Pharmaceutical applications of the ELANE gene as a target for heart failure and new uses of sivelestat

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the pharmaceutical use of the ELANE gene as a target for heart failure and a new use of sivelestat. The present invention can increase the ejection fraction in the heart failure stage, slow down myocardial cell apoptosis, and effectively restore cardiac function by overexpressing the ELANE gene in myocardial cells of pressure-load heart failure. It is clarified that ELANE can be used as a target gene in drug or gene therapy, and applied to the prevention, relief and / or treatment of pressure-load heart failure, providing a new strategy for the prevention and treatment of pressure-load heart failure. The present invention also provides a new use of sivelestat, which can significantly improve the cardiac function and myocardial fibrosis of mice with pressure-load-induced heart failure, is an effective drug for treating heart failure, and can be used to prepare drugs for treating heart failure.

Description

Technical Field

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

[0002] Heart failure (HF) is a complex, chronic clinical syndrome characterized by abnormal changes in cardiac structure and / or function due to various factors, resulting in impaired ventricular systolic pumping and / or diastolic filling function, leading to an inability of the heart to deliver sufficient blood volume for normal physiological function. The etiologies of HF are diverse, including ischemic cardiomyopathy, hypertensive heart disease, hereditary cardiomyopathies, and valvular heart disease. Ischemic cardiomyopathy, which causes primary myocardial damage, and hypertensive heart disease, which causes increased left ventricular afterload, are the two most common causes. In hypertensive heart disease, the pressure on the left ventricle increases due to increased peripheral circulatory resistance. To overcome the increased afterload and ensure normal pumping, the left ventricular myocardium undergoes compensatory growth, leading to pathological remodeling. However, as the disease progresses, the pathological remodeling worsens, and cardiac function gradually shifts from a compensated to a decompensated state, ultimately leading to HF. While the pathology of HF has been better understood and progress has been made in its treatment, HF is still a major concern. However, the precise underlying pathological mechanisms of cardiac pathological remodeling and heart failure remain to be further investigated.

[0003] Neutrophil elastase (ELANE, NE) is a serine protease encoded by the ELANE gene that primarily plays a 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 implicated in a variety of destructive and inflammatory diseases, including chronic and acute lung disease and atherosclerosis. Specifically, NE mRNA and protein are detected in human atherosclerotic plaques, and increased plasma NE activity is observed during the progression of atherosclerosis. Studies have also demonstrated a role for NE in atherosclerosis and injury-induced neointimal smooth muscle cell proliferation. However, whether NE participates in adaptive myocardial remodeling and regulation of myocardial function under conditions of cardiac pressure overload remains unknown.

[0004] Sivelestat is a marketed selective neutrophil elastase inhibitor and the first drug approved worldwide for the treatment of acute lung injury associated with systemic inflammatory response syndrome (SIRS). The daily dose for this disease is 4.8 mg / kg / day (0.2 mg / kg / h intravenously administered for 24 hours). Based on its highly selective inhibitory effect on neutrophil elastase, Sivelestat is expected to be used to treat a variety of diseases.

[0005] The use of sivelestat in treating heart diseases disclosed in the prior art is mainly related to sepsis-induced myocardial damage. There are no reports that sivelestat can be used to treat or alleviate other types of heart diseases, especially pressure-overload-induced heart failure. Summary of the Invention

[0006] To address the above issues, the present invention provides information on the pathophysiological significance of the ELANE gene or its encoded protein, NE, in pressure-overload heart failure and its use in the treatment of pressure-overload heart failure, with the goal of providing candidate targets for the development of heart failure therapeutics. Furthermore, the present invention provides information on the use of sivelestat or a pharmaceutically acceptable salt thereof in the preparation of a drug 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 treating 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 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 treating 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 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 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 ELANE gene expression.

[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 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 a pharmaceutically acceptable salt or a pharmaceutically acceptable derivative thereof.

[0038] Preferably, the substance is sivelestat.

[0039] Specifically, the pharmaceutical composition further 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 in 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-induced heart failure.

