaging results, the fluorescence of the exogenous synthetic micropeptide MP19 was located in the cells. The results show that the exogenous synthetic micropeptide MP19 has good transmembrane properties and can enter the cells to play a role, and can be used for exogenous administration.

[0103] Example 4: Effect of exogenous MP19 on lipid droplets in the cytoplasm of macrophages

[0104] Macrophages that engulf large amounts of fat produce foam cells, a significant cause of atherosclerosis and a potential contributor to heart disease and cerebral infarction. The cytoplasm of foam macrophages contains numerous lipid droplets, characteristic of atherosclerotic plaques. RAW264.7 cells (Shanghai Chenying Biotechnology Co., Ltd.) were induced to exhibit foam cell formation using 50 μg / mL ox-LDL (Yisheng Biotechnology (Shanghai) Co., Ltd., 20605ES05). After successful cell model establishment, different doses of the micropeptide MP19 (1 nM, 10 nM) were administered, with Atorvast atin (10 μM) used as a positive control. After 24 hours of drug treatment, changes in lipid droplet content in the cytoplasm were observed using Oil Red staining.

[0105] The results are shown in Figure 5. Exogenous administration of micropeptide MP19 can reduce the content of intracellular lipid droplets in foamed RAW264.7 cells, with a better effect than the positive control Atorvastatin. At the same time, MP19 combined with Atorvastatin can synergistically reduce the content of lipid droplets, indicating that micropeptide MP19 or its combination with Atorvastatin has potential therapeutic effects on diseases or symptoms such as atherosclerosis, coronary heart disease, ischemic stroke, and embolism caused by the detachment of lipid plaques.

[0106] Example 5: Effect of exogenous MP19 administration on intracytoplasmic lipid droplets in hepatocytes

[0107] AML12 cell stimulation with oleic acid and palmitic acid is a classic model for fatty liver, non-alcoholic hepatitis, and liver fibrosis. AML12 cells in the growth phase are used at a rate of 1x10⁻⁶ cells / cells. 5 Cells were seeded in 6-well plates and cultured in DMEM / F12 complete medium for 24 hours. Cells were then divided into several groups: a control group (no treatment), a negative control group (3% FBS, 225 μM oleic acid, 122.5 μM palmitic acid), a positive control group (3% FBS, 225 μM oleic acid, 122.5 μM palmitic acid, 10 μM MAtorvastatin), and experimental groups with different doses of MP19 peptide (3% FBS, 225 μM oleic acid, 122.5 μM palmitic acid, 1 nM / 10 nM MP19), each group treated for 24 hours.

[0108] Subsequently, changes in intracellular lipid droplet content were observed by Oil Red staining, or BODIPY 493 / 503 and Hoechst 33342 staining, respectively.

[0109] Oil red staining method is: PBS washing 2 times, 4% paraformaldehyde fixation for 30 min; PBS washing 2 times, 60% isopropanol rinse for 5 s, 60% oil red staining solution staining for 30 min; 60% isopropanol rinse for 5 s, PBS washing 2 times, observation under microscope.

[0110] BODIPY 493 / 503 and Hoechst33342 staining method is: PBS washing 2 times, 4% paraformaldehyde fixation for 30 min; PBS washing 2 times, BODIPY 493 / 503 staining solution staining for 20 min, PBS washing 2 times; Hoechst3342 staining solution staining for 5 min, PBS washing 2 times, observation under fluorescence microscope.

[0111] The results are shown in Figures 6 and 7. After exogenous administration of micropeptide MP19, the content of lipid droplets in the cytoplasm of AML12 cells decreased, indicating that it has good therapeutic effect on fatty liver, non-alcoholic hepatitis, liver fibrosis, cirrhosis and other diseases, and indicating that it has drug potential in the future.

[0112] Example 6: Effect of exogenous MP19 on oxidative damage of liver cells

[0113] The AML12 cells in the growth phase were plated at 5000 / well in a 6-well plate, and were given DMEM / F12 complete medium for adherent culture for 24 h. Then the cells were divided into negative group, positive drug control group (10 uM Atorvastatin), and micropeptide MP19 experimental group (10 nM MP19). After 24 h of treatment, the level of reactive oxygen species in the cells was analyzed by DCFH-DA staining.

