Application of melatonin and melatonin receptor agonist in prevention or treatment of valvular heart disease

Abstract

The invention provides application of melatonin and / or a melatonin receptor stimulant and / or a Gas6 protein inhibitor and / or an AXL receptor tyrosine kinase inhibitor in prevention and / or treatment of valvular heart disease. Specifically, the invention provides application of a first component and an optional second component in preparation of a medicine for preventing and / or treating diseases caused by heart valve mucus-like degeneration, the first component comprises melatonin and / or a melatonin receptor agonist, and the second component comprises an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor. The invention lays a foundation for research on heart valve mucus-like degeneration and diseases caused by the heart valve mucus-like degeneration, and has important clinical application and scientific research values.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of melatonin and melatonin receptor agonists in preventing or treating heart valve disease. Background Art

[0002] There are four valves in the heart: the mitral valve, tricuspid valve, aortic valve and pulmonary valve. The heart valve acts like a one-way valve. When the valve is open, blood flows forward, and when the valve is closed, blood flows back to ensure that the heart moves in the right direction when contracting. When the opening or closing of the heart valve is dysfunctional, it affects the normal flow of blood, which is called valvular heart disease. Valvular heart disease is one of the main causes of the global morbidity and mortality caused by cardiovascular disease. With the aging of the population, the incidence of valvular heart disease is still rising. According to statistics, by 2018, about 200 million people in the world suffered from this disease. Myxoid degeneration is a common pathological change of valvular heart disease, and cardiac ultrasound shows redundancy, thickening and prolapse of the valve leaflets. Early drug intervention for patients with valvular heart disease is an effective means to reduce the incidence of the disease, but to date, there is no specific drug for valvular heart disease itself. Valve replacement surgery or plasty surgery in the late stage of valvular heart disease is still the gold standard for the treatment of valvular heart disease.

[0003] The normal valve extracellular matrix (ECM) structure is divided into three layers: (1) fibrous layer: originates from the mitral annulus, faces the left ventricle, and is mainly composed of parallel dense collagen fibers; (2) spongy layer: covers the fibrous layer, is a loose connective tissue layer rich in glycosaminoglycans (GAGs), and contains a small amount of collagen; (3) atrial layer: extends from the left atrial endocardium to the valve leaflet, and is mainly composed of elastin. The valve surface is covered by endothelial cells, and the deep layer of the valve is valvular interstitial cells (VIC) originating from endocardial endothelial cells. VIC plays an important role in maintaining valve structure and function by regulating the dynamic balance between ECM synthesis and degradation. Under physiological conditions, VICs are quiescent, non-contractile fibroblast-like cells that maintain valve ECM homeostasis. However, under pathological stimuli such as aging and mechanical stress, VICs are activated and transdifferentiate into myofibroblast-like cells, expressing the smooth muscle-related contractile protein α-SMA (α-smooth muscleactin, α-SMA), increasing the synthesis and secretion of collagen and MMPs (smatrix metalloproteinases, MMPs), and excessive deposition of GAGs / proteoglycans, leading to ECM remodeling and ultimately causing valve myxoid degeneration and late valve prolapse, regurgitation or regurgitation.

[0004] At present, there is no specific drug treatment for myxoid degeneration of valves. Clinical medication is mainly used to control the complications caused by valve prolapse and regurgitation caused by valve lesions, such as beta-blockers for lowering blood pressure and heart rate, and antiarrhythmic drugs for controlling abnormal heart rhythm. Although surgical treatment can restore valve function, it cannot prevent the progression of valve lesions itself, and valve prolapse and regurgitation may occur again. Therefore, there is an urgent need for drugs that can effectively intervene in myxoid degeneration of valves to reduce the occurrence of heart valve disease. Summary of the invention

[0005] The present invention is accomplished based on the following findings of the inventors:

[0006] The inventors of the present invention have found that by applying melatonin to mice with myxoid degeneration of the mitral valve, the morphology of the mitral valve of the mice was significantly improved, and the collagen content (i.e., the degree of fibrosis) and the glycosaminoglycan content were significantly reduced. Applying melatonin to human valvular interstitial cells can effectively inhibit valvular interstitial cell activation, fibrosis-related gene expression and glycosaminoglycan content, and promote the expression of proteoglycan degrading enzyme-related genes in valvular interstitial cells. Furthermore, the inventors selected SR1078, an agonist of the melatonin receptor RORα protein, for research and administered it to valvular interstitial cells, and found that it can also effectively inhibit valvular interstitial cell activation and fibrosis-related gene expression. Thus, it is shown that melatonin and melatonin receptor agonists can effectively improve myxoid degeneration of the mitral valve, and have preventive and therapeutic purposes.

[0007] In addition, the inventors knocked down the Gas6 gene in the valvular interstitial cells, which can down-regulate the expression of fibrosis-related genes and reduce the content of proteoglycan degrading enzymes. By applying Gas6 protein to the valvular interstitial cells, the expression of fibrosis-related genes in the valvular interstitial cells will be promoted, and the AXL receptor tyrosine kinase inhibitor R428 can effectively block this effect of Gas6 protein. Therefore, it is shown that Gas6 protein inhibitors and AXL receptor tyrosine kinase inhibitors can effectively improve mitral valve myxoid degeneration, and can work together with melatonin and melatonin receptor agonists to better prevent and treat diseases caused by mitral valve myxoid degeneration, and have important clinical applications and scientific research value.

