Angiopoietin-like protein 7 inhibitor and application thereof

Abstract

The invention relates to an angiopoietin-like protein 7 inhibitor and application thereof, and belongs to the technical field of biological medicine. The invention provides an ANGPTL7 gene expression inhibitor and application thereof in order to solve the problems that existing heart failure treatment drugs mostly indirectly regulate a neuroendocrine system and are difficult to accurately intervene in myocardial fibrosis and reconstruct a core mechanism. The inhibitor is specific siRNA (small interfering Ribonucleic Acid) targeting ANGPTL7. According to the scheme, by directly inhibiting a key disease promoting factor ANGPTL7 in heart failure, myocardial cell damage can be effectively relieved, myocardial fibrosis is inhibited, and the heart function is improved. The invention provides a brand-new targeted treatment strategy with a clear mechanism for prevention and treatment of heart failure, and has an important clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to angiopoietin-like protein 7 inhibitors and their applications. Background Technology

[0002] Heart failure is the end stage of many cardiovascular diseases, characterized by complex pathogenesis, high morbidity, and high mortality, making it a major public health problem posing a serious threat to human health. The core pathological basis of heart failure mainly manifests as myocardial hypertrophy, myocardial fibrosis, and the resulting myocardial remodeling and continuous deterioration of cardiac function. Under the influence of long-term stress load, continuous activation of the neuroendocrine system, or inflammatory stimulation, cardiomyocytes undergo structural and functional remodeling, myocardial fibroblasts are abnormally activated and undergo phenotypic transdifferentiation, and abnormal extracellular matrix deposition leads to decreased myocardial compliance and impaired cardiac pumping function. Currently, drugs used clinically to treat heart failure mainly include angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, beta-blockers, and aldosterone receptor antagonists. These drugs can improve patients' clinical symptoms and survival prognosis to some extent. However, existing treatments mostly work indirectly by regulating the neuroendocrine system or hemodynamics, making it difficult to precisely intervene in the key molecular regulatory mechanisms of myocardial remodeling processes such as myocardial hypertrophy and myocardial fibrosis. Furthermore, long-term use still faces problems such as limited efficacy or adverse reactions. Therefore, finding new molecular targets and developing corresponding treatment strategies is of great significance for the prevention and treatment of heart failure.

[0003] Angiopoietin-like proteins (ANGPTLs) are a class of secreted proteins involved in vascular function regulation, metabolic homeostasis maintenance, and tissue remodeling. Members of this family have been reported to participate in various pathological and physiological processes, including extracellular matrix regulation, tissue fibrosis, and cellular stress responses. ANGPTL7, as an important member of the ANGPTL family, has been shown to be abnormally expressed in various tissues during pathological remodeling. For example, inhibiting abnormally high expression of ANGPTL7 in corneal tissue can effectively alleviate the progression of glaucoma. Furthermore, studies suggest that ANGPTL7 may participate in tissue remodeling by regulating fibroblast activation, collagen synthesis, and related signaling pathways. Currently, systematic research on the specific mechanisms of action of ANGPTL7 in myocardial remodeling and myocardial fibrosis is lacking, and the development and application of drugs targeting ANGPTL7 for the prevention and treatment of heart failure are not yet fully reported. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an angiopoietin-like protein 7 inhibitor and its application in the preparation of drugs that inhibit ANGPTL7 gene expression.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a group of angiopoietin-like protein 7 inhibitors, wherein the angiopoietin-like protein 7 inhibitor is siRNA, the siRNA comprising a sense strand and an antisense strand, and the siRNA comprising any one of the following three groups of compounds, wherein: (1) the sense strand nucleic acid sequence is shown in SEQ ID NO:1, and the antisense strand nucleic acid sequence is shown in SEQ ID NO:2; (2) the sense strand nucleic acid sequence is shown in SEQ ID NO:3, and the antisense strand nucleic acid sequence is shown in SEQ ID NO:4; (3) the sense strand nucleic acid sequence is shown in SEQ ID NO:5, and the antisense strand nucleic acid sequence is shown in SEQ ID NO:6. The present invention provides a cell comprising the angiopoietin-like protein 7 inhibitor described above; This invention provides a viral vector containing nucleic acid encoding the angiopoietin-like protein 7 inhibitor; The present invention provides a pharmaceutical composition for inhibiting ANGPTL7 gene expression, the pharmaceutical composition comprising the angiopoietin-like protein 7 inhibitor and a pharmaceutically acceptable carrier; This invention provides a method for inhibiting ANGPTL7 gene expression in cells. The angiopoietin-like protein 7 inhibitor is introduced into target cells, and the introduction method is selected from any one of viral vector-mediated introduction, liposome transfection, electroporation or microinjection. Preferably, the cells are human or non-human animal cardiomyocytes or cardiomyocytes; Furthermore, this invention provides the application of angiopoietin-like protein 7 inhibitors in the preparation of drugs that inhibit ANGPTL7 gene expression; Furthermore, the present invention provides the use of angiopoietin-like protein 7 inhibitors in the preparation of drugs for the prevention and / or treatment of heart failure; Preferably, the heart failure includes at least one of myocardial fibrosis, chemotherapy-related cardiomyopathy, and heart failure with preserved ejection fraction.