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

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

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

[0049] Figure 1 Aortic arch constriction (TAC)-induced heart failure increased neutrophil elastase (NE) levels in the serum and infiltrating hearts of mice. A shows the expression of NE protein in myocardial tissue of mice in each group detected by Western blotting; B shows the NE content in the blood of mice in different groups after 14 days of TAC; C shows the NE content in the hearts of mice in different groups after 14 days of TAC; D shows the NE expression in the hearts of mice in the sham-operated group and mice after 14 days of TAC detected by immunofluorescence; *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 improved cardiac function and cardiac hypertrophy in mice with TAC-induced heart failure. A shows the echocardiogram, heart size, and WGA staining of each group; B shows the myocardial survival rate of each group of mice; C shows the heart-to-body weight ratio of each group of mice; D shows the heart-to-tibia length ratio of each group of mice; E shows the ejection fraction of each group of mice; F shows the left ventricular fractional shortening of each group of mice; G shows the cross-sectional area of ​​myocardial cells of each group of mice; (HJ) shows 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 4Figure 3: NE knockout improves myocardial fibrosis in TAC-induced heart failure mice. A: Masson, Picrosirius red, and HE staining; B: Western blotting to detect the expression levels of Collagen I and TGF-β proteins in the myocardial tissue of each group of mice; C: semi-quantitative analysis of Collagen I protein (fold difference); D: semi-quantitative analysis of TGF-β protein (fold difference); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0053] Figure 5 Figure 3 shows that NE knockout improves cardiac inflammation in TAC-induced heart failure mice. A is the mRNA expression level of IL-1β in the myocardial tissue of each group of mice; B is the mRNA expression level of IL-6 in the myocardial tissue of each group of mice; C is the mRNA expression level of TNF-α in the myocardial tissue of each group of mice; D is 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.

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

[0055] Figure 7 Figure NE overexpression exacerbates the decreased cardiac function and cardiac hypertrophy in TAC-induced heart failure mice. A shows the echocardiogram, heart size, and WGA staining of each group; B shows the myocardial survival rate of each group of mice; C shows the heart-to-body weight ratio of each group of mice; D shows the heart-to-tibia length ratio of each group of mice; E shows the ejection fraction of each group of mice; F shows the left ventricular fractional shortening of each group of mice; G shows the cross-sectional area of ​​myocardial cells of each group of mice; (HJ) shows 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 8Figure 1: NE overexpression exacerbates myocardial fibrosis in TAC-induced heart failure mice. A: Masson, Picrosirius red, and HE staining. B: Western blotting analysis of the expression levels of Collagen I and TGF-β proteins in the myocardial tissues of mice in each group. C: Semi-quantitative analysis of Collagen I protein (fold difference) in B. D: Semi-quantitative analysis of TGF-β protein (fold difference). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0057] Figure 9 Figure 3. Overexpression of NE exacerbated chronic cardiac inflammation in TAC-induced heart failure mice. A is the mRNA expression level of IL-1β in the myocardial tissue of each group of mice; B is the mRNA expression level of IL-6 in the myocardial tissue of each group of mice; C is the mRNA expression level of TNF-α in the myocardial tissue of each group of mice; D is 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] Figure 10 The figures show the effects of different concentrations of sivelestat sodium on cardiac function in 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] Figure 11 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] Figure 12 The effects of different concentrations of sivelestat sodium on the size of cardiac myocytes in each group of mice, where A is WGA staining; B is the statistics of the cross-sectional area of ​​cardiac myocytes in each group of mice; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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

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

[0063] Figure 15 Figure 3 Effects of different concentrations of sivelestat sodium on liver and kidney function-related indicators of mice in each group, where 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 with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all 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 blotting, 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 mice of C57BL / 6J background and WT mice of the same background were purchased from Saiye Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). They were acclimated to a diet for 1 week before the start of the study. All mice were housed in an SPF laboratory animal facility, maintained at a room temperature of 22 ± 1°C and a humidity of 50 ± 10%, under artificial lighting cycles of 12 h light and 12 h dark per day, with free access to food and water.

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

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

[0071] Mice in the TAC-induced heart failure group underwent TAC surgery to establish a TAC-induced heart failure mouse model. The specific procedure was as follows: Mice were anesthetized with intraperitoneal injection of tribromoethanol. Once deeply anesthetized, the skin anterior to the trachea was incised, and the muscle tissue was bluntly dissected. The second rib was gently lifted and incised, then pulled to the sides to fully expose the aortic arch. Transverse aortic constriction between the right innominate and left common carotid arteries was completed using 6-0 nylon sutures and ligated with a 26-gauge needle. The sternum and skin were then sutured. Doppler echocardiography demonstrated a transaortic transection velocity greater than 4 m / s, confirming the success of the TAC procedure. Mice were transferred to a heating pad and closely monitored. All operators and analysts were blinded to randomization to avoid genotype bias. Mice in the sham-operated group underwent the same surgical procedures, but without the ligation procedure.