[0008] To this end, in one aspect of the present invention, the present invention proposes the use of a first component and an optional second component in the preparation of a medicament for preventing and / or treating valvular heart disease, wherein the first component includes melatonin and / or a melatonin receptor agonist, and the second component includes an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

[0009] In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: melatonin and / or a melatonin receptor agonist; an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

[0010] In another aspect of the present invention, the present invention provides a method for inhibiting the activation of valve interstitial cells in vitro, inhibiting the expression of fibrosis-related genes in valve interstitial cells, and reducing the content of glycosaminoglycans or proteoglycans in valve interstitial cells. According to an embodiment of the present invention, the method comprises: contacting valve interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

[0011] In another aspect of the present invention, the present invention provides a method for inhibiting gene expression in valvular interstitial cells in vitro, wherein the gene comprises at least one of the following: ACTA2 gene, collagen gene and CCN2 gene. According to an embodiment of the present invention, the method comprises: contacting the valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

[0012] In another aspect of the present invention, the present invention provides a method for promoting gene expression in valvular interstitial cells in vitro, wherein the gene comprises at least one of the following: ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene. According to an embodiment of the present invention, the method comprises: contacting the valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

[0013] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0015] Figure 1 A schematic diagram of H&E staining of a mitral valve according to an embodiment of the present invention is shown;

[0016] Figure 2 A schematic diagram of Movat staining of the mitral valve according to an embodiment of the present invention is shown;

[0017] Figure 3 An analysis diagram of the expression of the ACTA2 gene, COL1A1 gene and CCN2 gene in valvular interstitial cells under the action of melatonin according to one embodiment of the present invention is shown;

[0018] Figure 4 An analysis graph of the expression of ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene in valvular interstitial cells under the action of melatonin and an analysis graph of the concentration of GAGs in valvular interstitial cells are shown according to an embodiment of the present invention;

[0019] Figure 5 An analysis diagram of the expression levels of the ACTA2 gene, COL1A1 gene, and CCN2 gene in valvular interstitial cells under the action of the RORα agonist SR1078 and the RORA inverse agonist SR3335 according to one embodiment of the present invention is shown;

[0020] Figure 6The graph showing the GAGs concentration analysis of valvular interstitial cells under the action of RORα inverse agonist SR3335, melatonin and TGF-β1 according to one embodiment of the present invention is shown;

[0021] Figure 7 A Gas6 gene expression analysis diagram of valvular interstitial cells according to an embodiment of the present invention is shown;

[0022] Figure 8 Shown is a diagram of knockdown of Gas6 expression, COL1A1 gene expression and GAGs concentration analysis of valvular interstitial cells according to one embodiment of the present invention;

[0023] Fig. 9 The figure shows the expression analysis of COL1A1 gene and CTGF gene in valvular interstitial cells under the action of recombinant human Gas6 (rhGas6) and / or AXL inhibitor R428 according to one embodiment of the present invention;

[0024] Fig.10 The graph shows the expression levels of ACTA2 gene, COL1A1 gene and CCN2 gene and GAGs concentration analysis of valvular interstitial cells under the action of melatonin, RORα agonist SR1078, siRNA Gas6 and / or AXL inhibitor R428 according to one embodiment of the present invention. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0027] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0028] use

[0029] In one aspect of the present invention, the present invention proposes the use of a first component and an optional second component in the preparation of a drug for preventing and / or treating valvular heart disease, wherein the first component includes melatonin and / or a melatonin receptor agonist, and the second component includes an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

[0030] AXL receptor tyrosine kinase (AXL receptor in this article) belongs to the TAM family of receptor tyrosine kinase (RTK) (this family also includes TYRO3 / AXL / MER) and plays an important role in neural development, immune regulation, spermatogenesis, NK cell differentiation, platelet function and angiogenesis.

[0031] In some embodiments, the following components can be used to prepare a drug for preventing or treating heart valve disease:

[0032] Component 1: Melatonin;

[0033] Component 2: melatonin receptor agonist;

[0034] Component 3: melatonin and melatonin receptor agonists;

[0035] Component 4: at least one of melatonin and a melatonin receptor agonist and an AXL receptor inhibitor;

[0036] Component 5: at least one of melatonin and a melatonin receptor agonist and a Gas6 protein inhibitor;

[0037] Component 6: at least one of melatonin and a melatonin receptor agonist, an AXL receptor inhibitor and a Gas6 protein inhibitor.

[0038] According to an embodiment of the present invention, the heart valve includes a mitral valve or an aortic valve.

[0039] According to an embodiment of the present invention, the valvular heart disease includes a disease caused by myxomatous degeneration of the heart valve.

[0040] According to an embodiment of the present invention, the diseases include degenerative mitral valve disease, rheumatic heart disease, Marfan syndrome, Ehlers-Danlos syndrome, Loeys-Dietz syndrome, Aneurysm-osteoarthritis syndrome, Williams-Beuren syndrome, hypertrophic cardiomyopathy, valvular changes in infective endocarditis, Pseudoxanthoma elasticum, and Osteogenesis imperfecta.

[0041] According to an embodiment of the present invention, the degenerative mitral valve disease includes mitral regurgitation, mitral regurgitation, mitral valve prolapse syndrome, mitral stenosis, aortic stenosis and aortic regurgitation.

[0042] According to an embodiment of the present invention, the melatonin receptor includes RORα protein.