[0006] The beneficial effects of this invention are as follows: 1. This invention reveals for the first time the crucial role of ANGPTL7 in heart failure: Through various in vivo and in vitro heart failure models (such as myocardial fibrosis, doxorubicin cardiomyopathy, and heart failure with preserved ejection fraction), this invention systematically demonstrates for the first time that ANGPTL7 expression is significantly upregulated during the development and progression of heart failure, playing a key role in promoting disease progression. Unlike traditional anti-heart failure drugs that mainly act on the neuroendocrine or hemodynamic systems, this invention directly targets the core molecular mechanisms of myocardial remodeling and myocardial fibrosis, providing a novel intervention strategy and target for the precision treatment of heart failure. Overexpression of ANGPTL7 can significantly aggravate myocardial injury, fibrosis, and cardiac dysfunction; conversely, inhibiting ANGPTL7 expression through siRNA and other means can effectively alleviate cardiomyocyte apoptosis, cardiomyocyte hypertrophy, fibroblast activation, and fibrosis. Comprehensive experimental data from whole animal models (echocardiography, serum markers, pathological staining) to in vitro cell models (cardiomyocytes, myocardial fibroblasts) are provided, strongly demonstrating the effectiveness of targeting ANGPTL7.

[0007] 2. Heart failure is the end-stage of cardiovascular disease, with high morbidity and mortality. Current therapies have limited efficacy and side effects. This discovery provides a solid foundation for developing more effective and precise therapeutics. This target has shown effectiveness in various types of heart failure, including heart failure with preserved ejection fraction (HFpEF) and chemotherapy-related cardiomyopathy, with a broad potential patient population. Rigorous in vitro and in vivo experiments have demonstrated that inhibiting ANGPTL7 can significantly reduce myocardial damage, inhibit extracellular collagen deposition, reverse pathological myocardial remodeling, and improve cardiac function.

[0008] 3. The market prospects are broad, targeting a huge market for diseases such as heart failure that lack effective drugs. It points to a clear direction for the development of next-generation targeted drugs for heart failure, and has significant potential commercial value and clinical translation potential.