[0114] DCFH-DA staining method is: PBS washing 2 times, Rose UP group additionally adding 1:1000 Rose Up for 20 min, then PBS washing 2 times; adding 1:1000 DCFH-DA probe in each well, treating for 20 min, PBS washing twice, and observing under fluorescence microscope.

[0115] The results are shown in Figure 8. Exogenous administration of micropeptide MP19 can reduce the level of reactive oxygen species in AML12 cells, and the effect is better than that of the same dose of positive control Atorvastatin. The results again verify that micropeptide MP19 has therapeutic activity on diseases related to abnormal cholesterol metabolism, and further indicate that it has good therapeutic effect on fatty liver, non-alcoholic hepatitis, liver fibrosis, cirrhosis and other diseases, indicating that it has drug potential in the future.

[0116] Example 7: Effect of exogenous MP19 on migration of human umbilical vein endothelial cells

[0117] Different concentrations of micropeptide MP19 (0.1 nM, 1 nM, 10 nM) were used to treat HUVEC cells for 24 h, and Avastin (8 μM, 24 h) was selected as a positive control.

[0118] The results are shown in Figure 9. Exogenous administration of micropeptide MP19 can significantly inhibit the migration of HUVEC cells, and the effect is better than that of the same dose of positive control Avastin. The results confirm that micropeptide MP19 has potential therapeutic effect on age-related macular degeneration; at the same time, it also suggests that micropeptide MP19 has an inhibitory effect on tumor-related angiogenesis, thereby also having potential therapeutic effect on various tumors.

[0119] Example 8: Activity detection of MP19 substitution mutant

[0120] The substitution mutation of micropeptide MP19 was performed by exogenous chemical synthesis method (synthesized by Changzhou Kanglong Biological Technology Co., Ltd.), and the 3rd, 8th, 18th and 19th amino acids in the amino acid sequence of MP19 were mutated to alanine to obtain different mutants, and their amino acid sequences are shown in Table 2.

[0121] The content of LDL-C in the cells was determined according to the Eliret low-density lipoprotein cholesterol (LDL-C) colorimetric test kit (double reagent direct method), and the content of HDL-C in the cells was determined according to the Solabio high-density lipoprotein cholesterol (HDL-C) content detection kit instructions. The positive drug control group (10 μM Atorvastatin) is represented as "AT" in Figure 10.

[0122] The results are shown in Figure 10. After the AML12 cells (Shanghai Chenying Biological Technology Co., Ltd.) were treated with different micropeptide MP19 mutants for 24 h, the content of LDL-C in the cells was lower than that in the control cells at a dose of 1 nM, indicating that these mutants (MP19-3A, MP19-8A, MP19-18A, MP19-19A) all have potential activity in treating cholesterol metabolism related diseases.

[0123] At the same time, the results also found that the mutants obtained by mutating the 3rd, 8th and 18th positions can increase the content of HDL-C, while the mutant MP19-19A obtained by mutating the 19th amino acid cannot increase the content of HDL-C, indicating that mutant MP19-19A has certain cholesterol metabolism regulation activity, but its activity is weaker than that of other mutants.

[0124] The above results show that the substitution mutation of the 3rd, 8th, 18th and 19th positions of MP19 has little effect on its activity, so the micropeptide MP19 mutants obtained by substituting different amino acids at these four positions will still have activity.

[0125] Table 2 Sequences of micropeptides and their mutants

[0126] Example 9: Activity detection of MP19 truncated mutants

[0127] The micropeptide MP19 was truncated by exogenous chemical synthesis method (synthesized by Changzhou Kanglong Biotechnology Co., Ltd.), and different sites (including the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th) in the amino acid sequence of MP19 were truncated to obtain different mutants, and their amino acid sequences are shown in Table 3 below.

[0128] The content of LDL-C in the cells was determined according to the Eliret Low Density Lipoprotein Cholesterol (LDL-C) Colorimetric Test Kit (Double Reagent Direct Method), and the content of HDL-C in the cells was determined according to the Solabio High Density Lipoprotein Cholesterol (HDL-C) Content Test Kit instruction. The positive drug control group (10 μM Atorvastatin) is represented as "AT" in Figure 11.