[0043] According to an embodiment of the present invention, the melatonin receptor agonist includes a RORα protein agonist, and the RORα protein agonist includes SR1078 and / or RS-2982.

[0044] The structure of SR1078 is as follows:

[0045] The structure of RS-2982 is as follows:

[0046] As used herein, the term "agonist" refers to an agent that enhances activity. "Melatonin receptor agonist" means that the presence of the agonist can enhance melatonin receptor activity compared to the absence of the agonist. For example, the melatonin receptor activity in the presence of the agonist is about 1.2 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 50 times or more of the melatonin receptor activity in the absence of the agonist.

[0047] According to an embodiment of the present invention, the AXL receptor tyrosine kinase inhibitor comprises at least one of the following: an antibody that specifically recognizes the AXL receptor tyrosine kinase, a functional antagonist of the AXL receptor tyrosine kinase, a competitive inhibitor, and an agent that downregulates the expression of the AXL receptor tyrosine kinase.

[0048] In some embodiments, the AXL receptor tyrosine kinase inhibitor includes nacentinib and / or dubermatinib (TP-0903), specifically, nacentinib (bemcentinib) comprises R428 and BGB324.

[0049]

[0050] According to an embodiment of the present invention, the Gas6 protein inhibitor includes at least one of the following: an antibody that specifically recognizes the Gas6 protein, a functional antagonist of the Gas6 protein, a competitive inhibitor, and an agent that downregulates the expression of the Gas6 protein.

[0051] In this article, the term "AXL receptor tyrosine kinase inhibitor / Gas6 protein inhibitor" refers to an agent that can specifically reduce the activity of AXL receptor tyrosine kinase / Gas6 protein, which can be a protein, a small molecule compound, a polymer compound, or a nucleic acid agent, as well as an agent suitable for causing a specific mutation (such as gene editing). Among them, reducing the activity of AXL receptor tyrosine kinase / Gas6 protein can be compared with not applying the inhibitor, reducing the activity of AXL receptor tyrosine kinase / Gas6 protein in the cell by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, for example, the protein expression level of AXL receptor tyrosine kinase / Gas6 protein in the cell can be reduced by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%.

[0052] In this article, the term "antibody" refers to a binding protein that contains an AXL receptor tyrosine kinase / Gas6 protein binding site. It can be a monoclonal antibody or a polyclonal antibody. Specifically, those skilled in the art can separate and purify antibodies that specifically recognize a predetermined antigen by common means, such as by hybridoma means or by phage display means. In addition, according to an embodiment of the present invention, the "antibody" that can be used includes but is not limited to a double-chain antibody, a full-length antibody, a single-chain antibody, a multimeric antibody, a CDR-transplanted antibody or a small molecule antibody. It will be understood by those skilled in the art that humanized antibodies can also be used as drugs.

[0053] As used herein, the term "competitive inhibitor" refers to an inhibitor that produces competitive inhibition. It usually has structural similarity with the substrate of the inhibited AXL receptor tyrosine kinase / Gas6 protein and can compete with the substrate for the binding site on the AXL receptor tyrosine kinase / Gas6 protein, thereby producing a reversible inhibition of the AXL receptor tyrosine kinase / Gas6 protein.

[0054] According to an embodiment of the present application, the reagent for downregulating the expression of the AXL receptor tyrosine kinase / Gas6 protein includes at least one of antisense oligonucleotide ASO, small hairpin RNA shRNA and interfering RNA RNAi specific to the AXL receptor / Gas6 protein. Those skilled in the art can design the sequence of the corresponding nucleic acid reagent based on the gene sequence of the target molecule such as the AXL receptor / Gas6 protein by conventional means.

[0055] According to an embodiment of the present invention, the drug is used to reduce at least one of the following parameters: mitral valve thickness, mitral valve area, mitral valve collagen content, glycosaminoglycan content and proteoglycan content.

[0056] According to an embodiment of the present invention, the drug is used to inhibit valve interstitial cell activation, and / or glycosaminoglycan production, and / or proteoglycan production and / or fibrosis-related gene expression in valve interstitial cells.

[0057] As used herein, the term "proteoglycan" refers to a complex consisting of one or more glycosaminoglycan chains and one or more core proteins.

[0058] According to an embodiment of the present invention, the drug is used to inhibit the expression of at least one of the following genes in valvular interstitial cells: ACTA2 gene, collagen gene and CCN2 gene. ACTA2 gene is a marker gene for activation of valvular interstitial cells and can encode α-SMA protein. CCN2 gene is used to encode connective tissue growth factor CTGF. Collagen genes can specifically include COL1A1 gene, COL1A2 gene, COL3A1 gene, etc. By inhibiting the expression of the above genes, the increase in the expression of fibrosis-related genes caused by the activation of valvular interstitial cells can be inhibited.

[0059] According to an embodiment of the present invention, the drug is used to promote the expression of at least one of the following genes in valve interstitial cells: ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene. ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene are glycosaminoglycan (GAGs) degradation enzyme-related genes, encoding metallopeptidase containing thrombospondin 1, 5, 9 respectively. ADAMTS1, 5, 9 can promote the degradation of glycosaminoglycans and reduce the content of glycosaminoglycans in valve interstitial cells.