[0009] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0010] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1Electrophoresis diagrams showing significant upregulation of ANGPTL7 expression in heart failure tissues; Figure A: Protein expression level of ANGPTL7 in heart tissues of normal control mice and mice with isoproterenol-induced myocardial fibrosis, with GAPDH as an internal control; Figure B: Protein expression level of ANGPTL7 in heart tissues of normal control mice and mice with doxorubicin-induced cardiomyopathy, with GAPDH as an internal control; Figure C: Protein expression level of ANGPTL7 in heart tissues of normal control mice and HFpEF mice induced by a high-fat diet plus nitric oxide synthase inhibitor, with GAPDH as an internal control. Figure 2 ANGPTL7 overexpression exacerbates myocardial injury and remodeling in mice with doxorubicin-induced cardiomyopathy; ANGPTL7 overexpression exacerbates doxorubicin-induced cardiomyopathy. Figure A: Representative echocardiograms of each group of mice; Figures B and E: Statistical graphs of LVEF, LVFS, LVIDd, and LVIDs in each group of mice; Figure F: Levels of B-type brain natriuretic peptide (BNP) in each group of mice; Figure G: Levels of cardiac troponin T (cTnT) in each group of mice; Figure H: Levels of creatine kinase isoenzyme (CK-MB) in each group of mice; Figure I: Levels of lactate dehydrogenase (LDH) in each group of mice; Figure J: Sirius red staining of cardiac sections from each group of mice to assess myocardial fibrosis. Figure 3 ANGPTL7 overexpression exacerbates myocardial injury and remodeling in HFpEF mice; ANGPTL7 overexpression exacerbates heart failure with preserved ejection fraction. Figure A: Panoramic view of mice in each group; Figure B: Weekly weight statistics of mice in each group; Figure C: Glucose tolerance test of mice in each group; Figure D: Systolic blood pressure (SBP) statistics of mice in each group; Figure E: Diastolic blood pressure (DBP) statistics of mice in each group; Figure F: Lung weight of mice in each group; Figure G: Treadmill test to detect activity distance of mice in each group. Figure 4 ANGPTL7 silencing was used to alleviate doxorubicin-induced cardiomyocyte apoptosis. ANGPTL7 silencing also alleviated doxorubicin-induced myocardial injury. Figure A: Protein expression levels of apoptosis-related proteins Bax and Bcl2 in H9c2 rat cardiomyocytes treated with ANGPTL7-targeting siRNA followed by doxorubicin stimulation, with GAPDH as an internal control. Figures BC: Statistical graphs of protein expression levels of apoptosis-related proteins Bax and Bcl2 in each group. Figure D: TUNEL-stained fluorescence images of cardiomyocytes in each group. Figure E: Statistical graph of the percentage of TUNEL-positive cells. Figure 5To alleviate ANGPTL7 silencing in a "double-hit" model-induced cardiomyocyte hypertrophy; ANGPTL7 silencing alleviates cardiomyocyte hypertrophy. Figure A: H9c2 rat cardiomyocytes treated with ANGPTL7-targeting siRNA, followed by "PA+L-NAME" stimulation, and the protein expression levels of hypertrophy-related proteins ANP and BNP in each group, with GAPDH as an internal control; Figures BC: Statistical graphs of protein expression levels of hypertrophy-related proteins ANP and BNP in each group; Figure D: Phalloidin-stained fluorescence images of cardiomyocytes in each group; Figure E: Statistical graphs of relative area of ​​cardiomyocytes based on phalloidin staining. Figure 6 To investigate the effects of ANGPTL7 silencing on the proliferation, migration, and transdifferentiation of cardiac fibroblasts, and to alleviate ANGPTL7 activation and transdifferentiation of cardiac fibroblasts, Figure A shows representative images of α-SMA fluorescence staining in primary rat cardiac fibroblasts treated with ANGPTL7-targeting siRNA followed by TGF-β1 stimulation, indicating cell transdifferentiation; Figure B shows representative images of Col1 fluorescence staining in each component of the fibroblasts, indicating extracellular matrix proteins; Figure C shows representative images of EdU staining in each component of the fibroblasts, indicating cell proliferation; and Figure D shows representative images of scratch assays in each component of the fibroblasts, indicating cell migration. Detailed Implementation

[0011] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0012] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0013] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0014] Example 1 I. Upregulation of ANGPTL7 expression in tissues of an adriamycin-induced cardiomyopathy model (1) Doxorubicin cardiomyopathy mouse model: Male C57BL / 6 mice aged 8-12 weeks were selected and administered doxorubicin intraperitoneally at a dose of 5 mg / kg once a week for a cumulative dose of 20 mg / kg. The control group received an equal volume of physiological saline intraperitoneally. The body weight and general condition of the mice were monitored.

[0015] (2) Western blot detection of ANGPTL7 protein expression in cardiac tissue A suitable amount of mouse heart tissue was taken, lysis buffer was added, and the mixture was homogenized thoroughly on ice. The supernatant was then collected by centrifugation to obtain the total protein sample from the heart tissue. After determining the protein concentration using the BCA method, an equal volume of protein was mixed with loading buffer and boiled for denaturation. The sample was then added to the wells of a pre-prepared SDS-PAGE gel for electrophoresis to separate the proteins. After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then primary antibody against ANGPTL7 protein was added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed with TBST, and secondary antibody labeled with horseradish peroxidase was added and incubated at room temperature for 1 hour. After washing again, ECL chemiluminescence developer was added, and the membrane was exposed and developed in a gel imaging system to obtain band images of ANGPTL7 protein. ImageJ software was used for semi-quantitative analysis of the band gray values, and the results were corrected using a GAPDH internal reference protein to compare the relative expression levels of ANGPTL7 protein.

[0016] The results showed that, compared with the control group, the expression level of ANGPTL7 protein in the heart tissue of heart failure mice was significantly increased. Figure 1 .

[0017] II. ANGPTL7 overexpression exacerbates myocardial damage and worsening of cardiac function in doxorubicin cardiomyopathy. (1) Adeno-associated virus (AAV9) injection Select and prepare 6-8 week old adult C57BL / 6 mice. Remove the AAV9 virus stock solution carrying ANGPTL7 cDNA from -80℃ and thaw it slowly on ice. Dilute the virus to the required working concentration with sterile saline (typical dose is 5 × 10^11 vg / mouse, total injection volume controlled at approximately 200 μL). Then, place the mice in a restraint and warm the tail with 40-45℃ water for 1-2 minutes to dilate blood vessels. Using an insulin needle, puncture the caudal vein at an angle of less than 30 degrees and slowly and evenly inject the virus suspension. After injection, briefly apply pressure to stop bleeding and return the mice to their cages for observation. Typically, AAV9-mediated gene expression reaches a stable plateau 2-4 weeks after injection. At the predetermined time point, analyze mRNA (qPCR) and protein (Western Blot) samples from heart tissue to verify the overexpression efficiency of ANGPTL7. Two weeks after virus injection, establish an doxorubicin-induced myocardial injury model.