[0129] The results are shown in Figure 11. After the AML12 cells (Shanghai Chenying Biotechnology Co., Ltd.) were treated with different micropeptide MP19 mutants for 24 h, the content of LDL-C in the cells was lower than that in the control cells at a dose of 1 nM, indicating that these mutants (MP19-A, MP19-B, MP19-C, MP19-D, MP19-E, MP19-F) all had potential activity in treating cholesterol metabolism related diseases.

[0130] At the same time, it was also found that other mutants obtained by truncating the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 16th, 17th, 18th amino acids could increase the content of HDL-C, but the mutant MP19-E obtained by truncating the 13th-15th amino acids failed to increase the content of HDL-C, indicating that the mutant MP19-E had certain cholesterol metabolism regulation activity, but its activity was weaker than that of other mutants. In addition, this result also indicated that the 13th-15th amino acids were the conserved sequence of MP19 micropeptide to function.

[0131] Table 3 Sequences of micropeptides and their mutants

[0132] Example 10: Weight loss effect of exogenous MP19 on obese model mice

[0133] This example uses 6-week-old C57BL / 6J mice (purchased from Jiangsu Ailinfeng Biotechnology Co., Ltd.), all animals are adaptively fed in SPF level animal laboratory for 1 week, the environmental conditions include indoor temperature control at 20-24℃, daily temperature difference control within 4℃, relative air humidity maintained at 40-70%, 12 hours of light per day, 12 hours of darkness. After adaptive feeding, 60% HFD high-fat diet is fed for 12 weeks, and more than 42g body weight is used as the standard for successful modeling of obesity, and 48 mice with balanced body weight (±1.5g) are selected for grouping into drug experiments.

[0134] In addition to the blank group mice using wild type mice, the mice after successful modeling are randomly divided into seven groups (i.e. groups numbered 1-7 in Table 4 below):

[0135] High, medium and low dose MP19 groups, mice receive subcutaneous injection of micropeptide MP19 (dose see Table 4 below), frequency 7 times per week, experimental period is 8 weeks;

[0136] Semaglutide group, mice receive subcutaneous administration of semaglutide (0.082mg / kg), frequency once a week, experimental period is 8 weeks;

[0137] Semaglutide+MP19 combined administration group, mice receive subcutaneous administration of semaglutide (0.082mg / kg) and low dose MP19 (2mg / kg), frequency of semaglutide once a week and low dose MP19 once a day, experimental period is 8 weeks;

[0138] Tirzepatide group, mice receive subcutaneous administration of tirzepatide (3nmol / kg), frequency once a week, experimental period is 8 weeks;

[0139] Model control group and blank control group, mice are given normal saline and receive regular feeding.

[0140] During the experiment, all groups of mice have free access to water and food, and the mice are weighed three times a week to record body weight changes.

[0141] Table 4 Administration scheme

[0142] The weight changes of the mice in each group were statistically analyzed after the experiment was carried out for two weeks, and the results are shown in Figure 12. The results show that, compared with the model control group, the body weight of the mice in the MP19 group, the positive drug Semaglutide group, and the positive drug Tirzepatide group decreased significantly and there was a significant difference (p<0.05), and the weight loss effect of MP19 had a dose-dependent effect, which indicated that the micropeptide MP19, the positive drug Semaglutide, and the positive drug Tirzepatide had obvious inhibitory effect on the body weight of the mice; the results also showed that the weight loss effect of the Semaglutide+MP19 combination administration group was better than that of the MP19 low-dose group and the Semaglutide group, indicating that the micropeptide MP19 and Semaglutide had a synergistic weight loss effect in terms of weight loss effect; in addition, the body weight of the mice in the second week showed that the weight loss effect of the medium and high dose MP19 group was better than that of the positive control drugs Semaglutide and Tirzepatide, indicating that the micropeptide MP19 may have a better effect in the treatment of weight control and obesity.