[0060] According to an embodiment of the present invention, the drug is used to reduce the content of glycosaminoglycan and proteoglycan in valvular interstitial cells. Increased glycosaminoglycan and proteoglycan content is one of the important pathological changes of mitral valve myxoid degeneration. Reduced glycosaminoglycan can alleviate mitral valve myxoid degeneration lesions.

[0061] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug described herein to an individual in need.

[0062] In this article, the term "administration" refers to the introduction of a predetermined amount of material into a patient by a suitable manner. The medicine of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are expected, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, local, nasal, pulmonary and rectal administration, but the present invention is not limited to these exemplified modes of administration. However, due to oral administration, peptides are digested and peptide bonds are broken, so the active ingredient of the oral drug should be coated or formulated to prevent it from being degraded or destroyed in the stomach. Preferably, the medicine of the present invention can be administered with an injectable preparation. In addition, the medicine of the present invention can be administered using a specific device that transmits the active ingredient to the target cell.

[0063] The frequency and dosage of the drug of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient, and the type of drug as the active ingredient. According to some embodiments of the present invention, the daily dose can be divided into 1, 2 or more doses in a suitable form, so that it can be administered once, twice or more times over the entire time period, as long as the therapeutically effective amount is reached.

[0064] As used herein, the term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly improve certain symptoms associated with a disease or condition, i.e., an amount that provides a therapeutic effect for a given condition and dosing regimen. For example, in cancer treatment, a drug or compound that reduces, prevents, delays, inhibits, or blocks any symptom of a disease or condition should be therapeutically effective. A therapeutically effective amount of a drug or compound does not need to cure a disease or condition, but will provide treatment for the disease or condition, such that the onset of the disease or condition in an individual is delayed, stopped, or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or, for example, the disease or condition becomes less severe, or recovery is accelerated.

[0065] Pharmaceutical composition

[0066] In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: melatonin and / or a melatonin receptor agonist; an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor. The pharmaceutical composition of the present invention can effectively improve myxoid degeneration of heart valves and contribute to the prevention and treatment of heart valve disease.

[0067] According to an embodiment of the present invention, the melatonin receptor includes RORα protein.

[0068] According to an embodiment of the present invention, the melatonin receptor agonist includes a RORα protein agonist.

[0069] According to an embodiment of the present invention, the AXL receptor tyrosine kinase inhibitor comprises at least one of the following: an antibody that specifically recognizes the AXL receptor tyrosine kinase, a functional antagonist of the AXL receptor tyrosine kinase, a competitive inhibitor, and an agent that downregulates the expression of the AXL receptor tyrosine kinase.

[0070] According to an embodiment of the present invention, the Gas6 protein inhibitor includes at least one of the following: an antibody that specifically recognizes the Gas6 protein, a functional antagonist of the Gas6 protein, a competitive inhibitor, and an agent that downregulates the expression of the Gas6 protein.

[0071] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0072] In this article, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, the "pharmaceutically acceptable" of the present invention refers to the use in animals, especially humans, approved by the national government or listed in the Chinese Pharmacopoeia or other generally recognized pharmacopoeias.

[0073] In this context, "pharmaceutically acceptable excipients" are pharmaceutically or food-acceptable carriers, solvents, suspending agents or excipients used to deliver the active ingredients (melatonin and / or melatonin receptor agonists and AXL receptor inhibitors) in the pharmaceutical compositions of the present invention to animals or humans. Exemplary excipients may be liquid or solid, and include, but are not limited to, pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with the pharmacological compound. Examples of excipients include various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrins and starch.

[0074] The dosage form of the pharmaceutical composition is selected from one of a mixture, an injection, a tablet, a granule, a syrup, a capsule, an oral liquid, an aerosol and a spray. Thus, it is convenient to adopt different dosage forms according to different administration objects. For example, in order to facilitate administration, tablets, granules, syrups, capsules and oral liquids can be used, and the dosage form of the drug can be adjusted according to the absorption site of the drug and the release requirements of the drug, so as to improve the bioavailability of the drug and prolong the release time of the drug. For patients with severe conditions, an injection can be used to achieve the large-dose administration requirements, and avoid the influence of gastrointestinal circulation on the active ingredients of the drug.

[0075] It should be noted that the features and advantages described above for the uses are also applicable to the pharmaceutical composition and will not be repeated here.

[0076] method

[0077] In another aspect of the present invention, the present invention proposes a method for inhibiting the activation of valvular interstitial cells in vitro, inhibiting the expression of fibrosis-related genes in valvular interstitial cells, and reducing the glycosaminoglycan content or proteoglycan content in valvular interstitial cells. According to an embodiment of the present invention, the method comprises: contacting valvular interstitial cells with melatonin and / or a melatonin receptor agonist and an optional AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor. Thus, the method can effectively inhibit the activation of valvular interstitial cells, fibrosis or reduce the glycosaminoglycan content in valvular interstitial cells, which is helpful to carry out in-depth research on valvular interstitial cells.

[0078] In another aspect of the present invention, the present invention provides a method for inhibiting gene expression in valvular interstitial cells in vitro, wherein the gene includes at least one of the following: ACTA2 gene, collagen gene and CCN2 gene. According to an embodiment of the present invention, the method includes: contacting the valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor. Thus, the method can effectively inhibit the expression of the above genes in valvular interstitial cells, which is helpful for conducting research on the above genes and valvular interstitial cells.

