Application of small molecule compound in ischemic heart disease

Abstract

The invention discloses application of a small molecule compound in ischemic heart disease, and relates to the technical field of ischemic heart disease treatment. And the CBX7 inhibitor is [delta]-Amyrenone. The delta-Amyrenone can be used as an inhibitor of CBX7 protein, so that the cell cycle is regulated, cell apoptosis is inhibited, myocardial cell proliferation is promoted, and the delta-Amyrenone has a good application prospect in prevention and / or treatment of ischemic heart disease.

Description

Technical Field

[0001] The present invention relates to the technical field of ischemic heart disease treatment, and in particular to application of a small molecule compound in ischemic heart disease. Background Art

[0002] Myocardial infarction (MI) is a major cause of disability and death worldwide. Its pathogenesis is mainly related to the rupture of coronary atherosclerotic plaques or endothelial erosion, especially in elderly patients. Although current treatment strategies focus on myocardial reperfusion, the reperfusion process itself can trigger further death of myocardial cells (i.e., reperfusion injury), leading to progressive loss of cardiac function and heart failure. Due to the extremely low proliferation capacity of adult mammalian cardiomyocytes, existing treatments are difficult to effectively repair the large number of cardiomyocytes lost after myocardial infarction reperfusion (MI / R).

[0003] The occurrence of myocardial reperfusion injury involves various complex mechanisms, including calcium overload, inflammatory response, mitochondrial dysfunction and oxidative stress. These mechanisms work together to further aggravate myocardial cell loss, expand the area of ​​necrosis, and significantly increase the risk of myocardial fibrosis. As the fibrosis process intensifies, the structure and function of the heart gradually deteriorate, which may lead to left ventricular remodeling, cardiac function decline, and eventually develop into chronic heart failure. In addition, reperfusion injury may also cause serious arrhythmias, further endangering the patient's life safety. These complications significantly affect the overall effect of reperfusion therapy and become an important obstacle to improving the long-term prognosis of patients.

[0004] Recent studies have shown that by targeting and inhibiting the key factor CBX7 through gene editing, cardiomyocytes can be induced to re-enter a proliferative state, thereby reducing the degree of injury in young mice during ischemia-reperfusion injury. However, MI should pay more attention to the biological characteristics of the elderly. In addition, gene therapy is limited by the long clinical transformation cycle and high technical barriers. In contrast, the development of small molecule inhibitors targeting CBX7 has significant advantages such as strong specificity and clear mechanism of action, and is easier to achieve clinical transformation.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide the use of CBX7 inhibitors in ischemic heart disease to solve the above technical problems.

[0007] The present invention is achieved in that: The present invention provides an application of a small molecule compound in ischemic heart disease. The CBX7 inhibitor is delta-Amyrenone or its salt or isomer.

[0008] The present invention has the following beneficial effects: The present invention has identified an effective small molecule inhibitor of CBX7 through screening, confirmed that δ-Amyrenone has a targeting effect on CBX7 protein, revealed the connection mode and binding site of the two through computer simulation, and verified the direct interaction between δ-Amyrenone and CBX7 from a practical perspective through in vitro molecular interaction experiments.

[0009] δ-Amyrenone can promote proliferation and inhibit apoptosis of cardiomyocytes. The results of cell viability detection experiments and staining experiments show that the screened CBX7 inhibitor has a significant effect on promoting proliferation. At the same time, flow analysis and protein immunoblotting experimental fluid revealed that it has a significant effect on inhibiting cardiomyocyte apoptosis. In addition, the present invention also found that the small molecule inhibitor δ-Amyrenone has a regulatory effect on the cycle of ischemic reperfusion cardiomyocytes through flow analysis, protein and gene levels. Therefore, δ-Amyrenone can be used as an inhibitor of CBX7 protein, thereby regulating the cell cycle, inhibiting cell apoptosis, and promoting cardiomyocyte proliferation, and has good application prospects in the prevention and / or treatment of ischemic heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0011] Figure 1 The results of the expression of CBX7 protein in the infarct area of ​​the MI / R model in aged mice ((a) ECG changes in mice before and after infarction; (b) TTC staining of heart sections of MI / R mice; (c) qPCR was used to detect the expression level of CBX7 in myocardial infarction-reperfused heart tissue; (d) Immunofluorescence was used to observe the distribution of CBX7 in myocardial infarction-reperfused heart tissue; (e) Western blot was used to analyze the expression of CBX7 in myocardial infarction-reperfused heart tissue, scale bar = 200 μm; images represent n = 3 independent repeated experiments; data in the figure represent Mean±SD; significant difference: **p<0.01); Figure 2 The structural formulas of the top 10 small molecule compounds with the highest docking scores after high-throughput screening; Figure 3Schematic diagram of CBX7-δ-Amyrenone visualization using PyMOL software ((a) Visualization of the binding between CBX7 protein and δ-Amyrenone molecule using molecular docking software; (b) Prediction of the binding site between CBX7 and δ-Amyrenone and the interaction between them); Figure 4 The results of the systematic optimization process of CBX7 protein expression conditions are shown in Figure 2 ((a) The effect of different IPTG concentrations on the expression level of CBX7 protein after induction of the recombinant plasmid; (b) The expression changes of CBX7 protein after IPTG induction of the recombinant plasmid at different temperatures were investigated; (c) The effect of IPTG induction time on the expression of CBX7 protein in the recombinant plasmid was further investigated; (d) The optimization of the Tris washing step in the CBX7 protein purification process and the schematic diagram of determining the optimal imidazole elution concentration; (e) The purification effect of the recovered protein); Figure 5 To verify the experimental results of the interaction between CBX7 and δ-Amyrenone in vitro ((a) The binding strength between δ-Amyrenone and CBX7 at different concentrations was obtained through in vitro molecular interaction experiments; (b) The signal values ​​at key time points before the termination of the experiment were selected and the affinity fitting curve was drawn to deeply analyze the affinity characteristics between CBX7 and δ-Amyrenone); Figure 6 The experimental results of the promotion of AC16 cell viability and proliferation by δ-Amyrenone ((a) After 24 hours of treatment, the effect of specified concentration of δ-Amyrenone on AC16 cell proliferation was determined by CCK8 method; (b) After AC16 cells were treated with 20 μM δ-Amyrenone for 24 hours, the cell survival was evaluated by trypan blue exclusion method, and the survival ratio was calculated; (c) This part of the data is directly related to part (b), showing the counting and calculation results of the survival ratio in detail; (d) After the cells were treated with 20 μM δ-Amyrenone for 24 hours, the cell survival was counted by YO-PRO-1 fluorescence staining method, image scale = 200 μm, and the images shown are representative of three independent repeated experiments; all data are expressed as Mean±SD; significant differences are marked as: p<0.05， ***p<0.0001); Figure 7Figure 1 shows the experimental results of the inhibitory effect of δ-Amyrenone on AC16 cell apoptosis ((a) After AC16 cells were treated with 20 μM δ-Amyrenone for 24 hours, the cell apoptosis was analyzed by flow cytometry; (b) A statistical chart of the positive cell rate of the flow cytometry results in (a) was provided; (c) After AC16 cells were treated with 20 μM δ-Amyrenone for 24 hours, the cell apoptosis was observed using apoptosis and necrosis staining technology, image scale = 200 μm; (d) Western blot experiments were performed to further explore the changes in the expression levels of apoptosis-related proteins after δ-Amyrenone treatment; all images shown are representatives of three independent repeated experiments; data are presented in the form of Mean±SD, and significant differences are marked as ****p<0.0001); Figure 8 The results of the experiment of δ-Amyrenone promoting AC16 cell proliferation by regulating the cell cycle ((a) AC16 cells were treated with 20 μM δ-Amyrenone for 24 hours, and then the distribution of the cell cycle was analyzed by flow cytometry; (b) The statistical graph of the positive cell ratio of the flow cytometry results in (a) was shown; (c) The changes in the expression levels of cell cycle-related proteins after δ-Amyrenone treatment were detected by Western blot experiments to further verify its effect on the cell cycle; all images are from representatives of three independent repeated experiments; data are expressed as Mean±SD, and significant differences are marked as *p<0.05, **p<0.01, ***p<0.001); Fig. 9 The experimental results of δ-Amyrenone regulating CBX7 protein and then affecting cell cycle-related proteins ((a) After treating AC16 cells with a specific concentration (20 μM) of δ-Amyrenone for 24 hours, the expression pattern of cell cycle-related protein genes was analyzed by qPCR technology; (b) Western blot was used to detect the changes in cell cycle protein-related markers after δ-Amyrenone treatment; the data in the figure represent Mean±SD; significant difference: ****p<0.0001). DETAILED DESCRIPTION

