Pharmaceutical application of ELANE gene as heart failure target and new application of sivelestat

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to pharmaceutical application of ELANE gene as a heart failure target and new application of sivelestat. The ELANE gene is overexpressed in the cardiac muscle cells of the pressure-loaded heart failure, so that the ejection fraction in the heart failure stage can be increased, the apoptosis of the cardiac muscle cells can be retarded, and the cardiac function can be effectively recovered. It is clear that ELANE can serve as a target gene in medicine or gene therapy to be applied to prevention, relieving or / and treatment of the pressure-loaded heart failure, and a new strategy is provided for prevention and treatment of the pressure-loaded heart failure. The invention also provides a new application of the sivelestat, and the sivelestat can obviously improve the heart and heart functions and myocardial fibrosis conditions of a heart failure mouse induced by a pressure load, is an effective medicine for treating the heart failure, and can be used for preparing a medicine for treating and resisting the heart failure.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to pharmaceutical use of ELANE gene as a heart failure target and new use of sivelestat. Background Art

[0002] Heart failure refers to a complex chronic clinical syndrome caused by abnormal changes in cardiac structure and (or) function due to various reasons, resulting in ventricular systolic pumping and (or) diastolic filling dysfunction, which causes the heart output to be unable to meet the body's normal physiological function. There are many causes of heart failure, including ischemic cardiomyopathy, hypertensive heart disease, hereditary cardiomyopathy and valvular heart disease. Among them, ischemic cardiomyopathy that causes primary myocardial damage and hypertensive heart disease that causes increased left ventricular afterload are the two most important pathogenic factors. In hypertensive heart disease, due to increased peripheral circulation resistance, the pressure faced by the left ventricle also increases accordingly. In order to overcome the increase in afterload and ensure normal heart pumping, the left ventricular myocardium will grow compensatorily, leading to the occurrence of cardiac pathological remodeling. However, as the disease progresses, cardiac pathological remodeling continues to intensify, and cardiac function gradually changes from a compensated state to a decompensated state, eventually leading to heart failure. Although the pathological process of heart failure has been better understood and some progress has been made in treatment. However, the more precise underlying pathological mechanisms of cardiac pathological remodeling and heart failure still need further study.

[0003] Neutrophil elastase (ELANE, NE) is a serine protease encoded by the ELANE gene that plays a major role in the destruction of intracellular and extracellular pathogens. It has potent proteolytic activity against a variety of extracellular matrix proteins as well as a variety of non-matrix proteins such as cytokines / chemokines, cell surface proteins / receptors, and other functional soluble proteins. NE has been associated with a variety of destructive and inflammatory diseases, including chronic and acute lung diseases and atherosclerosis. Specifically, NE mRNA and protein can be detected in human atherosclerotic plaques, and increased plasma NE activity is observed during the development of atherosclerosis. Studies have also demonstrated the role of NE in atherosclerosis and injury-induced neointimal smooth muscle cell proliferation. However, whether NE is involved in the adaptive remodeling of the myocardium and the regulation of myocardial function under conditions of cardiac pressure overload is unknown.

[0004] Siveletastat is a selective neutrophil elastase inhibitor that has been marketed. It is the world's first drug approved for the treatment of acute lung injury with systemic inflammatory response syndrome. The daily dose for the treatment of this disease is 4.8 mg / kg / day (0.2 mg / kg / h intravenous administration for 24 hours). Based on its highly selective inhibitory effect on NE, Siveletastat is expected to be used to treat a variety of diseases.

[0005] The use of sivelestat in the treatment of heart diseases disclosed in the prior art is mainly related to myocardial damage induced by sepsis. There is no report that sivelestat can be used to treat or alleviate other types of heart diseases, especially heart failure induced by pressure load. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the pathophysiological significance of the ELANE gene or the protein NE encoded by it in pressure-overload heart failure and its application in the treatment of pressure-overload heart failure, in order to provide candidate targets for the development of heart failure therapeutic drugs. And provides the use of sivelestat or its pharmaceutically acceptable salt in the preparation of drugs for treating or alleviating heart failure.

[0007] In one aspect, the present invention provides pharmaceutical uses of substances that inhibit or silence neutrophil elastase activity in heart diseases.

[0008] Specifically, the substances include, but are not limited to: sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative;

[0009] and / or, ulinastatin or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof;

[0010] and / or, flavonoid aglycones and their glycosides and methylated, acetylated and hydroxylated derivatives.

[0011] Preferably, the substance is sivelestat or a pharmaceutically acceptable salt or a pharmaceutically acceptable derivative thereof.

[0012] More preferably, the substance is sivelestat.

[0013] Specifically, the heart disease includes, but is not limited to, coronary artery disease, arrhythmia, cardiomyopathy, heart failure, pericardial disease, hypertensive heart disease or cor pulmonale.

[0014] More specifically, the heart disease is heart failure.

[0015] More specifically, the heart disease is pressure overload-induced heart failure.

[0016] In another aspect, the present invention provides pharmaceutical uses of a substance that inhibits or silences neutrophil elastase gene expression in heart diseases.

[0017] Specifically, the substances include, but are not limited to: sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative;

[0018] and / or, ulinastatin or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof;

[0019] and / or, flavonoid aglycones and their glycosides and methylated, acetylated and hydroxylated derivatives.

[0020] Preferably, the substance is sivelestat or a pharmaceutically acceptable salt or a pharmaceutically acceptable derivative thereof.

[0021] More preferably, the substance is sivelestat.

[0022] Specifically, the heart disease is coronary artery disease, arrhythmia, cardiomyopathy, heart failure, pericardial disease, hypertensive heart disease or cor pulmonale.

[0023] More specifically, the heart disease is heart failure.

[0024] More specifically, the heart disease is pressure overload-induced heart failure.

[0025] In another aspect, the present invention provides use of sivelestat or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for treating heart disease.

[0026] Specifically, the heart disease is coronary artery disease, arrhythmia, cardiomyopathy, heart failure, pericardial disease, hypertensive heart disease or cor pulmonale.

[0027] More specifically, the heart disease is heart failure.

[0028] More specifically, the heart disease is pressure overload-induced heart failure.

[0029] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0030] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0031] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0032] Specifically, the dosage form of the drug includes but is not limited to: solution form, emulsion form or powder form.

[0033] In another aspect, the present invention provides a pharmaceutical composition for treating heart disease, comprising a substance that inhibits or silences the expression of the ELANE gene.