[0079] In another aspect of the present invention, the present invention proposes a method for promoting gene expression in valvular interstitial cells in vitro, wherein the gene includes at least one of the following: ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene. According to an embodiment of the present invention, the method includes: contacting the valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor. Thus, the method can effectively promote the expression of the above genes in valvular interstitial cells, which is helpful for conducting research on the above genes and valvular interstitial cells.

[0080] According to an embodiment of the present invention, the melatonin receptor includes RORα protein.

[0081] According to an embodiment of the present invention, the melatonin receptor agonist includes a RORα protein agonist.

[0082] According to an embodiment of the present invention, the AXL receptor tyrosine kinase inhibitor comprises at least one of the following: an antibody that specifically recognizes the AXL receptor tyrosine kinase, a functional antagonist of the AXL receptor tyrosine kinase, a competitive inhibitor, and an agent that downregulates the expression of the AXL receptor tyrosine kinase.

[0083] According to an embodiment of the present invention, the Gas6 protein inhibitor includes at least one of the following: an antibody that specifically recognizes the Gas6 protein, a functional antagonist of the Gas6 protein, a competitive inhibitor, and an agent that downregulates the expression of the Gas6 protein.

[0084] It should be noted that the features and advantages described above for the purposes are also applicable to the above methods and will not be repeated here.

[0085] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0086] Example 1 Animal Experiment

[0087] 1. Animal Model: 8-week-old male and female wild-type mice and Fbn1 C1039G / + Mice (i.e., Marfan syndrome mice).

[0088] 2. Prepare solution:

[0089] Melatonin solution: provide a melatonin stock solution containing 0.5 mg / μl (dissolution ratio: 0.5 mg melatonin vs. 1 μl DMSO), take 5 μl and add 995 μl PBS to obtain a melatonin solution with a concentration of 2.5 μg / μl (DMSO concentration is 5%).

[0090] Vehicle solution: PBS solution containing 5% DMSO.

[0091] 3. Experimental groups:

[0092] WT+Vehicle: Wild-type mice were administered with Vehicle solution.

[0093] WT+Melatonin: Wild-type mice were administered melatonin solution.

[0094] Fbn1 C1039G / + +Vehicle: Fbn1 C1039G / + The mice were administered with the Vehicle solution.

[0095] Fbn1 C1039G / + +Melatonin: Fbn1 C1039G / + Mice were administered melatonin solution.

[0096] 4. Administration:

[0097] Intraperitoneal injection, injection volume ≤ 200 μl / mouse.

[0098] Dosing time, frequency and cycle: Inject once daily at 9:00-10:00 am for 4 weeks.

[0099] Dosage: Melatonin solution / Vehicle solution 20 mg / kg·BW, BW: body weight.

[0100] 5. Efficacy evaluation method:

[0101] The mitral valves of mice fed for 4 weeks were stained with H&E ( Figure 1 ) and Movat staining ( Figure 2 ) to evaluate valve morphology, thickness, area, and collagen and glycosaminoglycans (GAGs) deposition.

[0102] like Figure 1 As shown, Fbn1 C1039G / + The anterior and posterior leaflets of the mitral valve in the +Vehicle group were significantly thicker and larger than those in the WT+Vehicle group. C1039G / + Mouse (Fbn1 C1039G / + + melatonin group) had a lower mitral valve than Fbn1 C1039G / + The size of the scrotum in the +Vehicle group mice was significantly reduced, and its thickness and area were similar to those of wild-type mice (n=5 / group).

[0103] like Figure 2 As shown, Fbn1 C1039G / + The anterior and posterior leaflets of the mitral valve in the +Vehicle group were significantly thicker and larger than those in the WT+Vehicle group, and the deposition of collagen (light red) and glycosaminoglycan (blue) was significantly increased. C1039G / + Mouse (Fbn1 C1039G / + + melatonin group) had a higher mitral valve thickness, area, collagen and glycosaminoglycan content than Fbn1 C1039G / + The mitral valve of mice in the +Vehicle group was significantly smaller, and the thickness, area, collagen and glycosaminoglycan content of the mitral valve were not significantly different from those of mice in the WT+Vehicle group (n=5 / group).

[0104] Example 2 Cell experiment

[0105] 1Cell type: human mitral interstitial cells

[0106] Culture method: tissue block adherence method, DMEM high-glucose medium containing 10% serum.

[0107] Final drug concentrations: melatonin: 25 μM, TGF-β1: 0.5 ng / ml, SR3335: 5 μM, SR1078: 5 μM.

[0108] 1.1 Determination of the inhibitory effect of melatonin on mitral interstitial cell phenotypic activation, fibrosis, and GAGs content

[0109] 1.1.1 Experimental groups: ① Vehicle group, ② TGF-β1 (promote cell activation) group, ③ Melatonin (melatonin) group, ④ Melatonin + TGF-β1 group.

[0110] 1.1.2 Cell treatment method: When the cell confluence rate reached 80%, serum-free DMEM medium containing 0.5% FBS was used to culture for 24 hours. Each group was treated as follows, and cell samples were collected after 48 hours.

[0111] ①Vehicle group: not processed.

[0112] ②TGF-β1 group: cells were co-incubated with TGF-β1.

[0113] ③Melatonin group: cells were co-incubated with melatonin.

[0114] ④Melatonin+TGF-β1 group: The cells were incubated with melatonin, and TGF-β1 was given after 1 hour for continued culture.