[0012] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0013] The present invention provides an application of a CBX7 inhibitor in preparing a drug for preventing and / or treating ischemic heart disease. The CBX7 inhibitor is delta-Amyrenone or a salt or isomer thereof.

[0014] δ-Amyrenone CAS No.: 20248-08-2, molecular formula C 30 H 48 O, the structural formula is as follows: .

[0015] The inventors used Discovery Studio software to implement a high-throughput screening strategy on the small molecule compound library provided by Taoshu Biotechnology, and successfully identified a variety of compounds that can effectively bind to the CBX7 protein. After screening, it was found that CBX7 and δ-Amyrenone have a strong binding affinity. Experiments show that δ-Amyrenone can act as an inhibitor of CBX7 protein, thereby regulating the cell cycle, inhibiting cell apoptosis, and promoting cardiomyocyte proliferation, and has good application prospects in the prevention and / or treatment of ischemic heart disease.

[0016] In a preferred embodiment of the present invention, the above-mentioned ischemic heart disease includes but is not limited to: any one of myocardial infarction, myocardial infarction reperfusion injury, ventricular fibrosis, myocarditis, arrhythmia, angina pectoris, heart failure, coronary heart disease and myocardial ischemia.

[0017] In a preferred embodiment of the present invention, the above-mentioned ischemic heart disease is myocardial infarction reperfusion injury.

[0018] In a preferred embodiment of the present invention, the above-mentioned drug has at least one of the following uses: (1) Promote the proliferation of cardiomyocytes; (2) Inhibit the expression of apoptotic proteins; (3) Slowing down the apoptosis of myocardial cells induced by hypoxia-reperfusion and / or ischemia-reperfusion; and (4) improving cell cycle abnormalities caused by hypoxia-reperfusion and / or ischemia-reperfusion.

[0019] In a preferred embodiment of the present invention, the above-mentioned use (4) includes: upregulating the level of at least one of the following genes and / or proteins: CDCA2, ARRB1, Sirt7, ALDH7A1 and FoxM1.

[0020] In a preferred embodiment of the present invention, the above-mentioned drug has at least one of the following uses: (1) Promote the repair of ischemic myocardial tissue; (2) Reduce myocardial infarction area; and (3) reduce cardiac fibrosis.

[0021] In a preferred embodiment of the present invention, the above-mentioned drug has at least one of the following uses: (1) Reduce the overexpression of G1 phase-related proteins in cardiomyocytes; (2) Increase the expression of S phase-related proteins in cardiomyocytes; (3) Promote the G1 / S phase transition of cardiomyocytes; (4) Promote the S / G2 phase transition of cardiomyocytes.

[0022] In a preferred embodiment of the present invention, the G1 phase-related protein is Cyclin D1 protein.

[0023] In a preferred embodiment of the present invention, the above-mentioned medicine further includes a pharmaceutically acceptable carrier.

[0024] In another embodiment, the medicament can be a preparation of tablets, pills, powders, lozenges, sachets, cachets, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions or sterile packaged powder injections.

[0025] In one embodiment, the active ingredient (δ-Amyrenone) is prepared into a drug or pharmaceutical composition so that it can release the active ingredient quickly, slowly or delayed after administration to a subject, for example, the active ingredient can be mixed with a carrier, diluted with a carrier or encapsulated in a carrier. The carrier, excipient and diluent can be a carrier, excipient and diluent that acts as an active ingredient, or a carrier, excipient and diluent that acts as a medium. In another embodiment, the drug or pharmaceutical composition in the above application can be a solid, semisolid or liquid material, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water syrup, methylcellulose, methyl and propyl paraffin, talc, magnesium stearate or liquid paraffin.

[0026] In another embodiment, the drug or pharmaceutical composition in the above application may further include a lubricant, a wetting agent, an emulsifying and suspending agent, and a preservative.

[0027] In a preferred embodiment of the present invention, the above-mentioned drug is administered by injection.

[0028] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0029] Example 1 This example provides an experiment to test the expression of CBX7 protein in the infarct area of ​​the MI / R model in aged mice.

[0030] 1. Experimental Animals SPF-grade C57 mice provided by Chengdu Dashuo were used as experimental subjects, regardless of gender, and continued to be cultured until the age of 2. To ensure the sanitary conditions of the animals, the experimental mice were kept in an environment with a 12-hour light-dark cycle control and free access to clean drinking water and high-quality feed. Animal ethics standards: Strictly follow the guidelines of the Experimental Animal Ethics Review Committee of Sichuan Provincial People's Hospital.