[0143] Example 11: Weight loss effect of exogenous MP19 on high-fat diet mice

[0144] In this example, 5-week-old C57BL6 / J mice (purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.) were used, and all animals were adaptively fed in a SPF level animal laboratory for 1 week. The environmental conditions included indoor temperature control at 20-24°C, daily temperature difference control within 4°C, relative air humidity maintained at 40-70%, 12 hours of light per day, and 12 hours of darkness. After adaptive feeding, the mice were fed with high-fat diet to simulate the process of normal body weight gain, and then the mice were grouped and administered after the body weight of the mice increased.

[0145] After the body weight of the mice increased, the mice were randomly divided into three groups: the Control group, in which the mice were fed with regular diet; the Atorvastatin group, in which the mice were administered with atorvastatin (10 mg / kg / d) by gavage, and the experimental period was 8 weeks; and the MP19 group, in which the mice were administered with micropeptide MP19 (1 mg / kg / d) by intraperitoneal injection, and the experimental period was 8 weeks. During the experiment, the mice had free access to water and food, and the mice were weighed once a week to record the body weight changes.

[0146] After 8 weeks of experiment, the weight changes of the mice in each group were statistically analyzed, and the results are shown in Figure 13. The results show that, compared with the Control group and the Atorvastatin group, the body weight of the mice in the MP19 group decreased significantly and there was a significant difference (p<0.05). This indicates that the micropeptide MP19 has obvious inhibitory effect on the body weight of the mice, and the effect is better than that of the positive control drug Atorvastatin, suggesting that the micropeptide MP19 may have a better effect in the treatment of weight control and obesity.

[0147] Example 12: The therapeutic effect of exogenous MP19 on atherosclerotic model mice

[0148] Purchased 5-week-old ApoE from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. - / - Mice were acclimatized for one week and fed a high-fat diet for 12 weeks to establish a mouse model of atherosclerosis. Simultaneously, 5-week-old C57BL6 / J mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., acclimatized for one week, and fed a high-fat diet for 20 weeks to establish a mouse model of atherosclerosis.

[0149] After meeting the experimental requirements, the mice were randomly divided into a solvent control group, a positive drug control group (Atorvastatin, Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number HY-B0589, dose 10 mg / kg / day), and different doses of MP19 treatment groups (dose of 0.5 mg / kg / day, 1 mg / kg / day, 2 mg / kg / day, 4 mg / kg / day, 8 mg / kg / day, and 16 mg / kg / day, respectively), and administered the drugs continuously for 8 weeks.

[0150] After the drug administration cycle was completed, the aortic plaque accumulation in mice was observed by Oil Red staining of cells: mice were sacrificed and the aorta was freed and fixed in 4% paraformaldehyde for 24 h; the tissue was removed, rinsed twice with PBS, immersed in Oil Red staining solution, and stained at 37℃ for 60 min; the tissue was soaked in 75% ethanol for 10 min to induce differentiation, rinsed twice with distilled water, and photographed.

[0151] The results are shown in Figure 14. After 8 weeks of treatment with different doses of micropeptide MP19 via intraperitoneal injection, the plaque accumulation in the arteries of mice was significantly reduced. These results indicate that micropeptide MP19 has a certain therapeutic effect on cholesterol metabolism-related diseases or symptoms such as atherosclerosis and embolism caused by the detachment of lipid plaques.

[0152] Example 13: The therapeutic effect of exogenous MP19 on atherosclerotic model mice

[0153] Purchased 5-week-old ApoE from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. - / -The mice were fed for 1 week, and the atherosclerosis model mice were modeled by feeding with high-fat feed for 12 weeks. The serum LDL-C content of the mice was determined by taking blood from the tail, and after meeting the experimental requirements, the mice were divided into a solvent control group (control), a positive drug control group (Atorvastatin, Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., Catalog No. HY-B0589, dose 10 mg / kg / day), and an MP19 treatment group (dose 1 mg / kg / day). The mice were continuously administered for 8 weeks, and after the administration period ended, the serum and liver LDL-C, HDL-C, TC, and TG contents of the mice were determined. The serum and tissue LDL-C content was determined according to the ELISA kit (double reagent direct method) for Low Density Lipoprotein Cholesterol (LDL-C) of Elabscience, the HDL-C content in the serum and liver tissue was determined according to the HDL-C content test kit of Solabio, the TC content in the serum and tissue was determined according to the TC colorimetric test kit (double reagent direct method) of Elabscience, and the TG content in the serum and tissue was determined according to the TG colorimetric test kit (double reagent direct method) of Elabscience.