[0115] 1.1.3 Evaluation method: Real-time PCR and western blot were used to detect the activation marker gene ACTA2 and its protein α-SMA ( Figure 3 A, B), fibrosis marker genes and corresponding proteins COL1A1 / type I collagen and CCN2 / CTGF (connective tissue growth factor) ( Figure 3 A, B), expression of GAGs degradation enzyme-related genes ADAMTS1 / 5 / 9 (metallopeptidase containing thrombospondin 1, 5, 9) ( Figure 4 A); BlyscanTM Sulfated Glycosaminoglycan assay to detect intracellular GAGs concentration ( Figure 4 B).

[0116] like Figure 3 As shown in the figure, real-time PCR and western blot results show that melatonin can directly inhibit the expression of the activation marker gene ACTA2 and its corresponding protein α-SMA of valvular interstitial cells, and at the same time, it can also partially block the activation effect of TGF-β1 on cell phenotype. Melatonin has the same inhibitory and blocking effects on intracellular fibrosis marker genes and their corresponding proteins COL1A1 / type I collagen and CCN2 / CTGF (connective tissue growth factor).

[0117] like Figure 4 As shown in the figure, the real-time PCR results showed that TGF-β1 inhibited the expression of proteoglycan degradation enzyme-related genes ADAMTS1 / 5 / 9 (metallopeptidase containing thrombospondin 1, 5, 9) in valvular interstitial cells, and melatonin could partially reverse the inhibitory effect of TGF-β1. The BlyscanTM Sulfated Glycosaminoglycan experiment detected the changes in the concentration of intracellular GAGs. TGF-β1 promoted the increase of intracellular GAGs content, and melatonin could partially inhibit the effect of TGF-β1.

[0118] 1.2 Determine whether melatonin exerts the above-mentioned biological inhibitory effects by activating RORα receptors

[0119] 1.2.1 Experimental Grouping:

[0120] RORα receptor inverse agonist group SR3335: ①Vehicle group, ②TGF-β1 group, ③Melatonin (melatonin) + TGF-β1 group, ④SR3335+Melatonin (melatonin) + TGF-β1 group.

[0121] RORα receptor agonist group SR1078: ⑤Vehicle group, ⑥TGF-β1 group, ⑦SR1078+TGF-β1 group, ⑧SR1078 group.

[0122] 1.2.2 Cell treatment method: When the cell confluence rate reached 80%, the cells were cultured in DMEM serum-free medium with 0.5% FBS for 24 hours. Each group was treated as follows, and cell samples were collected after 48 hours.

[0123] ①Vehicle group and ⑤Vehicle group: untreated.

[0124] ②TGF-β1 group and ⑥TGF-β1 group: the cells were co-incubated with TGF-β1.

[0125] ③Melatonin+TGF-β1 group: cells were incubated with melatonin, and TGF-β1 was given after 1 hour for continued culture.

[0126] ④SR3335+Melatonin+TGF-β1 group: cells were incubated with SR3335, then with melatonin after 0.5h, and then TGF-β1 was given after 1h for continued culture.

[0127] ⑦SR1078+TGF-β1 group: cells were incubated with SR1078, and TGF-β1 was given after 1 hour for continued culture.

[0128] ⑧SR1078 group: cells were co-incubated with SR1078.

[0129] 1.2.3 Evaluation method: Real-time PCR detection of valvular interstitial cell activation marker gene ACTA2 ( Figure 5 A, B), fibrosis marker genes / proteins COL1A1 and CCN2 ( Figure 5 A, B); BlyscanTM Sulfated Glycosaminoglycan assay to detect intracellular GAGs concentration ( Figure 6 ).

[0130] like Figure 5Real-time PCR results of A showed that RORα agonist SR1078 can directly inhibit the expression of valvular interstitial cell activation marker gene ACTA2, fibrosis marker genes COL1A1 and CCN2, and at the same time, it can also partially block the activation effect of TGF-β1 on cells.

[0131] like Figure 5 Real-time PCR results of B showed that RORα inverse agonist SR3335 could partially block the protective effect of melatonin on valvular interstitial cells, namely inhibiting the activation of valvular interstitial cells (ACTA2) and the increase in the expression of fibrosis genes (COL1A1 and CCN2).

[0132] like Figure 6 As shown, TGF-β1 promoted the increase of intracellular GAGs content, melatonin could partially inhibit the effect of TGF-β1, and RORα inverse agonist SR3335 blocked the effect of melatonin.

[0133] 2Cell type: human mitral interstitial cells

[0134] Culture method: tissue block adherence method, DMEM high-glucose medium containing 10% serum.

[0135] Final drug concentrations: TGF-β1: 0.5 ng / ml, R428: 2 μM, human recombinant Gas6 (rh Gas6): 500 ng / ml, final siRNAGas6 concentration: 50 nM.

[0136] The nucleotide sequence of siRNA is shown below, which can knock down Gas6 protein expression.

[0137] sense(5'-3'):CGCGGAAUCUGGUCAUCAAdTdT

[0138] antisense(5'-3'):UUGAUGACCAGAUUCCGCGdTdT

[0139] 2.1 Determine the inhibitory effect of knocking down Gas6 in mitral interstitial cells on cell phenotypic activation and other effects.

[0140] 2.1.1 Cell grouping: ① siRNA NC group, ② siRNAGas6 group, ③ siRNA NC+TGF-β1 group, ④ siRNAGas6+TGF-β1 group.