[0031] 2. Experimental Materials and Reagents Table 1 Main experimental materials and reagents

[0032]

[0033] 3. Experimental Methods 3.1 MI / R model construction First, the pleura was exposed and the thoracotomy was performed at the second and third intercostal spaces on the left side of the chest. After that, the thoracotomy was performed at the second and third intercostal spaces on the left side to expose the heart. A slipknot was tied at the origin of the left anterior descending coronary artery using 7-0 silk suture and the left anterior descending coronary artery was closed. During the operation, it was ensured that there was no obvious bleeding in the cardiac cavity and that the operation area was kept clean. Finally, the surgical area was sutured layer by layer to ensure that the tissue layers were fully adhered. After 40 minutes of ischemia, the coronary artery ligature was removed. At the end of the experimental period, the animals were euthanized using an overdose of isoflurane, and major organ tissues were removed for further analysis.

[0034] 3.2 Tissue collection and real-time quantitative PCR (RT-qPCR) Table 2 Primers for different genes

[0035] (1) Model construction and sample processing The mouse MI / R model was established according to 3.1. After 24 hours, the mice were anesthetized and humanely killed, and then the hearts were removed for experimental use.

[0036] (2) Cardiac region division and tissue sample collection The removed heart was divided into three areas according to the infarct area, the peripheral area and the non-infarct normal area, and equal amounts of tissue were collected from each area for subsequent experimental analysis.

[0037] (3) RNA extraction steps TRIzol reagent was used to extract RNA from the three tissues, and the total RNA was purified using the SteadyPure Tissue Cell Small RNA Extraction Kit.

[0038] (4) cDNA synthesis process The extracted RNA samples were reverse transcribed into cDNA using a reverse transcription kit.

[0039] (5) RT-qPCR detection RT-qPCR detection was performed using a qPCR kit. The reaction system was placed in a qPCR instrument, the temperature gradient was set according to the instructions, and the cycle threshold (Ct) of the PCR product was finally accurately determined.

[0040] 3.3 Tissue collection and western blotting (1) Tissue sample acquisition and lysis processing: The mouse MI / R model was constructed according to 3.1. After 24 hours, the mice were anesthetized and humanely killed, and then the heart was removed for experimental use. Samples were obtained from the infarcted area, marginal area and normal area of ​​the heart, grinding beads were added, and the tissue was further refined using a grinding device after RIPA lysis solution. The tissue was lysed in an ice bath, and the supernatant was obtained by centrifugation. The protein concentration was determined by the BCA method. Subsequently, the protein sample was diluted to 30 μg / μL with 5× loading buffer and an appropriate amount of RIPA lysis solution, heated in a 95°C metal bath for 10 minutes to denature it, and stored at -80°C after aliquoting for use.

[0041] (2) Electrophoresis: Prepare the gel according to the instructions of the kit. After the gel is stable, load the sample, set the voltage and start electrophoresis. End the electrophoresis when all the bands are separated.

[0042] (3) Transfer and blocking steps: Transfer the protein to the PVDF membrane in the transfer tank. After the transfer is completed, the membrane is blocked with 5% skim milk powder for 1-2 hours, and then the membrane is washed with PBST buffer on a decolorizing shaker for 5 minutes each time, for a total of 6 washes.

[0043] (4) Antibody binding reaction: Immerse the blocked membrane in the target primary antibody and shake it at 4°C overnight. Recover the primary antibody, wash with PBST for 5 minutes, 6 times, then place in the corresponding species secondary antibody, incubate at 37°C for 1-2 hours, and then wash the membrane again.

[0044] (5) Chemiluminescence: Prepare the developer according to the developer instructions. Place the PVDF membrane in the exposure machine, add the developer, and record and analyze the development results.

[0045] 3.4 Tissue collection and immunofluorescence (1) Process of obtaining, fixing and dehydrating heart samples: The MI / R model of aged mice was constructed according to 3.1. After 24 hours, the mice were anesthetized and humanely killed, and then the hearts were removed for experiments. The heart tissue was immersed in a 4% paraformaldehyde solution and fixed for 24 hours to ensure that the integrity of the tissue structure was maintained. Next, the heart tissue was dehydrated using a fully automatic tissue dehydration device.

[0046] (2) Paraffin embedding and thin section preparation of cardiac tissue: The dehydrated cardiac tissue needs to be further embedded in paraffin for subsequent sectioning. When sectioning, it is necessary to ensure that the thickness of the cardiac tissue slice is 5 microns to meet the specific needs of the experiment.

[0047] (3) Dewaxing and hydration: Place the paraffin sections into a fully automatic staining machine for baking and dewaxing to complete hydration.

[0048] (4) Antigen repair: Place the sections in a steam pressure cooker containing sodium citrate buffer (pH 6.0) for high temperature and high pressure repair for 5-10 minutes. After cooling naturally to room temperature, rinse the sections with PBS three times, 5 minutes each time.

[0049] (5) Penetration and blocking: The slices were treated with a 0.5% concentration of Triton X-100 solution to enhance the penetration of antibodies in tissues. Subsequently, the slices were blocked with goat serum for 30 minutes at room temperature to reduce the interference of nonspecific binding and ensure the accuracy of the experimental results.

[0050] (6) Primary antibody incubation: Add primary antibody according to the ratio in the instruction manual, cover the sliced ​​tissue, and incubate overnight in a humidified box at 4°C. Then wash the slices three times with PBS, 5 minutes each time.

[0051] (7) Secondary antibody incubation: Then add fluorescently labeled secondary antibody (select the corresponding secondary antibody according to the primary antibody host, and the dilution ratio is shown in the instructions), incubate at 37°C for 1 hour, and then wash again with PBS 3 times, 5 minutes each time.

[0052] (8) DAPI staining and sealing: Stain with DAPI for 20 minutes at 37°C to mark the cell nucleus. Wash with PBS three times, 5 minutes each time, to remove excess stain. Add an appropriate amount of sealing solution, cover the slice with a coverslip, and gently flatten it to prevent bubbles.

[0053] (9) Microscopic observation and photography: Observe the sections using a fluorescence microscope, use appropriate excitation wavelengths to capture the target fluorescence signal and nuclear staining signal in sequence, and save the images for subsequent analysis.

[0054] 3.5TTC staining (1) Sample collection: The mice were anesthetized and then killed, and their hearts were removed for experimental use.

[0055] (2) Preparation of TTC solution: Weigh 0.5 g of TTC powder and dissolve it in 50 mL of distilled water to prepare a 1% TTC solution.

[0056] (3) Staining: Wash the heart and cut it into slices with a thickness of 1 to 2 mm from the middle to the bottom of the heart. Place the slices in TTC solution, making sure they are immersed, and then incubate at 37°C for 30 min.