[0034] Specifically, the substances include, but are not limited to: sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative;

[0035] Or, ulinastatin or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof;

[0036] or, flavonoid aglycones and their glycosides and methylated, acetylated and hydroxylated derivatives.

[0037] Specifically, the substance is sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative.

[0038] Preferably, the substance is sivelestat.

[0039] Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0040] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0041] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0042] Specifically, the dosage form of the pharmaceutical composition is a solution dosage form, an emulsion form or a powder form.

[0043] The technical effects achieved by the present invention are:

[0044] (1) NE is highly expressed in serum and cardiac tissue of patients with pressure-overload heart failure.

[0045] (2) NE knockout can significantly alleviate cardiac function, myocardial fibrosis, myocardial hypertrophy and myocardial inflammation in mice with pressure-overload heart failure.

[0046] (3) AAV9-mediated NE overexpression can aggravate cardiac function, myocardial fibrosis, myocardial hypertrophy and myocardial inflammation in mice with pressure-overload heart failure.

[0047] (4) Sivelestat can alleviate pressure load-induced cardiac function in mice.

[0048] (5) Civelestat can alleviate myocardial fibrosis and hypertrophy in mice with pressure overload-induced heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Heart failure induced by aortic arch constriction (TAC) caused an increase in neutrophil elastase (NE) in the serum and heart infiltration of mice. A is the expression level of NE protein in the myocardial tissue of each group of mice detected by Western blotting; B is the NE content in the blood of different groups of mice after 14 days of TAC; C is the NE content in the hearts of different groups of mice after 14 days of TAC; D is the immunofluorescence detection of NE expression in the hearts of sham-operated mice and mice treated with TAC for 14 days; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0050] Figure 2 Detection of NE levels in the serum of NE knockout mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0051] Figure 3 NE knockout improves cardiac function and cardiac hypertrophy in TAC-induced heart failure mice, where A is the echocardiogram, heart size and WGA staining of each group; B is the myocardial survival rate of each group of mice; C is the heart-to-body weight ratio of each group of mice; D is the heart-to-tibia length ratio of each group of mice; E is the ejection fraction of each group of mice; F is the left ventricular short-axis shortening rate of each group of mice; G is the cross-sectional area of ​​myocardial cells of each group of mice; (HJ) are the mRNA expression levels of ANP, BNP and β-MHC in myocardial tissue of each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0052] Figure 4NE knockout improves myocardial fibrosis in TAC-induced heart failure mice, where A is Masson staining, Picrosirius red staining and HE staining; B is Western blotting to detect the expression levels of Collagen Ⅰ and TGF-β proteins in the myocardial tissue of each group of mice; C is the semi-quantitative analysis of Collagen Ⅰ protein (difference fold); D is the semi-quantitative analysis of TGF-β protein (difference fold); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0053] Figure 5 NE knockout improved cardiac inflammation in TAC-induced heart failure mice, where A is the mRNA expression level of IL-1β in myocardial tissue of each group of mice; B is the mRNA expression level of IL-6 in myocardial tissue of each group of mice; C is the mRNA expression level of TNF-α in myocardial tissue of each group of mice; D is the mRNA expression level of MCP1 in myocardial tissue of each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0054] Figure 6 The NE expression level in the serum of mice overexpressing NE was significantly increased; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0055] Figure 7 NE overexpression aggravates the decreased cardiac function and cardiac hypertrophy in TAC-induced heart failure mice, where A is the echocardiogram, heart size and WGA staining of each group; B is the myocardial survival rate of each group of mice; C is the heart-to-body weight ratio of each group of mice; D is the heart-to-tibia length ratio of each group of mice; E is the ejection fraction of each group of mice; F is the left ventricular short-axis shortening rate of each group of mice; G is the cross-sectional area of ​​myocardial cells of each group of mice; (HJ) are the mRNA expression levels of ANP, BNP and β-MHC in myocardial tissue of each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0056] Figure 8NE overexpression aggravates myocardial fibrosis in TAC-induced heart failure mice, where A is Masson staining, Picrosirius red staining and HE staining; B is Western blotting to detect the expression levels of Collagen Ⅰ and TGF-β proteins in the myocardial tissue of each group of mice; C is the semi-quantitative analysis of Collagen Ⅰ protein in B (difference fold); D is the semi-quantitative analysis of TGF-β protein (difference fold); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0057] Fig. 9 NE overexpression aggravated chronic cardiac inflammation in TAC-induced heart failure mice, where A was the mRNA expression level of IL-1β in the myocardial tissue of each group of mice; B was the mRNA expression level of IL-6 in the myocardial tissue of each group of mice; C was the mRNA expression level of TNF-α in the myocardial tissue of each group of mice; D was the mRNA expression level of MCP1 in the myocardial tissue of each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0058] Fig.10 The effects of different concentrations of sivelestat sodium on the cardiac function of mice with TAC-induced heart failure, where A is the echocardiogram of each group; B is the left ventricular ejection fraction of each group of mice; C is the left ventricular fractional shortening of each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0059] Fig.11 The figures show the effects of different concentrations of sivelestat sodium on the heart size of mice in each group, where A is the heart size of mice in each group; B is the heart-to-body weight ratio of mice in each group; C is the heart-to-tibia length ratio of mice in each group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0060] Fig.12 The effects of different concentrations of sivelestat sodium on the size of cardiac myocardial cells in each group of mice, where A is WGA staining; B is the statistics of the cross-sectional area of ​​cardiac myocardial cells in each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0061] Fig.13 The effects of different concentrations of sivelestat sodium on myocardial fibrosis in each group of mice were shown, including Masson staining, Picrosirius red staining and HE staining.

[0062] Fig.14 Figure 3 Effects of different concentrations of sivelestat sodium on myocardial fibrosis-related proteins in each group of mice. A is the expression levels of Collagen Ⅰ and TGF-β proteins in myocardial tissue of each group of mice detected by Western blotting; B is the semi-quantitative analysis of Collagen Ⅰ protein (difference fold); C is the semi-quantitative analysis of TGF-β protein (difference fold); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0063] Fig.15 The figures are the effects of different concentrations of sivelestat sodium on the liver and kidney function-related indices of mice in each group, among which, A is the serum AST level of mice in each group; B is the serum SER level of mice in each group; C is the serum ALT level of mice in each group; D is the serum BUN level of mice in each group; E is the serum UA level of mice in each group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0064] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0065] Example 1 Study on the expression of NE in the myocardium of TAC-induced heart failure mouse model

[0066] Western B1ot, Elisa and immunofluorescence were used to detect the expression level of NE in the myocardium of TAC-induced heart failure mice.