[0018] (2) Mouse cardiac function test Before echocardiography in mice, the mice are induced with isoflurane and maintained under 1.5-2.0% isoflurane anesthesia. They are then fixed in a supine position on a 37°C constant-temperature platform, and the fur on their chest area is shaved. Before ultrasound examination, ultrasound coupling agent is evenly applied to the shaved area. Standard two-dimensional images are obtained in the short-axis section (papillary muscle level) of the left ventricle parasternally. The left ventricular chamber diameter and wall thickness at end-systole and end-diastole are recorded using M-mode ultrasound, from which core cardiac function indicators such as left ventricular ejection fraction (LVEF) and fractional shortening (FS) are calculated. Figure 2 AE represents the cardiac function test results of AAV9-ANGPTL7 and AAV9-EV mice treated with doxorubicin. Compared with the saline control group, the doxorubicin-induced cardiomyopathy mice exhibited enlarged cardiac chambers and impaired cardiac systolic function, mainly manifested as increased left ventricular end-diastolic diameter and left ventricular end-systolic diameter, while ejection fraction and fractional shortening, indicators reflecting cardiac function, were significantly decreased. ANGPTL7 overexpression further exacerbated these cardiac function indicators.

[0019] (3) Detection of serum myocardial injury markers Whole blood samples were collected from mice via orbital sampling. After coagulation, the samples were centrifuged at 3000 rpm for 15 minutes at 4°C to obtain the supernatant serum. Subsequently, a commercially available enzyme-linked immunosorbent assay (ELISA) kit (CK-MB, BNP, cTnT, LDH) was used, strictly following the instructions: standards and diluted serum samples were added to microplates coated with specific antibodies. After incubation and washing, biotin-labeled detection antibodies, horseradish peroxidase-labeled streptavidin, and chromogenic substrates were added sequentially. Finally, the absorbance of each well was measured at a specific wavelength using an ELISA reader. The accurate concentrations of CK-MB, BNP, cTnT, and LDH in the serum were calculated using a standard curve plotted based on the standard concentration versus absorbance. Throughout the process, repeated freeze-thaw cycles should be avoided, and all steps should be performed on ice or at low temperatures to ensure the stability of the biomarkers. Results are as follows: Figure 2 The results showed that ANGPTL7 overexpression significantly increased the serum levels of myocardial injury markers in doxorubicin cardiomyopathy mice.

[0020] (4) Pathological examination The main procedures for preparing paraffin-embedded specimen sections include: trimming the heart → preparing the embedding frame → rinsing with running water → dehydration → clearing → wax infiltration → embedding → sectioning → spreading → air-drying or baking for later use.

[0021] The procedure for Sirius red staining of mouse heart tissue was as follows: Paraffin-embedded heart tissue sections were dewaxed with xylene, hydrated with graded ethanol, and rinsed with deionized water. 0.1% Sirius red staining solution (prepared with saturated picric acid aqueous solution) was added and incubated at room temperature in the dark for 60 minutes. After staining, the sections were quickly rinsed twice with 0.5% glacial acetic acid solution to remove non-specific staining. After routine dehydration and clearing, the sections were mounted with neutral resin. The staining results could be observed under a light microscope (collagen fibers appeared bright red, and myocardial tissue appeared yellow). Image analysis software could quantify the collagen volume fraction in specific regions. Results are as follows: Figure 2 The results showed that ANGPTL7 overexpression significantly increased the area of ​​cardiac fibrosis in doxorubicin cardiomyopathy mice.

[0022] III. ANGPTL7 inhibitors inhibit doxorubicin-stimulated cardiomyocyte apoptosis (1) Construction and transfection of ANGPTL7 siRNA in H9c2 rat cardiomyocytes To knock down ANGPTL7 in H9c2 cardiomyocytes, a siRNA targeting ANGPTL7 was constructed. The sense strand sequence of the siRNA is shown in SEQ ID NO:1 (5'-GCACCAAGGACAAGGACAA-3'), and the antisense strand sequence is shown in SEQ ID NO:2 (5'-TTGTCCTTGTCCTTGGTGC-3'). Using a liposome transfection reagent (such as Lipofectamine 3000), the siRNA and transfection reagent were first diluted separately with serum-free medium, incubated at room temperature for 5 minutes, then mixed, and incubated for 15-20 minutes to form a transfection complex. During this period, the cardiomyocytes were washed once with PBS and replaced with maintenance medium without antibiotics. Subsequently, the transfection complex was evenly added to the cell culture wells, gently shaken, and incubated at 37°C, 5% CO2 for 6 hours, then replaced with normal medium containing serum. After culturing for another 48-72 hours, the knockdown efficiency of ANGPTL7 was verified by qPCR or Western Blot.