[0057] (4) Observation and photography: Take out the slices and drain the liquid, arrange the slices in order and take photos with a camera.

[0058] To explore the expression changes of CBX7 after MI / R, this study established a model with aged mice and collected heart tissues 24 h after infarction. By comparing the electrocardiogram of mice before and after infarction, the ST segment of the electrocardiogram of the infarction model was elevated, indicating that the myocardial infarction model was successfully established ( Figure 1 This was also demonstrated by the TTC results of mouse heart slices 1 day later ( Figure 1 b). The expression of CBX7 was analyzed by qPCR, Western Blot and immunofluorescence detection. First, one day after the successful establishment of the model, myocardial tissues of the infarct (IZ), marginal (MZ) and normal area (NZ) were taken for RT-qPCR detection. The experimental results showed that compared with the normal myocardial tissue area, the myocardial tissue in the infarct area showed a significant upregulation trend in the mRNA expression of CBX7, with a P value of 0.0013 ( Figure 1 In the middle (c), the difference in gene transcription level was highlighted. In order to further explore this change, Western Blot technology was further used to verify the protein expression of CBX7. The results showed that the expression of CBX7 protein in the infarct area was significantly higher than that in the normal area ( Figure 1 In addition, the localization and expression intensity of CBX7 were observed by immunofluorescence technology, and it was found that the CBX7 fluorescence signal in the infarct area was significantly enhanced, such as Figure 1 As shown in middle d, this is consistent with the aforementioned mRNA and protein expression results.

[0059] In summary, the expression of myocardial CBX7 in aged mice changed significantly after MI / R, and this finding suggests that CBX7 may become a potential therapeutic target in the repair process of myocardial infarction.

[0060] Example 2 In this example, the information of CBX7 protein was obtained from the Uniport database, and the small molecule compound library was obtained from Taoshu Biotechnology, and the compounds with the most potential specific binding ability were screened out through Discovery Studio software. Subsequently, biological experiments were conducted to evaluate whether the small molecule compound could regulate the cell cycle of cardiomyocytes by inhibiting CBX7, thereby promoting the repair process of cardiomyocytes and inhibiting cell apoptosis, providing experimental basis for subsequent related research.

[0061] 1. Experimental Materials (1) Experimental cell line: AC16 human cardiomyocyte cell line was derived from ATCC (American Type Culture Collection).

[0062] (2) Experimental software: Table 3 Experimental software

[0063] (3) Experimental reagents Table 4 Main experimental materials and reagents

[0064] Common reagent preparation (1) Cell culture medium: Add 5.6 ml of double antibody and 56 ml of FBS to every 500 ml of basal culture medium DMEM / F-12.

[0065] (2) Culture medium of Escherichia coli: Add 10 g of trypsin, 5 g of yeast powder, and 10 g of sodium chloride to every 1000 ml of double distilled water, and then adjust the pH value of the solution to 7.2.

[0066] 2. Experimental Methods 2.1 Discovery Studio software high-throughput screening and AutoDock validation process (1) Obtaining CBX7 protein structure information: By accessing the Uniprot database (URL: https: / / www.uniprot.org / ), the 3D structure file of CBX7 protein was downloaded and then imported into Discovery Studio software. Next, the protein structure was preprocessed as necessary, including removing irrelevant ligands and redundant parts, adding hydrogen atoms, and performing energy minimization optimization steps. Finally, the active site of the protein was determined, completing the structural preprocessing.

[0067] (2) Integration and preparation of the small molecule compound library: After the introduction of Taoshu Biotechnology's small molecule compound library, a series of pretreatment processes were performed on it, including adding hydrogen atoms, removing salt ions, adjusting charge distribution, etc., and energy minimization calculations were performed by applying molecular force fields to generate small molecule three-dimensional structures suitable for molecular docking analysis.

[0068] (3) Perform simulated docking: After the small molecule and protein are prepared, click libdock in the Discovery Studio software to perform docking.

[0069] (4) Screening and comprehensive evaluation of candidate molecules: Small molecule compounds were screened based on criteria such as the rationality of the binding site, the minimum binding free energy, and the LibDockScore score, aiming to identify candidate molecules with the best binding potential and provide valuable candidates for the subsequent experimental verification stage.

[0070] (5) AutoDock verification: To further verify the binding mode and energy stability of the selected candidate compounds, AutoDock4 was used for molecular docking analysis. The protein and small molecule structure files were processed into PDBQT format, water molecules and impurities were removed, and polar hydrogen atoms were added. The active site was defined as a 126 Å × 126 Å × 126 Å cubic grid, and the grid spacing was set to 0.05 nm. The docking operation was performed under the Lamarckian Genetic Algorithm (LGA) conditions to obtain the binding posture and binding energy data of the candidate compounds and the CBX7 protein, and the complex with the lowest binding energy and stable binding mode was screened to provide a reference for further research.

[0071] 2.2 PyMOL software visualization of protein-small molecule compounds (1) Importing structure files: Import the optimal protein-small molecule complex structure file (PDB format) obtained by AutoDock4 docking into PyMOL software for visual analysis.

[0072] (2) Structural display of proteins and small molecules: Load protein structures and small molecule compounds in PyMOL, and adjust the color and display mode of the protein backbone, small molecule compounds and related ligands respectively. Commonly used display modes include line, sticks and surface modes to highlight the interaction between the binding position of small molecules and the active site of proteins.

[0073] (3) Analysis of binding sites: Use PyMOL's measurement tools to analyze the key interactions between small molecule compounds and protein active sites, including hydrogen bonds, hydrophobic interactions, π-π stacking, etc. By marking and highlighting these key interactions, an intuitive basis is provided for analyzing binding stability.

[0074] (4) Optimized display of secondary structure and binding mode: The secondary structure of the protein (such as α-helix and β-fold) is displayed as a ribbon (cartoon) mode, and the small molecule compound and protein binding region are displayed in a rod or surface mode to ensure that the overall structure is clear and well-organized.

[0075] (5) Marking of key residues: The key residues that bind to the small molecule and their numbers are marked, and the hydrogen bond distance or non-bonded interaction distance data are displayed to provide support for explaining the binding mechanism of the small molecule.

[0076] (6) Image and video export: Take screenshots of the three-dimensional structure of key binding sites from multiple angles and save them as high-resolution images for reporting and publication; or generate rotating animations to show the dynamic interaction effects of protein-small molecule complexes. Based on the visualization results, summarize the binding characteristics and action modes of small molecules and proteins, providing a basis for subsequent molecular optimization and functional verification.