[0067] 1.1 Selection and grouping of experimental mice

[0068] 8-10 week old SPF C57BL / 6J background mice and WT mice of the same background were purchased from Saiye Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). They were fed adaptively for 1 week before the start of the study. All mice were raised in an SPF laboratory animal center, with room temperature maintained at 22±1°C, humidity maintained at 50±10%, artificial cycle lighting, 12 hours of light and 12 hours of darkness per day, and free access to food and water.

[0069] The experimental mice were randomly divided into a sham operation group and a TAC-induced heart failure group. The TAC-induced heart failure group was further divided into four groups at 3 days, 14 days, 28 days and 56 days after surgery, with 6 mice in each group. The mice in the TAC-induced heart failure group underwent TAC surgery, while the mice in the sham operation group underwent the same surgery but without ligation.

[0070] 1.2 Construction of TAC-induced heart failure mouse model

[0071] The mice in the TAC-induced heart failure group underwent TAC surgery to establish a mouse model of TAC-induced heart failure. The specific operation steps were as follows: the mice were anesthetized by intraperitoneal injection of tribromoethanol. When the mice were deeply anesthetized, the skin in front of the trachea was incised and the muscle tissue was bluntly separated. The second rib was gently lifted and incised, pulled to both sides, and the aortic arch was fully exposed. The transverse aortic constriction between the right innominate and left common carotid arteries was tightened and ligated with a 26-gauge needle using 6-0 nylon sutures, and then the sternum and skin were sutured. Doppler echocardiography showed that the transaortic transection velocity was greater than 4m / s, confirming the success of the TAC surgery. The mice were transferred to a heating pad and closely monitored. All operators and analysts were blindly randomized to avoid mouse genotype preference. The mice in the sham operation group underwent the same surgical steps, but no ligation was performed.

[0072] 1.3 Western blotting and enzyme-linked immunosorbent assay (ELISA) detection of NE expression in the serum of TAC-induced heart failure mice

[0073] (1) Western blotting

[0074] The specific steps of using western blot to detect the expression of NE in the serum of each group of mice are as follows:

[0075] 1) Protein extraction and quantification

[0076] The protein lysate was added with protease inhibitors and phosphatase inhibitors at a ratio of 100:1 to prepare a protein extraction solution, and the mixture was mixed and placed on ice for later use.

[0077] Animal protein extraction: Take a small piece of tissue and put it into a centrifuge tube. Add 1 mL of protein extract for every 0.1 g of tissue, and then use a homogenizer to homogenize until the tissue fragments are micronized. Then put the tube on ice and let it lyse on ice for 20 minutes. Place the centrifuge tube in a centrifuge at 4°C and centrifuge at 12,000 g for 15 minutes. Then transfer the supernatant to a new centrifuge tube; take an appropriate amount of supernatant for protein concentration determination, and store the rest in a -80°C refrigerator.

[0078] Dilute the standard protein in the kit to a concentration of 0.5 mg / μL; calculate the number of standards and samples, and prepare an appropriate amount of BCA working solution for protein concentration determination; add 0, 2, 4, 8, 16, and 20 μL of the diluted standard protein to a 96-well plate, and then add different volumes of pure water to each well to make up to 20 μL; add the protein sample to be tested to the 96-well plate, and add pure water to make up to 20 μL; add 200 μL to each well. BCA working solution, incubate at 37℃ for 30 minutes; measure the absorbance at 562nm wavelength on a microplate reader; use the concentration of standard protein and the corresponding absorbance to make a standard curve, and calculate the protein concentration in the test sample according to the standard curve; according to the concentration of the sample test, configure a 20μL system (5×Loading buffer 4μL, and the remaining volume is supplemented with protein lysis buffer) according to each sample containing 20μg protein; heat at 100℃ for 10 minutes to denature the protein; the denatured protein is cooled on ice and directly loaded for electrophoresis, or stored in a -20℃ refrigerator for later use.

[0079] 2) SDS-PAGE

[0080] (1) Install the gel making device with a clean glass plate, prepare 10% separation gel solution and add it to the gel making tank, and let it stand at room temperature. After the separation gel solidifies, prepare 5% concentrated gel solution and quickly add it to the gel making tank. After filling, immediately insert the comb vertically, pay attention to avoid bubbles, and let it stand at room temperature again to wait for solidification. The ratio of separation gel and concentrated gel is as follows:

[0081]

[0082] Sample loading: Take the gel out of the gel tank and put it into the electrophoresis tank. Pour 1× electrophoresis buffer until the surface of the gel is covered. Slowly pull out the comb and add the molecular weight marker Maker and protein sample to each loading well in order;

[0083] Electrophoresis: Turn on the transmitter device switch and perform initial concentrated gel electrophoresis at a voltage of 60 V. After the sample enters the separation gel layer and the molecular weight markers are dispersed, adjust the voltage to 120 V and continue constant voltage electrophoresis. Stop the electrophoresis when the bromophenol blue reaches the bottom of the gel;

[0084] Transfer: Prepare a PVDF membrane (polyvinylidene fluoride membrane) of appropriate size and activate it in methanol before transfer. Place the sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad in the black and white folder in sequence. After fixing them, put the transfer folder into the electrotransfer tank. Under low temperature conditions, set the constant current to 300mA and the continuous transfer time to 60-90 minutes (the transfer time depends on the molecular weight of the target protein);

[0085] Membrane blocking: After the transfer is completed, the PVDF membrane is placed in a TBST solution containing 10% skim milk and shaken on a shaker for 60 minutes for blocking. The PVDF membrane is then washed three times in TBST buffer for 10 minutes each time;

[0086] Incubate with primary antibody: Cut out the corresponding band according to the molecular weight of the target protein. Add the diluted primary antibody (Neutrophil Elastase, Abcam, ab310335; Anti-LY6G, Servicebio, GB11229; Collagen I, Abcam, ab260043; TGF-β, Abcam, ab215715; Anti-GAPDH, Proteintech, HRP-60004), place on a shaker, and incubate overnight at 4°C. The next day, aspirate and recover the primary antibody, add TBST buffer and wash 3 times, 10 minutes each time;

[0087] Incubation with secondary antibody: The strips were placed in the diluted secondary antibody (Invitrogen, 31444, 31460), incubated on a shaker at room temperature for 1 hour, and then washed again with TBST buffer for 3 times;

[0088] Imaging exposure: incubate the strips with the prepared exposure solution, and then use the gel imaging system to collect images;

[0089] Image analysis: ImagJ software was used to analyze the grayscale values ​​of the target protein and the internal reference protein for protein level expression analysis.