[0154] The results are shown in Figure 15. After 8 weeks of intraperitoneal injection of the micropeptide MP19, the LDL-C and triglyceride contents in the serum of the mice were significantly reduced, and the HDL-C content in the liver was significantly increased. The results show that the micropeptide MP19 has activity in treating hyperlipidemia, atherosclerosis, fatty liver, liver fibrosis, liver cirrhosis, embolism caused by blood lipid plaque shedding, and other diseases or symptoms.

[0155] Example 14: Therapeutic effect of exogenous MP19 on atherosclerosis model mice

[0156] 5-week-old C57 mice were purchased from Jiangsu Jiju Pharmaceutical Biological Technology Co., Ltd., and were adaptively fed for 1 week. The mice were fed with high-fat feed for 20 weeks to establish an atherosclerosis model. The serum LDL-C content of the mice was determined by taking blood from the tail. After the serum LDL-C content met the experimental requirements, the mice were divided into a solvent control group (control), a positive drug control group (Atorvastatin, 10 mg / kg / day, Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., HY-B0589), and an MP19 treatment group (1 mg / kg / day). The mice were continuously administered for 8 weeks. After the administration period, the serum and liver LDL-C, HDL-C, TC, and TG contents of the mice were determined. The serum and tissue LDL-C content was determined according to the ELISA kit (double reagent direct method) for low-density lipoprotein cholesterol (LDL-C) of Elabscience. The serum and tissue HDL-C content was determined according to the HDL-C content test kit of Solerbio. The serum and tissue TC content was determined according to the TC colorimetric test kit (double reagent direct method) of Elabscience. The serum and tissue TG content was determined according to the TG colorimetric test kit (double reagent direct method) of Elabscience.

[0157] The results are shown in FIG. 16. The serum LDL-C content of the mice in the MP19 treatment group was significantly reduced, and the liver tissue triglyceride and total cholesterol contents were significantly reduced. The results show that the micropeptide MP19 has the potential to treat atherosclerosis and other cholesterol metabolism abnormality-related diseases.

[0158] At the same time, the main organ tissues of the mice administered for 8 weeks were subjected to HE staining and pathological analysis. The results are shown in FIG. 17. It was found that the micropeptide MP19 can alleviate the fatty degeneration in the liver tissue of the mice.

[0159] In summary, the results in Examples 12-14 show that the exogenous administration of the micropeptide MP19 can also have a significant impact on cholesterol metabolism, including increasing the HDL-C content and reducing the LDL-C and triglyceride contents. Therefore, the micropeptide MP19 has a good therapeutic effect on various cholesterol metabolism abnormality-related diseases or symptoms, including but not limited to atherosclerosis, hyperlipidemia, non-alcoholic hepatitis, fatty liver, liver fibrosis, liver cirrhosis, embolism caused by blood lipid plaque shedding, and the like.

[0160] At the same time, the results of Examples 12-14 also show that the micropeptide MP19 has a better therapeutic effect on cholesterol metabolism abnormality than the positive drug Atorvastatin. Atorvastatin is a first-line drug for treating atherosclerosis and hyperlipidemia that has been marketed in the clinic, further proving the clinical application value of the micropeptide MP19.

[0161] It is to be understood that the above description is intended to be illustrative and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reading the above description. The scope of the application should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with their full scope of equivalents. It is important to note that all variables and / or elements described herein can be combined in any combination desired to produce the compositions and methods within the scope of the application.

Claims

1. A polypeptide, characterized in that, The polypeptide has an amino acid sequence of any one of (i)-(iv) below: (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) at least one of an amino acid sequence obtained by deletion, substitution or insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (iii) at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1; (iv) at least one of a sequence containing the amino acid sequence PVS.

2. The polypeptide of claim 1, wherein the site of deletion comprises at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, and / or 18th amino acid in the amino acid sequence set forth in SEQ ID NO:

1.