[0077] 2.3 Recombinant protein expression and purification (1) Expression of CBX7 protein: The base sequence of human CBX7 protein was obtained from the database, His tag and Flag tag were added to the head and tail, respectively, cloned into a plasmid and loaded into E. coli, and then cultured. When the OD600 value of each 200 ml bacterial solution grew to an appropriate range of 0.6 to 0.8, IPTG at different concentrations of 0.1 mM to 1.0 mM was used to induce protein expression at 16°C for 12 hours.

[0078] (2) Verification process of protein expression effect: After the induction expression is completed, the bacterial precipitate is collected and ultrasonically lysed under ice bath conditions to obtain a lysate containing the target protein. Subsequently, 20 μL of 5× loading buffer is added to 100 μL of lysate and heated in a 95°C metal bath for 10 minutes to denature the protein. The proteins are separated by SDS-PAGE electrophoresis technology, and the induction effect under different IPTG concentrations is detected by Coomassie Brilliant Blue staining to determine the optimal IPTG concentration. At the same time, the temperature and time of induced expression were optimized using the same method.

[0079] (3) Purification process of CBX7 protein: Under the optimal induction conditions, the bacterial precipitate was collected again and subjected to ultrasonic lysis, and then the supernatant was obtained by centrifugation. The CBX7 protein with the His tag was captured using a Ni-NTA affinity chromatography column. The impurity protein was removed by 6 Tris washes, and then different concentrations of imidazole were used for elution to recover the CBX7 protein. The protein content in each concentration of imidazole was detected by gel electrophoresis to determine the optimal imidazole concentration.

[0080] (4) Collection and purification of CBX7 protein: After the eluate containing CBX7 protein is collected, it is concentrated using an ultrafiltration tube. In order to further improve the purity of the protein, the concentrate is loaded onto an SDS-PAGE gel, the band position is determined by electrophoresis separation and staining, and the band is cut off. The protein is recovered using a kit to extract a high-purity CBX7 protein. Finally, a purified CBX7 protein sample is obtained.

[0081] 2.4 Surface plasmon resonance molecular interaction experiments (1) Determination of protein concentration: The BCA method was used to measure the concentration of CBX7 protein.

[0082] (2) Biotinylation labeling: The CBX7 protein was biotinylated according to the kit instructions and then fixed to the sensor chip.

[0083] (3) Binding of CBX7 to the sensor: Place the SSA sensor in contact with the protein in (2) and solidify according to the instructions.

[0084] (4) Sampling treatment: Prepare small molecule solutions of different concentrations and blank controls and inject them sequentially (0-100 nM).

[0085] (5) Recording and analysis: Parameters were set according to standard procedures, and corresponding programs were set up to monitor signal changes during baseline, binding, and dissociation. The data were processed using analysis software to calculate the affinity constants of the two, thus providing strong data support for subsequent research.

[0086] 2.5 Construction of myocardial cell ischemia-reperfusion / hypoxia model (1) Cell culture: Select human cardiomyocytes AC16 and culture them in a suitable culture medium environment to ensure that the cells maintain a good growth state.

[0087] (2) Simulated ischemia treatment: To simulate ischemia, the culture medium of AC16 cells was replaced with serum-free medium, and the cells were placed in a three-gas incubator. By adopting a mixed gas culture method and introducing nitrogen to exhaust other gases in the incubator, an anaerobic environment was created. For the hypoxia group, complete culture medium was used, and the gas conditions were consistent with those of the ischemia group.

[0088] (3) Collection and analysis of cell samples: When the oxygen concentration in the incubator is monitored to be lower than 0.5%, the timer starts. After 2 hours, the culture conditions return to normal, which is reperfusion. After reperfusion for more than 24 hours, the cells are collected.

[0089] 2.6 CCK-8 assay for cell viability, trypan blue staining, and YP-1 staining (1) CCK-8 method: Myocardial cells were seeded into 96-well plates, and the reperfusion model was constructed according to the method described above. Then, culture medium with different concentrations of small molecules was added. After 24 hours, CCK-8 reagent was added and incubated at 37°C for 2 hours. Subsequently, the absorbance value at OD450 was measured, and the OD value was processed and analyzed using GraphPad software to quantify cell viability.

[0090] (2) Trypan blue staining: Cardiomyocytes were seeded into 6-well plates to construct a reperfusion model, and small molecule drugs were added according to the optimal concentration determined by the CCK8 method. After 24 hours, cells were labeled by trypan blue staining. The images were observed and recorded under a microscope, and the staining images were analyzed using ImageJ software to distinguish between live and dead cells. The quantitative data of cell survival rate were then statistically analyzed using GraphPad software.

[0091] (3) YO-PRO-1 staining: Myocardial cells were seeded into 6-well plates to construct a reperfusion model, and small molecule drugs were added according to the optimal concentration determined by the CCK8 method. After 24 hours, the cells were labeled with YO-PRO-1 staining. Fluorescence microscopy was then used to observe and capture images to visually display cell apoptosis.

[0092] 2.7 Flow cytometry detection of cell cycle (1) Preparation process of cardiomyocyte samples: A cardiomyocyte ischemia-reperfusion model was constructed according to the method shown in 2.5, and small molecule drug intervention was introduced. After 24 hours, the cells were digested and collected, washed with PBS and centrifuged to remove excess liquid, fixed with 70% ethanol for more than half an hour, and then resuspended.

[0093] (2) Sample staining steps: Add an appropriate amount of propidium iodide staining solution to each group of prepared cell samples, mix thoroughly, and incubate at 37°C in the dark for 30 minutes to ensure sufficient and uniform staining.

[0094] (3) Flow cytometry detection and data analysis method: A flow cytometer was used to detect the fluorescence signal and light scattering characteristics of cells under 488 nm excitation light. FlowJo software was used to conduct in-depth analysis of the collected data to reveal the changes in the cell cycle.

[0095] 2.8 Flow cytometry detection of cell apoptosis (1) Preparation process of cardiomyocyte samples: Follow the method shown in 2.5 to construct a cardiomyocyte ischemia-reperfusion model and apply small molecule drug treatment. After 24 hours of treatment, trypsinize the cells, wash with PBS and resuspend, count and collect 100,000 cells into a 1.5 mL centrifuge tube. After removing the supernatant by centrifugation, add 195 μL of Annexin V-FITC conjugate solution and gently resuspend the cells. (2) Cell apoptosis staining steps: Add 5 μl of Annexin V-FITC and 10 μl of propidium iodide staining solution to the sample in (1) according to the instructions. Incubate the mixture at room temperature (20-25°C) in the dark for 10 to 20 minutes. During this period, wrap it with aluminum foil to fully block light. After incubation, transfer the sample to ice to keep it cold to reduce false positives caused by continued staining.