[0090] (2) ELISA test

[0091] Blood was collected from the tail vein of the mouse and placed in a serum separation tube. After the clot was formed, the blood sample was centrifuged at 5000rpm and serum was collected after 10 minutes. The serum and sample diluent were diluted at a ratio of 1:9 and used for detection. The mouse heart was cut, and an appropriate amount of tissue homogenate was added. After high-speed homogenization, it was placed on ice for 20 minutes. Centrifuged at 4℃ / 18000g for 20 minutes, the precipitate was discarded, and the supernatant was used to detect the protein concentration for later use.

[0092] Take out the ELISA plate pre-coated with NE and the required reagents (Abcam, ab79962) and equilibrate to room temperature. First, add 50uL of diluted serum or tissue samples, standards and ddH2O (blank) to the wells, then add 50uL of antibody mixture, seal the wells, and incubate at room temperature for 1h on a plate shaker at 400rpm. Wash the wells 3 times with 350uL of wash buffer (10×Wash Buffer diluted in ddH2O), and gently tap the plate on a clean paper towel to remove excess liquid after each wash. Add 100μL of TMB solution to each well and incubate in the dark on a plate shaker set at 400rpm for 15min. Add 100μL of stop solution to each well. Shake the plate on a plate shaker for 1min to mix, record the absorbance value at 450nm, draw a standard curve graph and calculate the concentration of NE in the sample.

[0093] (3) Immunofluorescence detection

[0094] 1) Paraffin embedding and sectioning

[0095] Fixation: The collected heart samples were fixed in 4% paraformaldehyde solution at room temperature for more than 24 hours;

[0096] Dehydration: Place the fixed samples into the marked paraffin embedding box for dehydration. The dehydration process is as follows: 70% ethanol for 60 minutes → 80% ethanol for 30 minutes → 95% ethanol for 30 minutes → anhydrous ethanol I for 30 minutes → anhydrous ethanol II for 30 minutes;

[0097] Clearing: The dehydrated samples were cleared according to the following process: xylene I 60 min → xylene II 60 min;

[0098] Wax immersion: Soak the sample in melted soft wax (melting point 52-54°C) for 90 minutes, then soak it in hard wax (melting point 58-60°C) for 90 minutes;

[0099] Embedding: Pour a small amount of paraffin wax into the bottom of the embedding mold box, then take out the sample from the tissue embedding box and put it into the mold box. After the paraffin wax in the mold has cooled and solidified slightly, place the pre-marked embedding box on top of the mold. Then add liquid paraffin until the sample is completely submerged in the paraffin wax. Then transfer the embedding mold box containing the sample to the cooling table. After the paraffin block has completely cooled and solidified, take out the solidified wax block from the mold box.

[0100] Slicing: Place the wax block on the table of the slicer, adjust the angle between the wax block and the blade, and ensure that the paraffin block is stable and flat. Set the slice thickness of the slicer to 20μm for trimming. After trimming until the exposed sample can be clearly seen, set the slice thickness of the slicer to 5μm for slicing;

[0101] Spreading: Use tweezers to move the cut paraffin slices into a water bath at 43°C to allow them to fully spread out;

[0102] Baking slices: After the slices are fully unfolded, use a dry glass slide to pick up the slices and mark them, pre-bake them on a slice baking machine at 65°C for 1 hour, and then place them in a slice box for later use.

[0103] 2) After dewaxing, immerse the sections in antigen retrieval solution and perform antigen retrieval in a microwave oven at 95°C for 12 minutes.

[0104] 3) After the repair solution has cooled to room temperature, wash the sections in 1× PBS three times, 5 minutes each time;

[0105] 4) Wipe the PBS remaining on the slices with absorbent paper, draw a circle around each heart tissue with an oil pen, add an appropriate amount of 3% BSA to each tissue, and block at 37°C for 30 minutes;

[0106] 5) Shake off BSA, place the slices in a wet box, add NE primary antibody (Abcam, ab310335) and incubate overnight at 4°C;

[0107] 6) The next day, the sections were washed three times in 1× PBS for 5 minutes each time, and then Ly6G primary antibody (Wuhan Sewell Biotechnology Co., Ltd., GB11229) was added and incubated at 4°C for 8 hours;

[0108] 7) Wash with PBS 3 times, 5 minutes each time;

[0109] 8) Incubate with fluorescent secondary antibodies (abcam, ab150080, ab150077) at room temperature for 2 hours, protecting from light;

[0110] 9) After the secondary antibody incubation, wash with PBS three times, 5 minutes each time, and keep away from light;

[0111] 10) Add DAPI mounting medium, cover the slide with a coverslip, and store in a 4°C refrigerator away from light;

[0112] 11) Observe under a microscope and collect images.

[0113] 1.4 Identification of NE knockout mice

[0114] (1) Direct PCR solution and proteinase K were prepared into rat tail lysis solution at a ratio of 100:1;

[0115] (2) Cut 1-2 mm of the mouse tail for gene identification, put it into the prepared mouse tail lysis solution, first in a 55°C water bath overnight, and then in an 85°C water bath for 1 hour;

[0116] (3) Establish a 20 μL PCR reaction system for PCR. The PCR reaction system is as follows:

[0117]

[0118]

[0119] (4) Forward primer SEQ ID NO.1

[0120] (F1):5'-GGATGTCTGCTGAGTTTTCATTGG-3';

[0121] Reverse primer SEQ ID NO.2

[0122] (R1):5'-GAACATGGGATTTAGGAACAGAAACC-3';

[0123] Internal control PCR primer SEQ ID NO.3

[0124] F:5'-GCAGAAGAGGACAGATACATTCAT-3';

[0125] Internal control PCR primer SEQ ID NO.4

[0126] R:5'-CCTACTGAAGAATCTATCCCACAG-3'.