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

[0073] (1) Western blotting

[0074] The specific steps for detecting NE expression in the serum of each group of mice by western blot are as follows:

[0075] 1) Protein extraction and quantification

[0076] Prepare protein extraction solution by adding protein lysis buffer, protease inhibitors and phosphatase inhibitors at a ratio of 100:1, mix well and place on ice for use;

[0077] Animal protein extraction: Remove a small piece of tissue and place it in a centrifuge tube. Add 1 mL of protein extract per 0.1 g of tissue. Homogenize using a homogenizer until the tissue fragments are micronized. Place the tube on ice and allow it to lyse on ice for 20 minutes. Place the centrifuge tube in a 4°C centrifuge at 12,000 g for 15 minutes. Transfer the supernatant to a new centrifuge tube. Take an appropriate amount of the supernatant for protein concentration determination, and store the remaining amount in a -80°C freezer.

[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. Incubate with BCA working solution at 37°C for 30 minutes; measure the absorbance at a wavelength of 562 nm on a microplate reader; create a standard curve using the concentration of the standard protein and the corresponding absorbance, and calculate the protein concentration in the test sample based on the standard curve; based on the sample concentration to be tested, prepare a 20 μL system (5× Loading buffer 4 μL, and make up the remaining volume with protein lysis buffer) so that each sample contains 20 μg of protein; heat at 100°C for 10 minutes to denature the protein; cool the denatured protein on ice and directly load it for electrophoresis, or store it in a -20°C refrigerator until needed.

[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% concentration gel solution and quickly add it to the gel making tank. Once it is full, immediately insert the comb vertically, taking care to avoid bubbles. Let it stand at room temperature again and wait for solidification. The ratio of separation gel and concentration gel is as follows:

[0081]

[0082] Sample loading: Remove the gel from the casting tank and place it in the electrophoresis tank. Pour 1x electrophoresis buffer until the surface of the gel is covered. Slowly remove the comb and add the molecular weight marker Maker and protein sample to each well in the order shown.

[0083] Electrophoresis: Turn on the transmitter device switch and perform initial stacking gel electrophoresis at 60 V. Once 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 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 a sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad in the black and white cassette in sequence. After securing, place the transfer cassette into the electroporation tank. Under low temperature conditions, set a constant current of 300mA and continue the transfer for 60-90 minutes (the transfer time depends on the molecular weight of the target protein).

[0085] Membrane blocking: After transfer, place the PVDF membrane in TBST solution containing 10% skim milk and shake on a shaker for 60 minutes for blocking. Then wash the PVDF membrane in TBST buffer three times, 10 minutes each time.

[0086] Incubation 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, then wash three times with TBST buffer for 10 minutes each.

[0087] Incubation with secondary antibody: The strips were placed in diluted secondary antibody (Invitrogen, 31444, 31460) and incubated on a shaker at room temperature for 1 hour, followed by washing three times with TBST buffer.

[0088] Imaging exposure: Incubate the strips with the prepared exposure solution, and then use the gel imaging system to capture 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 mouse tail vein and placed in a serum separator tube. After clot formation, the blood sample was centrifuged at 5000 rpm for 10 minutes, and serum was collected. Serum and sample diluent were diluted 1:9 for testing. The mouse heart was excised and an appropriate amount of tissue homogenate was added. After high-speed homogenization, the tube was placed on ice for 20 minutes. Centrifuged at 4°C / 18000g for 20 minutes, the pellet was discarded, and the supernatant was used for protein analysis.

[0092] Remove the ELISA plate pre-coated with NE and the required reagents (Abcam, ab79962) and equilibrate to room temperature. First, add 50 μL of diluted serum or tissue sample, standard, and ddH2O (blank) to the wells. Then, add 50 μL of the antibody mixture, seal the plate, and incubate on a plate shaker at 400 rpm for 1 hour at room temperature. Wash the plate three times with 350 μL of wash buffer (10× Wash Buffer diluted in ddH2O), gently tapping the plate on a clean paper towel after each wash to remove excess liquid. Add 100 μL of TMB solution to each well and incubate on a plate shaker set to 400 rpm in the dark for 15 minutes. Add 100 μL of stop solution to each well. Shake the plate on a plate shaker for 1 minute to mix. Record the absorbance at 450 nm, plot a standard curve, and calculate the NE concentration 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 specimens into labeled paraffin embedding cassettes for dehydration. The dehydration process is as follows: 70% ethanol for 60 minutes → 80% ethanol for 30 minutes → 95% ethanol for 30 minutes → Absolute ethanol I for 30 minutes → Absolute ethanol II for 30 minutes.