[0096] (3) Flow cytometry detection and data: Flow cytometry was used to detect the FITC and PI wavelength data of the samples. FlowJo software was used to analyze the data and quantify cell apoptosis.

[0097] 2.9 Detection of cell apoptosis by fluorescence microscopy (1) Cell sample preparation process: Cardiomyocytes were seeded in a 96-well plate at a density of 3,000 cells per well. Subsequently, a cardiomyocyte ischemia-reperfusion model was constructed according to the method described in 2.5, and small molecule drugs were introduced for intervention. After the treatment lasted for 24 hours, the 96-well plate was centrifuged for 5 minutes to prepare for the subsequent staining step.

[0098] (2) Cell apoptosis staining process: Perform staining according to step (2) shown in 2.8.

[0099] (3) Fluorescence microscopy observation: After staining, the cells are observed using a fluorescence microscope and corresponding fluorescent images are taken to record the staining of cell apoptosis.

[0100] 2.10RT-qPCR (1) Sample processing: AC16 cells were seeded into 6-well plates and the reperfusion model was constructed as described in 2.5. After 24 hours of adding small molecules, the cells were collected.

[0101] (2) Extraction of total RNA: Extract total cellular RNA according to the kit instructions and measure the concentration and purity of the resulting RNA.

[0102] (3) The subsequent steps are carried out in accordance with the steps in 3.2 of Example 1.

[0103] Table 5 Primers for different genes

[0104] 2.11 Protein extraction and western blotting (1) Construction of cell model and pretreatment: Myocardial cells were inoculated into a 10 CM cell culture dish and the ischemia-reperfusion model was constructed according to the steps in 2.5. After adding small molecule drugs for 24 hours, cell samples were prepared for subsequent protein analysis experiments.

[0105] (2) Protein extraction: Wash the cells in (1) three times with PBS. Then, drain the PBS, add lysis buffer, scrape the cells repeatedly to obtain a homogenate and transfer it to a centrifuge tube. After standing on ice for 30 minutes, ultrasonic treatment is performed to break the cells. After centrifugation at 12000g for 20 minutes, the supernatant is collected without touching the tube wall. Then the concentration is determined by the BCA method, and the loading buffer and lysis buffer are added to adjust the total protein concentration, and the protein is denatured in a metal bath at 95°C for 10 minutes.

[0106] (3) For the subsequent steps, refer to 3.3 of Example 1.

[0107] 2.12 Statistical analysis The data obtained from the experiment were expressed as mean ± standard deviation and evaluated by statistical methods such as T test and one-way analysis of variance (ANOVA). In the statistical analysis results, a P value less than 0.05 was set as the criterion for significant difference, where * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001, all of which are used to indicate statistically significant differences.

[0108] 3. Experimental results 3.1 CBX7 inhibitor screening Using Discovery Studio software, a high-throughput screening strategy was implemented on the small molecule compound library provided by Taoshu Biotechnology, and a variety of compounds that can effectively bind to the CBX7 protein were successfully identified. They can all form a three-dimensional stable binding with the target protein. The most promising small molecule δ-Amyrenone was screened out based on LibdockScore for subsequent verification. Table 6 lists some information on the top 10 small molecule compounds with docking scores. Figure 2 The corresponding chemical structure is shown. The binding mode and energy stability of the candidate compound δ-Amyrenone were verified by AutoDock software. Among them, δ-Amyrnone and CBX7 have a binding energy of -11.61 kcal / mol and a ligand efficiency of -0.37 kcal / mol, indicating that the binding is very stable.

[0109] Table 6 The top 10 small molecule compounds with the highest docking scores after high-throughput screening

[0110] 3.2 Analysis of docking between δ-Amyrenone and CBX7 protein In order to further verify the molecular docking between δ-Amyrenone and CBX7, PyMOL software was used to visualize the relevant structures. After careful analysis, the results showed that CBX7 tightly wrapped and bound δ-Amyrenone (see Figure 3 ).exist Figure 3 In the figure, it can be clearly observed that δ-Amyrenone forms an efficient binding interaction with CBX7, and the key binding sites involve the isoleucine (ILE) residues at positions 6 and 48, valine (VAL) at positions 10 and 13, phenylalanine (PHE) at position 11, tryptophan (TRP) at position 32, and histidine (HIS) at position 47, forming multiple binding pockets. In terms of interaction force, δ-Amyrenone forms a stable binding with the amino acid residues ILE, TRP, and PHE through Alkyl-Alkyl interactions. In addition, δ-Amyrenone binds to VAL through Pi-Alkyl interaction forces, and interacts with HIS through Pi-Alkyl and Pi-Sigma interaction forces. These interactions play a key role in the binding stability of δ-Amyrenone with the target protein, indicating that these amino acid residues have an important influence on its potential biological activity. The distances of these interactions are 3.47 Å, 4.88 Å, 4.58 Å, 3.43 Å, 5.45 Å, 4.29 Å, 5.16 Å, 5.08 Å, 4.34 Å, 4.79 Å, 5.50 Å, and 5.30 Å. This docking analysis revealed the binding mode between δ-Amyrenone and CBX7, providing reference information and theoretical basis for subsequent biological verification experiments.

[0111] 3.3 Systematic optimization of CBX7 protein expression and purification conditions In order to explore the effects of IPTG concentration, induction time and culture temperature on the induced expression of CBX7 protein, a series of experiments including different IPTG concentration gradients (0 to 1 mM), time gradients (0 to 12 hours) and temperature gradients (16 ℃ to 37 ℃) were carefully designed using the controlled variable method. Subsequently, Coomassie Brilliant Blue staining was used to verify the experimental results. The experimental data clearly showed (see Figure 4 ), under the conditions of IPTG concentration of 0.2 mM, induction temperature of 22°C and duration of 10 hours, the expression level of CBX7 protein reached a peak (see the specific image for details). Figure 4 middle ac). The CBX7 protein was extracted using a Ni-NTA purification column and the target protein was isolated from the bacterial lysate. The optimal number of Tris washes was determined to be 5 times by staining (see Figure 4 d), because the impurity protein components had been removed in the fifth time. Further analysis showed that 20 mM imidazole was the optimal elution condition for the target protein. Therefore, in subsequent purification experiments, 20 mM imidazole eluent was selected to purify the target protein. After collecting the eluate containing CBX7 protein, it was concentrated using an ultrafiltration tube. In order to further improve the purity of the protein, the concentrate was loaded onto SDS-PAGE gel. After electrophoretic separation, high-purity CBX7 protein was extracted by gel cutting and recovery, and finally a purified CBX7 protein sample was successfully obtained (see for details). Figure 4 (e).