[0141] 2.1.2 Cell treatment method: When the cell confluence rate reached 80%, the cells were cultured in DMEM serum-free medium containing 0.5% FBS for 24 hours. Each group was treated as follows, and cell samples were collected after 48 hours.

[0142] ① siRNA NC group: cells were transfected with siRNA negative control (NC) sequence using siRNA transfection reagent.

[0143] ②siRNA Gas6 group: cells were transfected with siRNA Gas6 sequence using siRNA transfection reagent.

[0144] ③ siRNA NC+TGF-β1 group: cells were transfected with siRNA negative control (NC) sequence using siRNA transfection reagent, and TGF-β1 stimulation was given 24 hours later.

[0145] ④siRNAGas6+TGF-β1 group: cells were transfected with siRNA Gas6 sequence using siRNA transfection reagent, and TGF-β1 stimulation was given 24 hours later.

[0146] 2.1.3 Evaluation method: Real-time PCR and / or western blot detection of Gas6 knockdown in valvular interstitial cells ( Figure 7 A, B), expression of fibrosis marker protein COL1A1 ( Figure 8 A); BlyscanTM SulfatedGlycosaminoglycan assay to detect intracellular GAGs concentration ( Figure 8 B).

[0147] like Figure 7 Real-time PCR (A) and western blot (B) results showed that Gas6 was successfully knocked down by siRNA.

[0148] like Figure 8 The western blot results of A showed that after knocking down the expression of Gas6 in valvular interstitial cells, the expression of COL1A1 was downregulated. Figure 8 The results of BlyscanTM Sulfated Glycosaminoglycan experiment of B showed that the expression of Gas6 in valvular interstitial cells was knocked down in the detected cells, and the concentration of intracellular GAGs was inhibited.

[0149] 2.2 Determine the blocking effect of AXL receptor inhibitor R428 on Gas6 activation of valvular interstitial cells.

[0150] 2.2.1 Cell grouping: ① Vehicle group, ② R428 group, ③ rhGas6 group, ④ rhGas6+R428 group, ⑤ R428+Melatonin group.

[0151] 2.2.2 Cell treatment method: When the cell confluence rate reached 80%, the cells were cultured in DMEM serum-free medium containing 0.5% FBS for 24 hours. Each group was treated as follows, and the cell samples were collected after a further incubation of 48 hours.

[0152] ①Vehicle group: not processed.

[0153] ②R428 group: cells were co-incubated with R428.

[0154] ③rhGas6 group: cells were co-incubated with rhGas6.

[0155] ④rhGas6+R428 group: the cells were incubated with R428 for 0.5 h and then cultured with rhGas6.

[0156] 2.2.3 Evaluation method: Western blot was used to detect the inhibitory effect of R428 on the expression of COL1A1 and CTGF in valvular interstitial cells ( Fig. 9 ).

[0157] like Fig. 9 Western blot results showed that rhGas6 promoted the expression of COL1A1 and CTGF in valvular interstitial cells, and R428 could effectively block this effect of rhGas6.

[0158] 3. Cell type: human mitral interstitial cells

[0159] Culture method: tissue block adherence method, DMEM high-glucose medium containing 10% serum.

[0160] Final concentration of drug or siRNA: same as above

[0161] 3.1 Determine the combined inhibitory effect of melatonin or melatonin receptor and AXL inhibitor or Gas6 inhibitor (siRNA) on the biological activity of valvular interstitial cells.

[0162] 3.1.1 Cell grouping

[0163] Group 1: ①Vehicle, ②Melatonin, ③Melatonin+R428, ④SR1078+R428

[0164] Group 2: ① siRNA NC, ② siRNA NC+Melatonin, ③ siRNA Gas6+Melatonin, ④ siRNAGas6+SR1078

[0165] 3.1.2 Cell treatment: When the cell confluency rate reached 80%, the cells were cultured in DMEM serum-free medium containing 0.5% FBS for 24 h. Each group was treated as follows.

[0166] ①Vehicle: Not processed.

[0167] ②Melatonin: incubate the cells with melatonin for 48 hours.

[0168] ③Melatonin+R428: incubate the cells with R428 for 48 hours.

[0169] ④SR1078+R428: incubate the cells with SR1078 and R428 for 48 hours.

[0170] ⑤siRNA NC: cells were transfected with siRNA NC sequence for 72h.

[0171] ⑥siRNA NC+Melatonin: 24 hours after cells were transfected with siRNA NC sequence, melatonin was added and cultured for another 48 hours.

[0172] ⑦siRNA Gas6+Melatonin: 24 hours after cells were transfected with siRNA Gas6 sequence, melatonin was added and cultured for another 48 hours.

[0173] ⑧siRNA Gas6+SR1078: 24 hours after cells were transfected with siRNA Gas6 sequence, SR1078 was added and cultured for another 48 hours.

[0174] 3.13 Evaluation method: real-time PCR detection of valvular interstitial cell activation marker gene ACTA2 ( Fig.10 A, B), fibrosis marker genes COL1A1 and CCN2 ( Fig.10 A, B); BlyscanTM Sulfated Glycosaminoglycan assay to detect intracellular GAGs concentration ( Fig.10 C, D).

[0175] like Fig.10 Real-time PCR results of A and B showed that Melatonin+R428(A), SR1078+R428(A), siRNAGas6+Melatonin(B) and siRNA Gas6+SR1078(B) could jointly inhibit the expression of ACTA2, a marker gene for activation of valvular interstitial cells, and COL1A1 and CCN2, marker genes for fibrosis.