[0097] Clearing: After dehydration, the samples were cleared according to the following process: xylene I 60 minutes → xylene II 60 minutes;

[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. Then, remove the sample from the tissue embedding cassette and place it in the mold. After the paraffin in the mold has cooled slightly and solidified, place the pre-marked embedding cassette on top of the mold. Next, add liquid paraffin until the sample is completely submerged in the paraffin. Then, transfer the embedding mold containing the sample to a cooling table. After the paraffin block has completely cooled and solidified, remove the solidified wax block from the mold.

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

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

[0102] Baking: After the slices are fully unfolded, use a dry glass slide to pick up the slices and mark them. Pre-bake them on a 65°C slide baker for 1 hour, and then place them in a slice box for 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 three times in 1× PBS for 5 minutes each.

[0105] 4) Wipe the remaining PBS on the slices with absorbent paper. Use a marker to draw a circle around each heart tissue. 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 sections in a humidified chamber, and 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 incubated with Ly6G primary antibody (Wuhan Sewell Biotechnology Co., Ltd., GB11229) at 4°C for 8 hours.

[0108] 7) Wash with PBS three 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 secondary antibody incubation, wash with PBS three times, 5 minutes each time, protecting from light;

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

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

[0113] Identification of 1.4NE 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 genetic identification and place it in the prepared mouse tail lysis solution. First, place it in a 55°C water bath overnight, and then lyse it 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 with 150 mL of 0.5× TBE solution and place in a microwave oven. Heat on high heat until dissolved. Cool at room temperature for 5 minutes, then add 10 μL of green fluorescent nucleic acid dye. Shake gently and pour into a gel mold with a comb inserted. Let stand at room temperature for 30 minutes.

[0128] (6) Install the electrophoresis apparatus, pour in 0.5× TBE electrophoresis buffer, 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 approximately 25 minutes;

[0130] (8) Exposure and color development with nucleic acid exposure instrument;

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

[0132] 1.5 Experimental animal grouping and model construction

[0133] Twenty 8-week-old male NE KO mice and 20 WT mice of the same background were randomly divided into 4 groups according to the random number table method, with 10 mice in each group. The groups were 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] Mice in the AAV-GFP-sham and AAV-GFP-TAC groups received tail vein injections of an AAV-GFP control plasmid two weeks prior to TAC. Mice in the AAV-NE-TAC group received tail vein injections of an AAV9 vector-encapsulated NE overexpression plasmid two weeks prior to TAC. Serum NE levels were measured by ELISA two weeks later to verify the efficacy of viral transfection.

[0143] 1.6 Mouse cardiac ultrasound examination

[0144] Post-transcatheter cardiac function in mice was assessed using a Vevo2100 small animal ultrasound imaging system. To minimize irritation to the mice, the mouse chest hair was removed with depilatory cream one day before the ultrasound examination, fully exposing the skin over the heart. One week after TAC surgery, left ventricular short-axis B-mode and M-mode echocardiograms were obtained with an MS400C ultrasensitive probe while the mice were awake for subsequent analysis. Cardiac ultrasound parameters included heart rate, left ventricular anterior and posterior wall thickness during systole and diastole, left ventricular end-systolic and end-diastolic volumes, ejection fraction, and fractional shortening. The average values ​​of five consecutive cardiac cycles were used for final statistical analysis.

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

[0146] (1) Dewax paraffin sections to water using the following steps: xylene I for 30 minutes → xylene II for 30 minutes → anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → 95% ethanol for 5 minutes → 80% ethanol for 5 minutes → distilled water for 5 minutes;

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

[0148] (3) Differentiate in 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 cleared with alcohol using the following steps: 95% ethanol for 30 seconds → anhydrous ethanol for 1 minute → anhydrous ethanol II for 1 minute → xylene I for 15 minutes → xylene II for 30 minutes;

[0151] (6) Seal the slides 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) Dewax paraffin sections to water;

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

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

[0157] (4) The sections were cleared using the following steps: xylene I for 15 minutes → xylene II for 30 minutes;

[0158] (5) Seal the slides 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) Dewax paraffin sections to water;

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

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

[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, add WGA working solution for 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, seal the slides with coverslips, and store in a refrigerator at 4°C 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: Mouse heart tissue samples were taken from a -80°C freezer, cut into rice-sized pieces, and placed in a 1.5 mL EP tube.