[0112] 3.4 Molecular Interactions between CBX7 and δ-Amyrenone in vitro The interaction mechanism between CBX7 and δ-Amyrenone was discovered through in vitro molecular interaction experiments. The experimental results showed that the two exhibited rapid binding and dissociation kinetics, and the binding strength increased significantly with the increase of δ-Amyrenone concentration (see Figure 5 Through detailed data analysis, the dissociation constant Kd value of CBX7 and δ-Amyrenone was calculated to be 49.65 nM, which strongly proves that there is a strong binding affinity between them (see Figure 5 (b)

[0113] Although current experimental results reveal that they have high affinity, more extensive and in-depth research is needed to fully and deeply analyze their potential pharmacological effects.

[0114] 3.5δ-Amyrenone promotes AC16 cell proliferation in vitro In order to explore the effect of δ-Amyrenone on cardiomyocyte proliferation, CCK-8 experimental method was used. The experimental results showed (see Figure 6 In (a), different concentrations of δ-Amyrenone showed different effects on the activity of cardiomyocytes after ischemia-reperfusion treatment, among which 20 μM was the best concentration. Compared with the ischemia-reperfusion group, the activity of cardiomyocytes was significantly increased by 13.87% ± 7.87% after treatment with 20 μM δ-Amyrenone. Based on this finding, 20 μM was selected as the working concentration of δ-Amyrenone in subsequent experiments.

[0115] In addition, in order to further evaluate the pro-proliferation effect of δ-Amyrenone, CCK-8 experiments were performed at the same concentration (20 μM) on the other 9 small molecule compounds with the highest docking scores in Table 6. The results showed that among all the candidate molecules, δ-Amyrenone still exhibited the best pro-proliferation activity (see Figure 6 (b)

[0116] In order to further verify the proliferation effect at this concentration, trypan blue and YO-PRO-1 staining experiments were performed. The trypan blue staining results showed that the cardiomyocytes in the 20 μM δ-Amyrenone treatment group proliferated significantly (see Figure 6 Meanwhile, a significant decrease in fluorescence intensity was observed in the YO-PRO-1 staining experiment (see Figure 6 (d) This suggests that the changes in the cell state in the δ-Amyrenone-treated group may be related to the enhancement of proliferation activity. These experimental results further strengthen the potential role of δ-Amyrenone in promoting cardiomyocyte proliferation.

[0117] 3.6 Analysis of δ-Amyrenone regulating cardiomyocyte apoptosis Apoptosis, as a core mechanism for regulating cell fate, plays an indispensable role in maintaining tissue and organ homeostasis. In order to further explore the specific effects of δ-Amyrenone on cardiomyocyte proliferation, flow cytometry was performed to evaluate cell apoptosis. Experimental data revealed that the cell apoptosis rate in the normal (control) group was maintained at a low level of 5.15% ± 0.13%. However, in cells undergoing hypoxia-reperfusion treatment and ischemia-hypoxia-reperfusion treatment, the apoptosis rates climbed to 6.60% ± 0.24% and 15.91% ± 0.77%, respectively. However, in the cells of the ischemia-hypoxia-reperfusion group after δ-Amyrenone intervention, the apoptosis rate was significantly inhibited, reduced to 10.42% ± 0.25% (see details). Figure 7 In addition, the fluorescence image results ( Figure 7 Figure c) also confirms this result.

[0118] Changes in apoptosis are usually associated with apoptosis-related proteins. WB was used to explore changes in the expression patterns of related proteins. In the constructed ischemia-reperfusion injury model, it was observed that the expression levels of c-caspase-3 and c-PARP, two key apoptosis proteins, were significantly upregulated compared with the normal group. However, in the δ-Amyrenone-treated group, the expression levels of these two proteins showed a significant downward trend. At the same time, it is worth noting that compared with the ischemia-reperfusion group, the expression of Bcl-2 and Bax proteins in the δ-Amyrenone-treated group also showed a downward trend (see details). Figure 7This series of experimental results strongly indicate that δ-Amyrenone significantly slows down the process of cell apoptosis by effectively inhibiting the expression of apoptosis-related proteins, thus playing a positive protective role.

[0119] 3.7 Analysis of δ-Amyrenone regulating cardiomyocyte cell cycle Studies have shown that CBX7 protein inhibits cardiomyocyte proliferation by regulating the cell cycle process. The effects of small molecule δ-Amyrenone on the human cardiomyocyte cell cycle were investigated by flow cytometry. Figure 8 As shown in a and b. The proportion of each phase of the cell cycle of normal cardiomyocytes is: G1 phase 42.57% ± 0.94%, S phase 36.77% ± 0.78%, and G2 phase 16.73% ± 0.58%. In contrast, hypoxia-reperfusion treatment caused cells to be mainly blocked in the G1 phase, and its proportion increased to 45.4% ± 0.67%. For cardiomyocytes intervened by small molecule δ-Amyrenone, the proportion of the G1 phase of the cycle was 40.53% ± 0.71%, the S phase was 36.7% ± 0.57%, and the G2 phase increased to 20.1% ± 0.08%. These experimental results suggest that δ-Amyrenone treatment can improve the abnormal cell cycle caused by hypoxia-reperfusion, reduce the arrest of cells in the G1 phase, and may promote the smooth transition of cells from the G1 phase to the S phase and G2 phase, thereby contributing to the normal progress of the cell cycle. Based on these findings, it was concluded that δ-Amyrenone regulates the cell cycle by acting on CBX7 protein, alleviates the functional damage of cardiomyocytes under hypoxia-reperfusion conditions, and thus promotes the proliferation of AC16 cells.

[0120] In order to further verify the regulatory effect of δ-Amyrenone on CBX7 protein, a Western blot experiment was performed (see Figure 8c). The experimental results showed that compared with normal cardiomyocytes, the expression level of Cyclin D1 protein (protein associated with G1 phase) was significantly increased in the hypoxia-reperfusion group and the ischemia-hypoxia-reperfusion group. However, in the ischemia-reperfusion group after δ-Amyrenone treatment, the expression of Cyclin D1 decreased significantly, which suggested that δ-Amyrenone could reduce the overexpression of this protein. The expression of CDK2 protein (protein associated with S phase) decreased in the hypoxia-reperfusion and ischemia-hypoxia-reperfusion groups compared with the normal group; while in the δ-Amyrenone treatment group, the expression of CDK2 recovered. It is worth noting that the expression level of Cyclin A2 protein (protein associated with G2 phase) in the δ-Amyrenone treatment group was the highest among the four groups. These protein imprinting results are consistent with the conclusions obtained from the previous flow cytometry analysis (i.e., promoting the smooth transition of cardiomyocytes from the G1 phase to the S phase and G2 phase), indicating that the small molecule δ-Amyrenone can inhibit the function of CBX7 protein, thereby regulating the cell cycle and promoting the proliferation of ischemia-reperfusion cardiomyocytes.