[0127] (5) Mix 3 mg of agarose and 150 mL of 0.5× TBE solution and place in a microwave oven, heating on high heat until dissolved. After cooling at room temperature for 5 minutes, add 10 μL of green fluorescent nucleic acid dye, shake gently, pour into the gel casting mold with a comb inserted, and let stand at room temperature for 30 minutes;

[0128] (6) Install the electrophoresis apparatus, pour in 0.5× TBE electrophoresis solution, slowly pull out the comb, and add the molecular weight marker Maeker and sample to each well in sequence;

[0129] (7) Electrophoresis was performed at 140 V for about 25 minutes;

[0130] (8) Nucleic acid exposure instrument exposure and color development;

[0131] (9) Band interpretation: The single band at 413 bp is for NE KO mice, the single band at 455 bp is for WT mice, and the double bands at 413 bp and 455 bp are for heterozygous mice.

[0132] 1.5 Experimental animal grouping and model construction

[0133] Twenty male NE KO mice and 20 WT mice of the same background at the age of 8 weeks were selected and randomly divided into 4 groups according to the random number table method, with 10 mice in each group. The groups were divided as follows:

[0134] (1) WT sham operation group (WT-sham operation)

[0135] (2) WT TAC model group (WT-TAC)

[0136] (3) NE KO control group (NE KO-sham operation)

[0137] (4) NE4 KO TAC model group (NE KO-TAC)

[0138] Thirty 6-week-old C57BL / 6J male WT mice were randomly divided into three groups according to the random number table method, with 12 mice in each group. The groups were as follows:

[0139] (1) AAV-GFP sham operation group (AAV-GFP-sham operation)

[0140] (2) AAF-GFP model group (AAV-GFP-TAC)

[0141] (3) AAF-NE model group (AAV-NE-TAC)

[0142] The AAV-GFP-sham and AAV-GFP-TAC mice were injected with AAV-GFP control plasmid in the tail vein 2 weeks before TAC, and the AAV-NE-TAC group mice were injected with AAV9 vector-encapsulated NE overexpression plasmid in the tail vein 2 weeks before TAC. Two weeks later, the serum NE level of mice was detected by ELISA to verify the effect of viral transfection.

[0143] 1.6 Ultrasound examination of mouse heart

[0144] The cardiac function of mice after TAC surgery was evaluated using the Vevo2100 small animal ultrasound imaging system. To reduce stimulation to the mice, the hair on the chest of the mice was removed with a depilatory cream one day before the ultrasound test to fully expose the skin of the heart area on the chest. One week after TAC surgery, in the awake state of the mice, the left ventricular short-axis B-mode and M-mode echocardiograms of the mice were obtained using the MS400C ultrasensitive probe for subsequent analysis. The detection indicators of mouse cardiac ultrasound include: heart rate, left ventricular anterior and posterior wall thickness during systole and diastole, left ventricular end-systolic and end-diastolic volumes, ejection fraction and shortening fraction. The average values ​​of 5 consecutive cardiac cycles were selected for the final data statistics.

[0145] 1.7 Hematoxylin-eosin (H&E) staining

[0146] (1) Dewaxing of paraffin sections to water, the steps are as follows: xylene I 30 min → xylene II 30 min → anhydrous ethanol I 5 min → anhydrous ethanol II 5 min → 95% ethanol 5 min → 80% ethanol 5 min → distilled water 5 min;

[0147] (2) Hematoxylin staining for 5 minutes and rinsing with distilled water for 1 minute;

[0148] (3) Differentiate with 1% hydrochloric acid alcohol solution for 5 seconds, rinse with distilled water for 1 minute, and soak in tap water for 10 minutes;

[0149] (4) Eosin staining for 5 minutes (the exact time depends on the degree of staining of the tissue);

[0150] (5) The stained sections were dehydrated and transparentized with alcohol, with the following steps: 95% ethanol for 30 seconds → anhydrous ethanol I minute → anhydrous ethanol II for 1 minute → xylene I for 15 minutes → xylene II for 30 minutes;

[0151] (6) Seal the slices with neutral gum and place them in a fume hood to dry;

[0152] (7) Observe under a microscope and collect images.

[0153] 1.8 Sirius red staining

[0154] (1) Dewaxing of paraffin sections to water;

[0155] (2) Wipe away the water around the tissue and stain with Sirius red for 1 hour;

[0156] (3) Soak the stained sections in anhydrous ethanol I and anhydrous ethanol II for 1 minute each;

[0157] (4) The sections were transparentized as follows: xylene I for 15 min → xylene II for 30 min;

[0158] (5) Seal the slices with neutral gum and place them in a fume hood to dry;

[0159] (6) Observe under a microscope and collect images.

[0160] Wheat germ agglutinin (WGA) fluorescence staining

[0161] (1) Dewaxing of paraffin sections to water;

[0162] (2) Place the sections in 1× Tris-EDTA antigen retrieval solution and boil at high temperature for 5 minutes in a microwave oven for antigen retrieval;

[0163] (3) After the repair solution has cooled naturally to room temperature, the sections are washed three times in 1× PBS, each time for 5 minutes;

[0164] (4) Wipe the remaining PBS on the slices with absorbent paper, draw a circle around each heart tissue with a pen, add an appropriate amount of 3% BSA to each tissue, and block at 37°C for 30 minutes;

[0165] (5) After removing BSA, use WGA working solution for drop staining and incubate at 37°C in the dark for 1 hour;

[0166] (6) Wash with PBS three times, 5 minutes each time;

[0167] (7) Add DAPI mounting medium, cover the slide with a coverslip, and store in a 4°C refrigerator away from light;

[0168] (8) Observe under a microscope and collect images.

[0169] 1.9 Real-time quantitative fluorescence PCR

[0170] Extraction of total RNA

[0171] (1) Sample preparation: Take out mouse heart tissue samples from the -80°C freezer, cut them into rice-sized pieces and place them in a 1.5 mL EP tube;

[0172] (2) Tissue lysis: Add 1 mL of Trizol to each tube and use an electric homogenizer to homogenize the tissue at high speed to destroy the cell membrane and nuclear membrane and release nucleic acids;

[0173] (3) RNA isolation: Place the homogenized lysate on ice, add 200 μL of chloroform, cover the tube tightly, shake up and down vigorously to mix, let stand on ice for 15 minutes, and then centrifuge at 12,000 g for 15 minutes at 4°C;

[0174] (4) DNA and protein removal: Take out the EP tube, transfer the upper liquid phase to a new 1.5 mL EP tube, add 500 μL of isopropanol to each EP tube, mix by inverting, let stand on ice for 10 minutes, and then centrifuge at 12000g for 15 minutes at 4°C. After centrifugation, a small amount of white precipitate can be seen at the bottom of the tube, and the supernatant is carefully discarded;

[0175] (5) RNA washing: Add 1 mL of 75% ethanol to each tube to wash the RNA, and centrifuge at 12,000 g for 15 min at 4°C;

[0176] (6) RNA drying: Carefully discard the supernatant, use a pipette to remove as much residual ethanol solution as possible, then turn the EP tube upside down and leave it at room temperature to dry the precipitate for 20 minutes;

[0177] (7) RNA dissolution: add appropriate amount of DEPC water to dissolve;

[0178] (8) Measurement of RNA concentration: Use Nanodrop spectrophotometer to measure RNA concentration and purity and store at -80°C for long-term storage.