[0023] (2) Western blot detection of apoptosis-related protein expression in cardiomyocytes The results are as follows Figure 4 The results showed that the ANGPTL7 inhibitor significantly alleviated the abnormal expression of apoptosis-related proteins in H9c2 rat cardiomyocytes induced by doxorubicin.

[0024] (3) TUNEL fluorescence staining to assess the degree of apoptosis Cell slides were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS, and permeabilized with 0.2% Triton X-100 for 10 minutes. After washing with PBS again, 50 μL of TUNEL reaction mixture (containing terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP) was added to each sample, and the slides were incubated in a humidified chamber at 37°C for 60 minutes. Positive controls (pretreated with DNase I) and negative controls (without TdT enzyme) were also included to ensure experimental specificity. After incubation, the slides were thoroughly washed with PBS, and DAPI staining was added for nuclear counterstaining. The slides were then mounted with anti-fluorescence quenching mounting medium. Cells were observed and counted under a fluorescence microscope: TUNEL-positive cells showed green fluorescence, indicating apoptotic cells with DNA breaks. The apoptosis rate was calculated by comparing the total number of nuclei with the total number stained with blue DAPI. Results are as follows: Figure 4 The results showed that the ANGPTL7 inhibitor significantly alleviated doxorubicin-induced apoptosis of H9c2 rat cardiomyocytes.

[0025] Example 2 I. Upregulation of ANGPTL7 expression in tissues of a heart failure model with preserved ejection fraction (1) Mouse model of heart failure with preserved ejection fraction Male C57BL / 6 mice aged 8-12 weeks were selected and fed a high-fat diet (fat energy ratio 60%), with Nω-nitro-L-arginine methyl ester (L-NAME, 0.5 g / L) added to their drinking water. The control group received a normal diet and regular water. Body weight and general condition were monitored.

[0026] (2) Western blot detection of ANGPTL7 protein expression in cardiac tissue A suitable amount of mouse heart tissue was taken, lysis buffer was added, and the mixture was homogenized thoroughly on ice. The supernatant was then collected by centrifugation to obtain the total protein sample from the heart tissue. After determining the protein concentration using the BCA method, an equal volume of protein was mixed with loading buffer and boiled for denaturation. The sample was then added to the wells of a pre-prepared SDS-PAGE gel for electrophoresis to separate the proteins. After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then primary antibody against ANGPTL7 protein was added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed with TBST, and secondary antibody labeled with horseradish peroxidase was added and incubated at room temperature for 1 hour. After washing again, ECL chemiluminescence developer was added, and the membrane was exposed and developed in a gel imaging system to obtain band images of ANGPTL7 protein. ImageJ software was used for semi-quantitative analysis of the band gray values, and the results were corrected using a GAPDH internal reference protein to compare the relative expression levels of ANGPTL7 protein.

[0027] The results showed that, compared with the control group, the expression level of ANGPTL7 protein in the heart tissue of mice with preserved ejection fraction heart failure was significantly increased. Figure 1 .

[0028] II. ANGPTL7 overexpression exacerbates ejection fraction-preserving heart failure in mice (1) Non-invasive blood pressure measurement in mice When using the non-invasive tail-cuff blood pressure measurement system for mice, the animal should first be placed in a constant-temperature chamber at 37-38°C for 10-15 minutes to preheat and dilate the tail artery. Then, an awake but gently restrained mouse is placed in the restraints, allowing its tail to pass naturally through the inflatable sensing tail cuff and plethysmography sensor. After starting the automatic measurement program, the system temporarily blocks blood flow to the tail by inflation and detects the pulse wave recovery point during slow deflation, recording the corresponding systolic blood pressure, diastolic blood pressure, and mean arterial pressure. To obtain reliable data, measurements should be taken continuously at a fixed time each day for 3-5 days, recording at least 10 valid readings each time and averaging them. The environment should be kept quiet throughout the measurement process to minimize stress interference. Results showed that ANGPTL7 overexpression significantly increased systolic and diastolic blood pressure levels in HFpEF mice.