[0172] (2) Tissue lysis: Add 1 mL of Trizol to each tube and homogenize the tissue at high speed using an electric homogenizer 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, cap the tube tightly, shake vigorously up and down to mix, let it stand on ice for 15 minutes, and then centrifuge at 12000g for 15 minutes at 4°C;

[0174] (4) DNA and protein removal: Remove the EP tube and 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 12,000 g for 15 minutes at 4°C. After centrifugation, a small amount of white precipitate will be visible at the bottom of the tube. Carefully discard the supernatant.

[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 minutes at 4°C;

[0176] (6) RNA drying: Carefully discard the supernatant and use a pipette to remove as much residual ethanol as possible. Then, turn the EP tube upside down and place 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) RNA concentration measurement: Use Nanodrop spectrophotometer to determine 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 as follows: 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 of eight-well centrifuge tubes according to the number of samples, set up two replicate wells for each sample, add 10 μL of the following reagents to each well to prepare the reaction system, mix well, and centrifuge the liquid to the bottom of the tube. Reaction procedure: 95°C for 30 seconds; 95°C for 5 seconds, 60°C for 34 seconds, 40 cycles; 95°C for 15 seconds, 60°C 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 1 μL ddH2O 3.3 μL ROX II 0.2μL total 10 μL

[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 a myocardial hypertrophy and heart failure model in mice. It has the characteristics of simple operation, low mortality, and high reproducibility. In the present invention, in order to minimize the interference of factors such as weight, gender, and age, all mice used were male, and their age and weight were very similar. Western blotting was used to detect changes in NE expression in serum at different time points after TAC. The results are shown in Figure 2. Figure 1Figure A shows that NE protein expression levels increased significantly 14 days after TAC. Therefore, all subsequent experiments were performed at 14 days after TAC. Elisa assay was used to detect changes in serum NE levels in mice after TAC. The results showed that compared with the sham operation group, TAC significantly increased serum NE levels 14 days after TAC ( Figure 1 B in Figure 1). Similar conclusions were obtained by detecting the NE content in the heart tissue of mice after TAC by ELISA ( Figure 1 C). Furthermore, immunofluorescence analysis of neutrophil and neutrophil infiltration in cardiac tissues in the sham-operated and TAC groups revealed a significant increase in neutrophil infiltration and neutrophil fluorescence intensity 14 days after TAC compared with the sham-operated group. These results demonstrate that neutrophil expression levels in serum and cardiac tissue were significantly upregulated 14 days after TAC, and that significant neutrophil infiltration was observed 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 used 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 the NE KO mice were successfully constructed. 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 Figures 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] Echocardiography was performed on mice treated with TAC using a Vevo 3100 system equipped with an MS400C probe. 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 Figure 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 of heart sections of mice in each group 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, the mRNA levels of myocardial hypertrophy-related makers 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 HJ in).

[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. Figure 4 As shown in Figure A, WT mice showed 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 fibrosis-related proteins Collagen I and TGF-β in myocardial tissue of NE KO mice were significantly lower than those of WT mice ( Figure 4 BC in the NE group), indicating 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 mice in each group. The results are shown in Figure 2. Figure 5 As shown, 14 days after TAC surgery, cardiomyocyte IL-1β, IL-6, TNF-α, and MCP1 gene mRNA expression levels in WT mice were significantly increased, while NE KO mice were able to significantly reduce the TAC-induced increase in cardiomyocyte IL-1β, IL-6, TNF-α, and MCP1 gene mRNA expression levels. These results indicate that NE knockout significantly improves 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, and AAV-mediated green fluorescent protein (GFP) was used as a control. As expected, AAV9-NE significantly increased the serum NE level ( 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 At the same time, compared with AAV-GFP-TAC heart failure mice, AAV9-NE aggravated TAC-induced cardiac dysfunction, as shown by further reductions in EF and FS ( Figure 7 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 HJ in).

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

[0202] Depend on Figure 8 As shown in Figure 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 proteins. Figure 8 As shown in Figure 3B, 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] Figure 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 mice of C57BL / 6J background and WT mice of the same background were purchased from Saiye Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). They were acclimated to a diet for 1 week before the start of the study. All mice were housed in an SPF laboratory animal facility, maintained at a room temperature of 22 ± 1°C and a humidity of 50 ± 10%, under artificial lighting cycles of 12 h light and 12 h dark per day, with free access to food and water.