[0179] RNA reverse transcription to synthesize cDNA

[0180] (1) Thaw RNA and all reagents on ice;

[0181] (2) Add the sample according to the above system and mix well. Set the reaction conditions: 42°C for 60 minutes, 70°C for 5 minutes, and perform reverse transcription to obtain cDNA.

[0182] Real-time quantitative PCR (qRT-PCR)

[0183] (1) Take an appropriate amount of 200 μL eight-row centrifuge tubes according to the number of samples, set up two duplicate wells for each sample, add the following reagents to each well to prepare 10 μL of the reaction system, mix well and centrifuge the liquid to the bottom of the tube. Reaction program: 95℃ for 30 seconds; 95℃ for 5 seconds, 60℃ for 34 seconds, 40 cycles; 95℃ for 15 seconds, 60℃ for 1 minute;

[0184] The PCR reaction system is shown in the table below:

[0185] Reagents dose SYBR Green premix (Takara, RR820A) 5μL Primer (F+R) 0.5μL cDNA 1μL ddH2O 3.3μL ROX II 0.2μL total 10μL

[0186] The primer information is as follows:

[0187]

[0188]

[0189] (2) Data analysis: GAPDH was used as the internal reference gene and the expression level of the target gene was 2 ﹣△△Ct Formula calculation.

[0190] 1.10 Results and Analysis

[0191] 1.10.1 TAC-induced heart failure causes an increase in serum and cardiac infiltrating neutrophil elastase NE

[0192] TAC surgery is used to establish a heart failure model in mice. This surgery is a classic method for establishing myocardial hypertrophy and heart failure models in mice, and has the characteristics of simple surgical operation, low mortality, and high repeatability. In the present invention, in order to minimize the interference of relevant factors such as weight, gender, and age, all mice used are male, and their age and weight are very similar. Western Blotting was used to detect the changes in NE expression in serum at different time points after TAC. The results are shown in Figure 2. Figure 1A in the figure shows that NE protein expression increased significantly 14 days after TAC. Therefore, all subsequent experiments were performed at 14 days after TAC. The ELISA method was used to detect the changes in NE content in the serum of mice after TAC. The results showed that compared with the sham operation group, TAC 14 days later could significantly increase the NE content in the serum ( Figure 1 B in the figure). Similar conclusions were obtained by using Elisa to detect the NE content in the heart tissue of mice after TAC ( Figure 1 C in the figure). In addition, immunofluorescence was used to detect the changes in neutrophils and NE in the heart tissue of the sham operation group and after TAC. The results showed that compared with the sham operation group, neutrophil infiltration in the heart tissue increased significantly 14 days after TAC, and the NE fluorescence intensity increased significantly. The above results show that 14 days after TAC, the NE expression level in serum and heart tissue was significantly upregulated, and there was significant neutrophil infiltration in the heart.

[0193] 1.10.2NE knockout significantly improves cardiac function and myocardial hypertrophy in TAC-induced heart failure mice

[0194] After observing that NE protein expression levels increased significantly 14 days after TAC surgery, it was suggested that NE may be involved in the development of TAC-induced heart failure. Therefore, the present invention uses NE gene knockout mice (NE KO) to further explore the specific role of NE increase in TAC-induced heart failure. Before TAC surgery, the NE content in serum was first detected to determine whether NE KO mice were successfully constructed. The results are as follows Figure 2 , NE expression in the serum of NE KO mice was almost completely confirmed, indicating that NE KO mice were successfully constructed.

[0195] Fourteen days after TAC surgery, the mortality of mice in the WT-sham, NE KO-sham, WT-TAC, and NE KO-TAC groups was recorded. Figure 3 Figure A shows that the survival rate of NE KO mice after TAC was significantly increased compared with wild-type TAC (WT-TAC). The heart-to-body weight ratio and heart-to-tibia length ratio were also recorded. Figure 3 As shown in B and C, compared with WT mice, NE KO mice had significantly reduced TAC-induced increases in heart-to-body weight ratio and heart-to-tibia length ratio.

[0196] The Vevo3100 system with MS400C probe was used to perform echocardiography on mice after TAC induction. The ventricular diameter and wall thickness during systole and diastole were determined by measuring the two-dimensional muscle pattern at the level of the papillary muscles, and the ejection fraction (EF) and short-axis contraction fraction (FS) were calculated. Figure 3As shown in DF, compared with WT mice, NE KO mice were able to significantly increase EF and FS after TAC, indicating that NE KO can improve the cardiac function of mice after TAC. WGA staining was performed on the heart sections of mice in each group, and the results showed that 14 days after TAC, the cross-sectional area of ​​myocardial cells in NE KO mice was significantly lower than that in WT mice ( Figure 3 In addition, 14 days after TAC surgery, the mRNA levels of myocardial hypertrophy-related maker ANP, BNP, and β-MHC genes in the myocardial tissue of NE KO mice were significantly lower than those in the WT control group ( Figure 3 in HJ).