[0121] 3.8 δ-Amyrenone regulates cardiomyocyte proliferation via SIRT7 / ALDH7A1 / FOXM1 proteins After exploring the effect of δ-Amyrenone on CBX7 protein, we further verified the expression changes of related genes in order to reveal its potential regulatory mechanism on cardiomyocyte proliferation. The results showed that after δ-Amyrenone treatment, the expression of genes such as CDCA2, ARRB1, FOXM1, SIRT7 and ALDH7A1 all changed significantly ( Fig. 9). Specifically, the expression of CDCA2, ARRB1, FOXM1, SIRT7 and ALDH7A1 genes were all upregulated after the action of δ-Amyrenone. Further literature analysis showed that CDCA2 can promote the G1 / S phase transition by upregulating and activating CCND1 / CDK4 / 6 and CCNE1 / CDK2, thereby promoting the proliferation of liver cancer cells and enhancing their cloning ability. At the same time, CDCA2 inhibits apoptosis in a p53 / p21-dependent manner by inhibiting the p38 MAPK pathway and activating the JNK / c-Jun pathway, and acts on the migration of p53 mutant Huh7 cells by promoting epithelial-mesenchymal transition. Experiments have found that δ-Amyrenone can upregulate the expression of CDCA2, thereby promoting the proliferation of cardiomyocytes. Other studies have shown that ARRB1 drives the transition of the cell cycle G1-S phase in liver cancer. In other words, δ-Amyrenone has the ability to upregulate the expression of ARRB1, thereby promoting the transition of cells from the G1 phase to the S phase. This mechanism of action may help promote the growth and repair of damaged myocardium. Sirt7 protein plays a role in the progression of non-small cell lung cancer, promoting tumor progression by promoting G1 / S phase transition, epithelial-mesenchymal transition, and activating AKT and ERK1 / 2 signaling pathways. After cardiomyocyte injury, Sirt7 expression decreases, but after δ-Amyrenone treatment, Sirt7 expression returns to normal levels, which may be one of its mechanisms for promoting cardiomyocyte proliferation. After cardiomyocyte ischemia-reperfusion, ALDH7A1 expression is downregulated, and ALDH7A1 has been shown to promote the transition of the G1-S phase. δ-Amyrenone treatment helps to increase ALDH7A1 expression. FoxM1 is an important cell cycle regulator that controls the expression of genes required for G1 / S and S / G2 phase transitions. After δ-Amyrenone treatment, FoxM1 protein expression is upregulated, further promoting the proliferation of damaged cardiomyocytes. In summary, δ-Amyrenone regulates the cell cycle through multiple signaling pathways and promotes cardiomyocyte proliferation. In addition to the signaling pathway mediated by CBX7 protein, δ-Amyrenone also promotes G1 / S and G2 phase transitions by upregulating genes / proteins such as CDCA2, ARRB1, Sirt7, ALDH7A1, and FoxM1, thereby promoting cardiomyocyte proliferation. This suggests that δ-Amyrenone promotes the repair and regeneration of damaged myocardium through multiple synergistic mechanisms.

[0122] In summary, the present invention successfully identified δ-Amyrenone as an effective small molecule inhibitor of CBX7: through a rigorous screening process, it was confirmed that δ-Amyrenone has a targeting effect on the CBX7 protein. Computer simulation revealed the connection mode and binding site of the two, and in vitro molecular interaction experiments verified the direct interaction between δ-Amyrenone and CBX7 from a practical perspective.

[0123] The selected small molecule δ-Amyrenone can promote proliferation and inhibit apoptosis of cardiomyocytes: the results of cell viability detection experiments and staining experiments showed that the selected inhibitor has a significant effect in promoting proliferation. At the same time, flow cytometry and protein immunoblotting experiments revealed that it has a significant effect in inhibiting cardiomyocyte apoptosis.

[0124] Through flow cytometry analysis, protein and gene levels, it was found that the small molecule inhibitor δ-Amyrenone has a regulatory effect on the cycle of ischemia-reperfusion cardiomyocytes.

[0125] In summary, δ-Amyrenone can act as an inhibitor of CBX7 protein, thereby regulating the cell cycle, inhibiting cell apoptosis, and promoting cardiomyocyte proliferation, and has good application prospects in the prevention and / or treatment of ischemic heart disease.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a CBX7 inhibitor in the preparation of a medicament for preventing and / or treating ischemic heart disease, characterized in that: The CBX7 inhibitor is δ-Amyrenone or its salt or isomer.

2. The use according to claim 1, characterized in that: The ischemic heart disease is selected from any one of myocardial infarction, myocardial infarction reperfusion injury, ventricular fibrosis, myocarditis, arrhythmia, angina pectoris, heart failure, coronary heart disease and myocardial ischemia.

3. The use according to claim 2, characterized in that: The ischemic heart disease is myocardial infarction reperfusion injury.

4. The use according to any one of claims 1 to 3, characterized in that: The drug has at least one of the following uses: (1) Promote the proliferation of cardiomyocytes; (2) Inhibit the expression of apoptotic proteins; (3) Slowing down the apoptosis of myocardial cells induced by hypoxia-reperfusion and / or ischemia-reperfusion; and (4) improving cell cycle abnormalities caused by hypoxia-reperfusion and / or ischemia-reperfusion.

5. The use according to claim 4, characterized in that: The use (4) includes: upregulating the level of at least one of the following genes and / or proteins: CDCA2, ARRB1, Sirt7, ALDH7A1 and FoxM1.

6. The use according to any one of claims 1 to 3, characterized in that: The drug has at least one of the following uses: (1) Promote the repair of ischemic myocardial tissue; (2) Reduce myocardial infarction area; and (3) reduce cardiac fibrosis.

7. The use according to any one of claims 1 to 3, characterized in that: The drug has at least one of the following uses: (1) Reduce the overexpression of G1 phase-related proteins in cardiomyocytes; (2) Increase the expression of S phase-related proteins in cardiomyocytes; (3) Promote the G1 / S phase transition of cardiomyocytes; (4) Promote the S / G2 phase transition of cardiomyocytes.

8. The use according to claim 7, characterized in that The G1 phase-related protein is Cyclin D1 protein.

9. The use according to any one of claims 1 to 3, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

10. The use according to any one of claims 1 to 3, characterized in that: The drug is administered by injection.

Images