[0029] (2) Treadmill experiment for mice Before the experiment, mice were placed in a treadmill aisle for three days for acclimatization training to familiarize them with the environment. During the formal test, the mice were placed on the treadmill track, initially set at low speed and zero incline. Subsequently, the treadmill speed and / or incline were increased every 2-3 minutes according to a pre-set protocol until the mice reached exhaustion (defined as remaining at the rear of the electric shock zone for 10 consecutive seconds without being able to return to the running area). The total running time, maximum speed, and maximum running distance from start to exhaustion were recorded as core endurance indicators. Throughout the experiment, the environment was kept quiet and at a constant temperature, and the mice's maximum oxygen consumption was monitored simultaneously using a gas analysis system to more accurately assess cardiopulmonary reserve. Results are as follows: Figure 3 The results showed that HFpEF mice overexpressing ANGPTL7 had significantly reduced exercise endurance.

[0030] (3) Glucose tolerance test Before the experiment, mice were fasted for 6-8 hours. Baseline blood glucose levels (0 minutes) were measured using a glucometer, followed by intraperitoneal injection of glucose solution (2 g / kg). Blood samples were collected from the tail vein of mice at 15, 30, 60, 90, and 120 minutes after glucose administration, and blood glucose concentrations were immediately measured at each time point. Finally, a blood glucose curve was plotted with time on the x-axis and blood glucose concentration on the y-axis to assess the mice's glucose clearance capacity and glucose tolerance. A quiet environment was maintained throughout the experiment to minimize stress interference with blood glucose levels. Results are as follows: Figure 3 The results showed that glucose tolerance was further impaired in HFpEF mice overexpressing ANGPTL7.

[0031] III. ANGPTL7 inhibitors inhibit cardiomyocyte hypertrophy (1) Construction of HFpEF double-hit in vitro model Healthy H9c2 rat cardiomyocytes were harvested and treated with the nitric oxide synthase inhibitor L-NAME (200 μM) to simulate cellular oxidative stress. Simultaneously, the cells were treated with saturated free fatty acid palmitic acid (PA, 100 μM, pre-prepared by binding with fatty acid-free BSA) to simulate lipotoxic damage under metabolic disturbances. After treatment, cell viability, cardiomyocyte cross-sectional area, and levels of myocardial injury markers were measured to verify that this double-hit model successfully induced metabolic stress and damage in cardiomyocytes.

[0032] (2) Construction and transfection of ANGPTL7 siRNA in H9c2 rat cardiomyocytes The method is as described in Example 1. The sense strand sequence of the siRNA is shown in SEQ ID NO:3 (5'-GAATCTCTGGAGTGTACAA-3'), and the antisense strand sequence is shown in SEQ ID NO:4 (5'-TTGTACACTCCAGAGATTC-3').

[0033] (3) Western blot detection of mast-related protein expression in cardiomyocytes The method is as described in Example 1. The results are as follows: Figure 5 The results showed that ANGPTL7 inhibitors significantly alleviated the abnormal expression of mast-related proteins in the cardiomyocytes of H9c2 rats treated with "PA+L-NAME".

[0034] (4) Phalloidin staining to assess cardiomyocyte area After gently rinsing the slides containing cardiomyocytes with PBS, fix them with 4% paraformaldehyde at room temperature for 15 minutes; rinse three times with PBS, 5 minutes each time. Add 0.1% Triton X-100 for 5 minutes to increase membrane permeability. After rinsing again with PBS, add fluorescently labeled phalloidin working solution (1:200) diluted with PBS containing 1% BSA, and incubate at room temperature in the dark for 60 minutes. After incubation, rinse three times with PBS in the dark to remove unbound dye. Add anti-fluorescence quenching mounting medium containing DAPI for nuclear counterstaining, and mount the slides. Observation under a fluorescence microscope: phalloidin specifically binds to F-actin, making the cytoskeleton fibers appear green, clearly showing the sarcomere arrangement and morphological structure of cardiomyocytes. Results are as follows. Figure 5 The results showed that ANGPTL7 inhibitors significantly alleviated cardiomyocyte hypertrophy in H9c2 rats treated with "PA+L-NAME".

[0035] Example 3 I. Upregulation of ANGPTL7 expression in myocardial fibrosis tissue (1) Isoproterenol mouse model of myocardial fibrosis: Male C57BL / 6 mice aged 8-12 weeks were selected. The mice were fixed, and after disinfecting the skin on the back of the neck, the skin was pinched up, and the needle was inserted horizontally into the subcutaneous tissue to slowly inject isoproterenol dissolved in physiological saline. The control group was given an equal amount of physiological saline. The condition of the mice (activity, fur, respiration) was observed, and the weight was recorded.