[0197] 1.10.3NE knockout significantly improves myocardial fibrosis and inflammation in TAC-induced heart failure mice

[0198] 14 days after TAC surgery, the hearts of mice in the WT-sham operation group, NE KO-sham operation group, WT-TAC group, and NE KO-TAC group were stained with Masson, Sirius red (PSR), and HE. The results of Masson, Sirius red (PSR), and HE staining of mouse hearts are shown in Figure 4 As shown in Figure A, WT mice had significantly increased myocardial fibrosis 14 days after TAC, while NE KO mice were able to significantly alleviate TAC-mediated myocardial fibrosis. In addition, after 14 days of TAC, the expression levels of CollagenⅠ and TGF-β proteins in myocardial tissue fibrosis-related proteins in NE KO mice were significantly lower than those in WT mice ( Figure 4 BC in the figure indicates that NE KO can significantly alleviate TAC-induced myocardial fibrosis. The q-PCR method was used to detect the mRNA expression levels of inflammatory factors in myocardial cells of each group of mice. Figure 5 As shown in the figure, 14 days after TAC surgery in WT mice, the expression levels of IL-1β, IL-6, TNF-α and MCP1 gene mRNA in cardiomyocytes increased significantly, while NE KO mice were able to significantly reduce the increase in the expression levels of IL-1β, IL-6, TNF-α and MCP1 gene mRNA in cardiomyocytes caused by TAC. The above results show that NE knockout significantly improves the cardiac function, cardiac hypertrophy, cardiac inflammation and myocardial fibrosis in mice with TAC-induced heart failure.

[0199] 1.10.4NE overexpression aggravates cardiac dysfunction and myocardial hypertrophy in TAC-induced heart failure mice

[0200] To further explore the role of NE in TAC-induced heart failure, WT mice were injected with adeno-associated virus (AAV)-mediated NE (AAV9-NE) overexpression via tail vein injection, and AAV-mediated green fluorescent protein (GFP) was used as a control. As expected, AAV9-NE was able to significantly increase the level of NE in serum compared with AAV-GFP ( Figure 6 ). The effects of AAV9-NE on cardiac function and myocardial hypertrophy in TAC-induced heart failure mice were further studied. Compared with AAV-GFP-TAC, AAV-mediated NE overexpression further increased the mortality rate, heart-to-body weight ratio, and heart-to-tibia length ratio in TAC-induced heart failure mice ( Figure 7 Meanwhile, compared with AAV-GFP-TAC heart failure mice, AAV9-NE aggravated TAC-induced cardiac dysfunction, as shown by further decreases in EF and FS ( Figure 7 DF in Figure 5). WGA staining results showed that AAV-NE treatment further aggravated TAC-induced cardiac hypertrophy ( Figure 7 This result was also confirmed by the further increase in the expression levels of myocardial hypertrophy makers such as ANP, BNP, and β-MHC gene mRNA ( Figure 7 in HJ).

[0201] 1.10.5NE overexpression exacerbates myocardial fibrosis and inflammation in TAC-induced heart failure mice

[0202] Depend on Figure 8 As shown in A, Masson staining, PSR staining and HE staining showed that 14 days after TAC, myocardial fibrosis in the AAV-NE-TAC group was further aggravated compared with the AAV-GFP-TAC group. Western blotting was used to detect the expression levels of myocardial fibrosis markers Collagen I and TGF-β protein. Figure 8 As shown in B, compared with the AAV-GFP-TAC group, the expression levels of Collagen Ⅰ and TGF-β proteins in the AAV-NE-TAC group were further increased, indicating that fibrosis was aggravated. Figure 8 C in Figure 8 Semi-quantitative results of B in .

[0203] Fig. 9 The results showed that compared with the AAV-GFP-TAC group, the mRNA expression levels of IL-1β, IL-6, TNF-α, and MCP1 genes in cardiomyocytes in the AAV-NE-TAC group were further increased, and cardiomyocyte inflammation was aggravated.

[0204] Example 2 New Uses of Sivelestat

[0205] 2.1 Selection of experimental mice

[0206] 8-10 week old SPF C57BL / 6J background mice and WT mice of the same background were purchased from Saiye Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). They were fed adaptively for 1 week before the start of the study. All mice were raised in an SPF laboratory animal center, with room temperature maintained at 22±1°C, humidity maintained at 50±10%, artificial cycle lighting, 12 hours of light and 12 hours of darkness per day, and free access to food and water.

[0207] 2.2 Construction of TAC-induced heart failure mouse model

[0208] The mice in the TAC-induced heart failure group underwent TAC surgery to establish a mouse model of TAC-induced heart failure. The specific operation steps were as follows: the mice were anesthetized by intraperitoneal injection of tribromoethanol. When the mice were deeply anesthetized, the skin in front of the trachea was incised and the muscle tissue was bluntly separated. The second rib was gently lifted and incised, pulled to both sides, and the aortic arch was fully exposed. The 26-gauge needle was tightened and ligated with a 6-0 nylon suture to complete the transverse aortic constriction between the right innominate and left common carotid arteries, and then the sternum and skin were sutured. Doppler echocardiography showed that the transaortic transection velocity was greater than 4m / s, confirming the success of the TAC surgery. The mice were transferred to a heating pad and closely monitored. All operators and analysts were blindly randomized to avoid mouse genotype preference. The mice in the sham operation group underwent the same surgical steps, but no ligation was performed.

[0209] 2.3 Sivelestat sodium (hereinafter referred to as Sive) was prepared into a mother solution with dimethyl sulfoxide (DMSO) as solvent, and Sivelestat sodium: PEG300: Tween-20: 0.9% NaCl were added in a ratio of 1:8:1:10 in this order. Each time, the mixture was mixed to ensure that the liquid was clear before proceeding to the next step. One week after TAC surgery, the mice were evaluated for arch blood flow by small animal ultrasound. The mice with successful construction were randomly divided into five groups: sham surgery + solvent (group a), TAC + solvent (group b), TAC + Sive 12.5 mg / kg (group c), TAC + Sive 25 mg / kg (group d), and TAC + Sive 50 mg / kg (group e). The solvent treatment (i.e., DMSO: PEG300: Tween-20: 0.9% NaCl is 1:8:1:10, 200 μL / mouse) was followed by intraperitoneal injection, once a day, for 7 consecutive weeks. In the 8th week, the cardiac function of the mice was evaluated by small animal ultrasound. After the evaluation, the mice in each group were euthanized, and the samples were collected, weighed, and subsequent experiments were performed.

[0210] 2.4 Ultrasound detection of mouse heart

[0211] The cardiac function of mice after TAC surgery was evaluated using the Vevo2100 small animal ultrasound imaging system. To reduce stimulation to the mice, the hair on the chest of the mice was removed with a depilatory cream one day before the ultrasound test to fully expose the skin of the heart area on the chest. One week after TAC surgery, in the awake state of the mice, the left ventricular short-axis B-mode and M-mode echocardiograms of the mice were obtained using the MS400C ultrasensitive probe for subsequent analysis. The detection indicators of mouse cardiac ultrasound include: heart rate, left ventricular anterior and posterior wall thickness during systole and diastole, left ventricular end-systolic and end-diastolic volumes, ejection fraction and shortening fraction. The average values ​​of 5 consecutive cardiac cycles were selected for the final data statistics.