3. The polypeptide of claim 2, wherein the site of deletion is selected from at least one of the following groups: (1) the 1st, 2nd and 3rd amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (2) the 4th, 5th and 6th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (3) the 7th, 8th and 9th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (4) the 10th, 11th and 12th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; (5) the 13th, 14th and 15th amino acids in the amino acid sequence set forth in SEQ ID NO: 1; and / or (6) the 16th, 17th and 18th amino acids in the amino acid sequence set forth in SEQ ID NO:

1.

4. The polypeptide of claim 1, wherein the site of substitution comprises at least one of the 3rd, 8th, 18th and / or 19th amino acid in the amino acid sequence set forth in SEQ ID NO:

1.

5. The polypeptide of claim 4, wherein the site of substitution is selected from at least one of the following groups: (1) the 3rd amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is mutated from G to A; (2) the 8th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; (3) the 18th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is mutated from S to A; and / or (4) the 19th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is mutated from L to A.

6. The polypeptide of any one of claims 1-5, having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-11.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotide encodes the polypeptide of any one of claims 1-6.

8. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 7.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, or the recombinant vector of claim 8.

10. The pharmaceutical composition of claim 9, wherein: The pharmaceutical composition further comprises a glucagon-like peptide-1 receptor agonist; preferably, the glucagon-like peptide-1 receptor agonist is Semaglutide; or The pharmaceutical composition further comprises an HMG-CoA reductase inhibitor; preferably, the HMG-CoA reductase inhibitor is Atorvastatin.

11. Use of the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 or 10 in the preparation of a medicament for preventing, alleviating, or treating a disease or a symptom.

12. The use of claim 11, wherein the disease or symptom comprises a disease related to abnormal cholesterol metabolism. Preferably, the disease or symptom comprises obesity, atherosclerosis, age-related macular degeneration, hyperlipidemia, non-alcoholic hepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, tumor angiogenesis, coronary heart disease, ischemic stroke, embolism caused by blood lipid plaque shedding.

13. Use of the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 or 10 in the preparation of a medicament for modulating cholesterol metabolism or ameliorating abnormal cholesterol metabolism.

14. The use of claim 13, wherein the modulating cholesterol metabolism or ameliorating abnormal cholesterol metabolism comprises at least one of the following group: (1) reducing the content of triglyceride and total cholesterol in cells, serum, or tissues; (2) reducing the content of low density lipoprotein cholesterol (LDL-C) in cells, serum, or tissues; (3) increasing the content of high density lipoprotein cholesterol (HDL-C) in cells, serum, or tissues; (4) reducing lipid accumulation in cells; (5) reducing the level of oxidative damage of cells; (6) inhibiting vascular endothelial cell migration; (7) reducing arterial plaque accumulation.

15. Use of the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 or 10 in the preparation of a medicament for reducing body weight.

16. A method of preventing, alleviating or treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-15. The method comprises administering the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 or 10 to a subject in need thereof.

17. The method of claim 16, wherein the disease or condition comprises a disease associated with abnormal cholesterol metabolism. Preferably, the disease or condition comprises obesity, atherosclerosis, age-related macular degeneration, hyperlipidemia, non-alcoholic hepatitis, fatty liver, liver fibrosis, liver cirrhosis, liver cancer, tumor angiogenesis, coronary heart disease, ischemic stroke, embolism caused by blood lipid plaque shedding.

18. A method of modulating cholesterol metabolism or ameliorating a disorder of cholesterol metabolism, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-17. The method comprises administering to a subject in need thereof the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 or 10.

19. The method of claim 18, wherein the modulating or ameliorating cholesterol metabolism comprises at least one of the following group: (1) reducing the content of triglyceride and total cholesterol in cells, serum or tissues; (2) reducing the content of low density lipoprotein cholesterol (LDL-C) in cells, serum or tissues; (3) increasing the content of high density lipoprotein cholesterol (HDL-C) in cells, serum or tissues; (4) reducing lipid accumulation in cells; (5) reducing the level of oxidative damage in cells; (6) inhibiting vascular endothelial cell migration; (7) reducing arterial plaque accumulation.

20. A method for reducing the weight of a subject, characterized in that, The method comprises administering to a subject in need thereof the polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the recombinant vector of claim 8, or the pharmaceutical composition of claim 9 or 10.

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