[0036] (2) Western blot detection of ANGPTL7 protein expression in cardiac tissue A suitable amount of mouse heart tissue was taken, lysis buffer was added, and the mixture was homogenized thoroughly on ice. The supernatant was then collected by centrifugation to obtain the total protein sample from the heart tissue. After determining the protein concentration using the BCA method, an equal volume of protein was mixed with loading buffer and boiled for denaturation. The sample was then added to the wells of a pre-prepared SDS-PAGE gel for electrophoresis to separate the proteins. After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then primary antibody against ANGPTL7 protein was added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed with TBST, and secondary antibody labeled with horseradish peroxidase was added and incubated at room temperature for 1 hour. After washing again, ECL chemiluminescence developer was added, and the membrane was exposed and developed in a gel imaging system to obtain band images of ANGPTL7 protein. ImageJ software was used for semi-quantitative analysis of the band gray values, and the results were corrected using a GAPDH internal reference protein to compare the relative expression levels of ANGPTL7 protein.

[0037] The results showed that, compared with the control group, the expression level of ANGPTL7 protein in the heart tissue of myocardial fibrosis mice was significantly increased. Figure 1 .

[0038] II. ANGPTL7 inhibitors inhibit the activation and transdifferentiation of cardiac fibroblasts. (1) Isolation and culture of primary rat cardiomyocytes Preparing for New Students 1 3-day Sprague In Dawley suckling mice, the heart was rapidly removed under aseptic conditions. Blood was washed away with pre-cooled PBS, and the atria and great vessels were excised, preserving the ventricular myocardium. The ventricular tissue was minced and placed in trypsin digestion solution for multiple rounds (usually 5-6 rounds) on a 37°C shaker. The supernatant after each digestion was neutralized with culture medium containing 20% ​​fetal bovine serum. The collected cell suspension was centrifuged at 1500 rpm for 8 min, and the supernatant was discarded. All collected cell pellets were resuspended in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. Cells were seeded in 100 mm culture dishes and allowed to adhere for 90 min. Unadhered cells were discarded, and fresh culture medium was used for further culture.

[0039] The sense strand sequence of siANGPTL7 used in this embodiment is shown in SEQ ID NO:5 (5'-GGTGTTCTGTGACATGGAA-3'), and the antisense strand sequence is shown in SEQ ID NO:6 (5'-TTCCATGTCACAGAACACC-3').

[0040] (2) TGF-β1 stimulation activates primary rat cardiomyocytes Primary rat cardiac fibroblasts cultured to passages 2-4 and in good growth condition were seeded into appropriate culture plates. When the cell confluence reached 70-80%, the medium was replaced with a low-serum medium containing 0.5% or 1% fetal bovine serum for synchronization culture for 16 hours. The medium was then replaced with fresh low-serum medium containing recombinant human TGF-β1 (10 ng / mL), and a negative control group containing only an equal volume of solvent was established. The cells were then cultured at 37°C in a 5% CO2 incubator. Depending on the experimental objective, the experiment was terminated at different time points after stimulation (e.g., 24, 48, and 72 hours), and the cells were collected.

[0041] (3) Immunofluorescence staining to assess the fluorescence intensity of α-SMA and Col1 Cell slides were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After washing three times with PBS, 0.1% Triton X-100 was added for permeabilization for 10 minutes. After another PBS wash, the slides were blocked with 3% BSA at room temperature for 30 minutes. The blocking solution was discarded, and primary antibodies (anti-α-SMA, Collagen I antibody, dilution ratio 1:200) prepared with dilution buffer were added, and the slides were incubated overnight at 4°C. The next day, the primary antibody was recovered, washed three times with PBS, and the corresponding species-specific fluorescent secondary antibody was added. The slides were incubated at room temperature in the dark for 1 hour. After washing with PBS in the dark, the cell nuclei were counterstained with DAPI-containing mounting medium and the slides were mounted. The slides were observed and photographed under a fluorescence microscope. The results are as follows: Figure 6 The results showed that ANGPTL7 inhibitors significantly alleviated the activation of primary rat cardiomyocytes and extracellular matrix expression.