[0212] 2.5 Wheat germ agglutinin (WGA) fluorescence staining

[0213] The method is the same as in Example 1.

[0214] 2.6 Sirius red staining

[0215] The method is the same as in Example 1.

[0216] 2.7 Hematoxylin-eosin (H&E) staining

[0217] The method is the same as in Example 1.

[0218] 2.8 Western Blot Detection

[0219] The method is the same as in Example 1.

[0220] 2.9 Results and Analysis

[0221] 2.9.1 Sivelestat sodium significantly alleviates cardiac function in mice with heart failure induced by TAC

[0222] All mice used in AC surgery were male and of similar age and weight. The Vevo3100 system with MS400C probe was used to perform echocardiography on mice after TAC induction. The systolic and diastolic ventricular diameters and wall thickness were determined by measuring the two-dimensional muscle pattern at the level of the papillary muscles, and the ejection fraction (EF) and short-axis contraction fraction (FS) were calculated. Fig.10 As shown in the figure, compared with the sham operation group, the cardiac function of mice after TAC was significantly deteriorated, which was manifested by a significant decrease in EF and FS, while treatment with different concentrations of sivelestat sodium could significantly increase EF and FS after TAC, indicating that sivelestat sodium can improve the cardiac function of mice after TAC surgery.

[0223] 2.9.2 Sivelestat sodium significantly improves cardiac hypertrophy in TAC-induced heart failure mice

[0224] Compared with the sham-operated group, the hearts of mice after TAC were significantly enlarged ( Fig.11 A), the heart-to-body weight ratio and heart-to-tibia length ratio increased significantly ( Fig.11 B, C), and treatment with different concentrations of sivelestat sodium significantly reduced TAC-induced heart size, heart-to-body weight ratio, and heart-to-tibia length. WGA staining results also showed that compared with the TAC group, the heart cross-sectional area was significantly reduced after treatment with different concentrations of sivelestat sodium ( Fig.12 A, B in it).

[0225] 2.9.3 Sivelestat sodium significantly improves cardiac tissue pathological changes in mice with TAC-induced heart failure

[0226] Compared with the sham-operated group, the myocardial arrangement of mice after TAC was disordered and myocardial fibrosis was significantly increased ( Fig.13 After treatment with different concentrations of sivelestat sodium, TAC-induced cardiac fibrosis was significantly reduced ( Fig.13 AC in).

[0227] 2.9.4 Sivelestat sodium significantly improved the expression levels of myocardial fibrosis-related proteins in heart tissue of TAC-induced heart failure mice

[0228] Compared with the sham operation group, the expression levels of myocardial fibrosis-related proteins Collagen Ⅰ and TGF-β in the heart tissue of mice after TAC were significantly increased, indicating that myocardial fibrosis occurred in the mice after TAC. Treatment with different concentrations of sivelestat sodium significantly reduced the increase in the expression levels of myocardial fibrosis-related proteins Collagen Ⅰ and TGF-β induced by TAC ( Fig.14 ).

[0229] 2.9.5 No adverse reactions occurred in the liver and kidney function of mice in each group at the supplementary dose of sivelestat sodium

[0230] Depend on Fig.15 It can be seen from the AEs in the experiment that the supplementary dose of sivelestat sodium did not affect the liver and kidney functions of the mice in each group.

Claims

1. Pharmaceutical use of substances that inhibit or silence the activity of neutrophil elastase in heart diseases.

2. The use according to claim 1, characterized in that The substance is sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative; and / or, ulinastatin or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof; and / or, flavonoid aglycones and their glycosides and methylated, acetylated and hydroxylated derivatives.

3. The use according to claim 2, characterized in that: The substance described is sivelestat.

4. The use according to any one of claims 1 to 3, characterized in that: The heart disease is coronary artery disease, arrhythmia, cardiomyopathy, heart failure, pericardial disease, hypertensive heart disease or cor pulmonale.

5. The use according to claim 4, characterized in that The heart disease is heart failure.

6. The use according to claim 5, characterized in that The heart disease is pressure overload-induced heart failure.

7. Pharmaceutical use of substances that inhibit or silence the expression of neutrophil elastase genes in heart diseases.

8. The use according to claim 7, characterized in that The substance is sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative; and / or, ulinastatin or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof; and / or, flavonoid aglycones and their glycosides and methylated, acetylated and hydroxylated derivatives.

9. The use according to claim 8, characterized in that The substance described is sivelestat.

10. The use according to claim 7, characterized in that The heart disease is coronary artery disease, arrhythmia, cardiomyopathy, heart failure, pericardial disease, hypertensive heart disease or cor pulmonale.

11. The use according to claim 10, characterized in that The heart disease is heart failure.

12. The use according to any one of claims 7 to 11, characterized in that: The heart disease is pressure overload-induced heart failure.

13. Use of sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative in the preparation of a medicament for treating heart disease.

14. The use according to claim 13, characterized in that The heart disease is coronary artery disease, arrhythmia, cardiomyopathy, heart failure, pericardial disease, hypertensive heart disease or cor pulmonale.

15. The use according to claim 14, characterized in that The heart disease is heart failure.

16. The use according to any one of claims 13 to 15, characterized in that: The heart disease is pressure overload-induced heart failure.

17. The use according to claim 16, characterized in that The medicine also includes pharmaceutically acceptable excipients.

18. The use according to claim 17, characterized in that The dosage form of the medicine is solution dosage form, emulsion form or powder form.

19. A pharmaceutical composition for treating heart disease, characterized in that: Contains a substance that inhibits or silences the expression of the ELANE gene.

20. The pharmaceutical composition according to claim 19, characterized in that The substance is sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative; Or, ulinastatin or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable derivative thereof; or, flavonoid aglycones and their glycosides and methylated, acetylated and hydroxylated derivatives.

21. The pharmaceutical composition according to claim 19, characterized in that The substance is sivelestat or its pharmaceutically acceptable salt or its pharmaceutically acceptable derivative.

22. The pharmaceutical composition according to any one of claims 19 to 21, characterized in that The substance described is sivelestat.

23. The pharmaceutical composition according to claim 20 further comprises a pharmaceutically acceptable excipient.

24. The use according to claim 23, characterized in that The dosage form of the pharmaceutical composition is solution dosage form, emulsion form or powder form.

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