[0176] like Fig.10The results of BlyscanTM Sulfated Glycosaminoglycan experiments in C and D showed that Melatonin+R428(C), SR1078+R428(C), siRNAGas6+Melatonin(D) and siRNA Gas6+SR1078(D) could jointly inhibit the intracellular GAGs concentration.

[0177] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of a first component and an optional second component in the preparation of a medicament for preventing and / or treating valvular heart disease, wherein the first component comprises melatonin and / or a melatonin receptor agonist, and the second component comprises an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

2. The use according to claim 1, characterized in that The heart valve comprises a mitral valve or an aortic valve; Optionally, the heart valve disease includes a disease caused by myxoid degeneration of heart valves; Optionally, the disease comprises degenerative mitral valve disease, rheumatic heart disease, Marfan syndrome, Ehlers-Danlos syndrome, Loess-Dietz syndrome, aneurysm-osteoarthritis syndrome, Williams syndrome, hypertrophic cardiomyopathy, valvular changes in infective endocarditis, pseudoxanthoma elasticum, and osteogenesis imperfecta; Optionally, the degenerative mitral valve disease includes mitral regurgitation, mitral regurgitation, mitral valve prolapse syndrome, mitral stenosis, aortic stenosis and aortic regurgitation.

3. The use according to claim 1, characterized in that The melatonin receptor includes RORα protein; Optionally, the melatonin receptor agonist comprises a RORα protein agonist; Optionally, the AXL receptor tyrosine kinase inhibitor comprises at least one of the following: an antibody that specifically recognizes the AXL receptor tyrosine kinase, a functional antagonist of the AXL receptor tyrosine kinase, a competitive inhibitor, and an agent that downregulates the expression of the AXL receptor tyrosine kinase; Optionally, the Gas6 protein inhibitor includes at least one of the following: an antibody that specifically recognizes the Gas6 protein, a functional antagonist of the Gas6 protein, a competitive inhibitor, and an agent that downregulates the expression of the Gas6 protein.

4. The use according to claim 1, characterized in that The drug is used to reduce at least one of the following parameters: mitral valve thickness, mitral valve area, mitral valve collagen content, glycosaminoglycan content and proteoglycan content; Optionally, the drug is used to inhibit valve interstitial cell activation, and / or glycosaminoglycan production, and / or proteoglycan production, and / or fibrosis-related gene expression in valve interstitial cells; Optionally, the drug is used to inhibit the expression of at least one of the following genes in valve interstitial cells: ACTA2 gene, collagen gene and CCN2 gene; Optionally, the drug is used to promote the expression of at least one of the following genes in valve interstitial cells: ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene; Optionally, the medicament is used to reduce the content of glycosaminoglycans and proteoglycans in valve interstitial cells.

5. A pharmaceutical composition, characterized in that include: melatonin and / or melatonin receptor agonists; AXL receptor tyrosine kinase inhibitors and / or Gas6 protein inhibitors.

6. The pharmaceutical composition according to claim 5, characterized in that The melatonin receptor includes RORα protein; Optionally, the melatonin receptor agonist comprises a RORα protein agonist; Optionally, the AXL receptor tyrosine kinase inhibitor comprises at least one of the following: an antibody that specifically recognizes the AXL receptor tyrosine kinase, a functional antagonist of the AXL receptor tyrosine kinase, a competitive inhibitor, and an agent that downregulates the expression of the AXL receptor tyrosine kinase; Optionally, the Gas6 protein inhibitor includes at least one of the following: an antibody that specifically recognizes the Gas6 protein, a functional antagonist of the Gas6 protein, a competitive inhibitor, and an agent that downregulates the expression of the Gas6 protein.

7. A method for inhibiting the activation of valvular interstitial cells in vitro, inhibiting the expression of fibrosis-related genes in valvular interstitial cells, and reducing the content of glycosaminoglycan or proteoglycan in valvular interstitial cells, characterized in that: The method comprises contacting valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

8. A method for inhibiting gene expression in valvular interstitial cells in vitro, wherein the gene comprises at least one of the following: ACTA2 gene, collagen gene and CCN2 gene, characterized in that: The method comprises contacting valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

9. A method for promoting gene expression in valvular interstitial cells in vitro, wherein the gene comprises at least one of the following: ADAMTS1 gene, ADAMTS5 gene and ADAMTS9 gene, characterized in that: The method comprises contacting valvular interstitial cells with melatonin and / or a melatonin receptor agonist and optionally an AXL receptor tyrosine kinase inhibitor and / or a Gas6 protein inhibitor.

10. The method according to any one of claims 7 to 9, characterized in that: The melatonin receptor includes RORα protein; Optionally, the melatonin receptor agonist comprises a RORα protein agonist; Optionally, the AXL receptor tyrosine kinase inhibitor comprises at least one of the following: an antibody that specifically recognizes the AXL receptor tyrosine kinase, a functional antagonist of the AXL receptor tyrosine kinase, a competitive inhibitor, and an agent that downregulates the expression of the AXL receptor tyrosine kinase; Optionally, the Gas6 protein inhibitor includes at least one of the following: an antibody that specifically recognizes the Gas6 protein, a functional antagonist of the Gas6 protein, a competitive inhibitor, and an agent that downregulates the expression of the Gas6 protein.

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