[0042] (4) EdU staining to assess cell proliferation Cultured fibroblasts were co-incubated with culture medium containing EdU (final concentration typically 10 μM) for 2 hours to label cells in the DNA synthesis phase. After incubation, the culture medium was discarded, and the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes. After PBS washing, 0.5% Triton X-100 was added for permeabilization for 20 minutes. After washing again with PBS, click reaction mixture (containing fluorescently labeled azide, reaction buffer, and catalyst) was added to each well, and the cells were incubated at room temperature in the dark for 30 minutes. After the reaction, the cells were washed three times with PBS, and DAPI-containing mounting medium was added for nuclear counterstaining. Under a fluorescence microscope, EdU-positive cell nuclei showed red fluorescence; the cell proliferation rate was calculated by calculating the proportion of EdU-positive nuclei to the total DAPI-positive cell nuclei. Results are as follows: Figure 6 The results showed that ANGPTL7 inhibitors significantly inhibited the proliferation of primary rat cardiac fibroblasts.

[0043] (5) Cell scratch assay to assess cell migration Primary rat cardiac fibroblasts were seeded at an appropriate density in six-well plates. When cell confluence approached 100%, three parallel straight lines were drawn perpendicularly to the bottom of the plate using a 200 μL sterile pipette tip along a ruler at a uniform speed. The cells were gently washed twice with PBS to remove detached cells and then replaced with maintenance medium containing 1% fetal bovine serum. The scratched areas were immediately marked under a microscope, and images were taken at 0 hours. The cells were then returned to the incubator for further culture. Images of the scratches were taken at the same location at different time points after culture. Finally, the scratch width was measured using ImageJ software, and the migration ability of cardiac fibroblasts was assessed by calculating the scratch closure rate. Results are as follows: Figure 6 The results showed that ANGPTL7 inhibitors significantly inhibited the migration ability of primary rat cardiac fibroblasts.

[0044] The above examples demonstrate that ANGPTL7 exhibits significant upregulation in various induced heart failure models. Using AAV9 to induce ANGPTL7 overexpression in mouse myocardium, cardiac function was assessed via echocardiography, and myocardial hypertrophy, fibrosis, and apoptosis-related indicators were detected through histological staining. Molecular biology analysis of myocardial injury markers revealed that ANGPTL7 overexpression exacerbates pathological myocardial remodeling and fibrosis. In cultured H9c2 rat cardiomyocytes, siRNA intervention targeting ANGPTL7 significantly reduced cardiomyocyte damage, apoptosis, and pathological myocardial hypertrophy. ANGPTL7 silencing also significantly inhibited the proliferation, migration, and transdifferentiation of primary rat cardiomyocytes. In conclusion, inhibiting ANGPTL7 gene expression suggests its potential application in the prevention and treatment of heart failure and its pathological characteristics.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An angiopoietin-like protein 7 inhibitor characterized in that: The angiopoietin-like protein 7 inhibitor is an siRNA, the siRNA comprises a sense strand and an antisense strand, and the siRNA is composed of any one of the following three groups of compounds, wherein: (1) the nucleic acid sequence of the sense strand is shown as SEQ ID NO: 1, and the nucleic acid sequence of the antisense strand is shown as SEQ ID NO: 2; (2) the nucleic acid sequence of the sense strand is shown as SEQ ID NO: 3, and the nucleic acid sequence of the antisense strand is shown as SEQ ID NO: 4; (3) the nucleic acid sequence of the sense strand is shown as SEQ ID NO: 5, and the nucleic acid sequence of the antisense strand is shown as SEQ ID NO:

6.

2. A cell, characterized by: The cell comprises the angiopoietin-like protein 7 inhibitor of claim 1.

3. A viral vector, characterized by: The viral vector comprises a nucleic acid encoding the angiopoietin-like protein 7 inhibitor of claim 1.

4. A pharmaceutical composition for inhibiting the expression of an ANGPTL7 gene, characterized by: The pharmaceutical composition comprises the angiopoietin-like protein 7 inhibitor of claim 1 and a pharmaceutically acceptable carrier.

5. A method for inhibiting the expression of an ANGPTL7 gene in a cell, the method comprising: introducing the angiopoietin-like protein 7 inhibitor of claim 1 into a target cell by any one of a viral vector-mediated introduction, a liposome transfection, an electroporation, or a microinjection.

6. The method of claim 5, wherein: The cell is a myocardial cell or a myocardial fibroblast cell of a human or a non-human animal.

7. Use of the angiopoietin-like protein 7 inhibitor of claim 1 in the preparation of a medicament for inhibiting the expression of an ANGPTL7 gene.

8. Use of the angiopoietin-like protein 7 inhibitor of claim 1 in the preparation of a medicament for preventing and / or treating heart failure.

9. Use according to claim 8, characterized in that: The heart failure comprises at least one of myocardial fibrosis, a chemotherapy drug-related cardiomyopathy, and a heart failure with preserved ejection fraction. The heart failure comprises at least one of myocardial fibrosis, a chemotherapy drug-related cardiomyopathy, and a heart failure with preserved ejection fraction.

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