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

[0208] Mice in the TAC-induced heart failure group underwent TAC surgery to establish a TAC-induced heart failure mouse model. The specific procedure was as follows: Mice were anesthetized by intraperitoneal injection of tribromoethanol. When the mice were deeply anesthetized, the skin anterior to the trachea was incised, and the muscle tissue was bluntly dissected. The second rib was gently lifted and incised, pulled to the sides, and the aortic arch was fully exposed. A 26-gauge needle was used to tighten and ligate the transverse aorta between the right innominate and left common carotid arteries using 6-0 nylon sutures, followed by sternal suture and skin suture. Doppler echocardiography demonstrated a transaortic transection velocity greater than 4 m / s, confirming the success of the TAC procedure. Mice were transferred to a heating pad and closely monitored. All operators and analysts were blinded to randomization to avoid genotype bias. Mice in the sham-operated group underwent the same surgical procedures, but no ligation was performed.

[0209] 2.3 Sivelestat sodium (hereinafter referred to as Sive) was prepared into a mother solution using dimethyl sulfoxide (DMSO) as solvent. Sivelestat sodium: PEG300: Tween-20: 0.9% NaCl were added in the ratio of 1:8:1:10 in this order. Mix thoroughly each time to ensure that the liquid was clear before proceeding to the next step. One week after TAC surgery, mice underwent small animal ultrasound to assess arcuate blood flow. Mice with successful TAC 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 mice were treated with solvent (DMSO:PEG300:Tween-20:0.9% NaCl in a ratio of 1:8:1:10, 200 μL / mouse) and then administered via intraperitoneal injection once daily for 7 consecutive weeks. At week 8, cardiac function was assessed by small animal ultrasound. After the evaluation, mice in each group were euthanized, and samples were collected, weighed, and used for subsequent experiments.

[0210] 2.4 Mouse cardiac ultrasound examination

[0211] Post-transcatheter cardiac function in mice was assessed using a Vevo2100 small animal ultrasound imaging system. To minimize irritation to the mice, the mouse chest hair was removed with depilatory cream one day before the ultrasound examination, fully exposing the skin over the heart. One week after TAC surgery, left ventricular short-axis B-mode and M-mode echocardiograms were obtained with an MS400C ultrasensitive probe while the mice were awake for subsequent analysis. Cardiac ultrasound parameters included heart rate, left ventricular anterior and posterior wall thickness during systole and diastole, left ventricular end-systolic and end-diastolic volumes, ejection fraction, and fractional shortening. The average values ​​of five consecutive cardiac cycles were used for final statistical analysis.

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

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

[0214] 2.6 Sirius red staining

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

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

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

[0218] 2.8 Western Blot Detection

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

[0220] 2.9 Results and Analysis

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

[0222] All mice used in AC surgery were male and of similar age and weight. Echocardiography was performed on mice after TAC induction using the Vevo3100 system with an MS400C probe. 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 10 As shown in the data, 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. However, treatment with different concentrations of sivelestat sodium could significantly increase the 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 larger ( Figure 11 A), the heart-to-body weight ratio and heart-to-tibia length ratio increased significantly ( Figure 11 B, C), and different concentrations of sivelestat sodium treatment significantly reduced TAC-induced heart size, heart-to-body weight ratio, and heart-to-tibial length. WGA staining results also showed that compared with the TAC group, different concentrations of sivelestat sodium treatment significantly reduced the heart cross-sectional area ( Figure 12 A, B in ).

[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 ( Figure 13 AC in the TAC), and different concentrations of sivelestat sodium treatment significantly reduced TAC-induced cardiac fibrosis ( Figure 13 AC in).

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

[0228] Compared with the sham operation group, the expression levels of myocardial fibrosis-related proteins CollagenⅠ 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 CollagenⅠ and TGF-β induced by TAC ( Figure 14 ).

[0229] 2.9.5 No adverse effects occurred in the liver and kidney functions of mice in each group at the supplemental dose of sivelestat sodium

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

Claims

1. Use of a substance that inhibits or silences neutrophil elastase activity in the preparation of a medicament for treating heart failure induced by pressure overload, characterized in that: The substance is sivelestat sodium.

2. The use according to claim 1, characterized in that The medicine also includes pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that The dosage form of the medicine is solution dosage form, emulsion form or powder form.

4. Use of a substance that inhibits or silences neutrophil elastase gene expression in the preparation of a medicament for treating pressure-overload-induced heart failure, characterized in that: The substance is sivelestat sodium.

5. The use according to claim 4, characterized in that The medicine also includes pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that The dosage form of the medicine is solution dosage form, emulsion form or powder form.

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