Application of a Small Molecule Compound in Ischemic Heart Disease

Abstract

The present invention discloses the application of a small molecule compound in ischemic heart disease, relating to the technical field of the treatment of ischemic heart disease. The CBX7 inhibitor is δ-Amyrenone. δ-Amyrenone can act as an inhibitor of the 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.

Description

Technical Field

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

[0002] Myocardial infarction (MI) is a major disabling and lethal disease globally. Its pathogenesis is mainly related to the rupture of coronary atherosclerotic plaques or endothelial erosion, especially with a higher incidence in elderly patients. Although the current treatment strategy focuses on myocardial reperfusion, the reperfusion process itself can trigger further death of cardiomyocytes (i.e., reperfusion injury), leading to progressive loss of cardiac function and heart failure. Due to the extremely low proliferative capacity of adult mammalian cardiomyocytes, existing treatment methods 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 act together to further exacerbate cardiomyocyte loss, expand the necrotic area, 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, decline in cardiac function, and ultimately develop into chronic heart failure. In addition, reperfusion injury may also trigger severe arrhythmias, further endangering the life safety of patients. These concurrent problems significantly affect the overall effect of reperfusion therapy and become an important obstacle restricting the improvement of the long-term prognosis of patients.

[0004] Recent studies have shown that targeted inhibition of the key factor CBX7 by gene editing can induce cardiomyocytes to re-enter the proliferative state, thereby reducing the degree of injury in young mice during ischemia-reperfusion injury. However, the disease of MI should pay more attention to the biological characteristics of the elderly population. In addition, gene therapy methods are limited by the long clinical translation cycle and high technical threshold. In contrast, developing small molecule inhibitors targeting CBX7 has significant advantages such as strong specificity and clear action mechanism, and is more likely to achieve clinical translation.

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

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

[0007] The present invention is implemented as follows:

[0008] The present invention provides the application of a small molecule compound in ischemic heart disease, and the CBX7 inhibitor is δ-Amyrenone or its salt, isomer.

[0009] The present invention has the following beneficial effects:

[0010] Through screening, the present invention identified an effective small molecule inhibitor of CBX7, confirmed that δ-Amyrenone has a targeting effect on the CBX7 protein, revealed the binding mode and binding site between the two through computer simulation, and verified the direct interaction between δ-Amyrenone and CBX7 through in vitro molecular interaction experiments.

[0011] δ-Amyrenone can promote the proliferation and inhibit the apoptosis of cardiomyocytes. The results of cell viability detection experiments and staining experiments both show that the screened CBX7 inhibitor has a significant effect on promoting proliferation. At the same time, flow analysis and protein immunoblotting experiments also revealed its significant effect on inhibiting cardiomyocyte apoptosis. In addition, through flow analysis, protein and gene levels, the present invention found that the small molecule inhibitor δ-Amyrenone has a regulatory effect on the cell cycle of ischemic reperfusion cardiomyocytes. Therefore, δ-Amyrenone can be used as an inhibitor of the CBX7 protein, thereby regulating the cell cycle, inhibiting cell apoptosis, promoting cardiomyocyte proliferation, and having good application prospects in the prevention and / or treatment of ischemic heart disease. Brief Description of the Drawings

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0013] Figure 1 Results diagram of the expression of CBX7 protein in the infarcted area of the elderly mouse MI / R model ((a) Electrocardiogram changes in mice before and after infarction; (b) TTC staining of heart sections of MI / R mice; (c) Detection of the expression level of CBX7 in the heart tissue of myocardial infarction reperfusion by qPCR; (d) Observation of the distribution of CBX7 in the heart tissue of myocardial infarction reperfusion by immunofluorescence method; (e) Analysis of the expression of CBX7 in the heart tissue of myocardial infarction reperfusion by Western blot, scale bar = 200 μm; the images represent n = 3 independent repeated experiments; the data in the figure represent Mean±SD; significant difference: **p<0.01);

[0014] Figure 2 Structural formula of the top 10 small molecule compounds in docking scores after high-throughput screening;

[0015] Figure 3Schematic diagram of visualizing CBX7-δ-Amyrenone with PyMOL software ((a) Visualizing the binding of CBX7 protein and δ-Amyrenone molecule using molecular docking software; (b) Predicting the binding site between CBX7 and δ-Amyrenone and the interaction force between them);

[0016] Figure 4 Results graph of the systematic optimization process of CBX7 protein expression conditions ((a) Effects of different IPTG concentrations on the expression level of CBX7 protein after induction of recombinant plasmid; (b) Exploring the expression changes of CBX7 protein after IPTG induction of recombinant plasmid at different temperatures; (c) Further investigating the effect of IPTG induction time on the expression of CBX7 protein in recombinant plasmid; (d) Schematic diagram of the optimization of the Tris washing step and determination of the optimal imidazole elution concentration in the CBX7 protein purification process; (e) Recycling the protein purification effect);

[0017] Figure 5 Experimental results of in vitro verification of the interaction between CBX7 and δ-Amyrenone ((a) The binding strength between different concentrations of δ-Amyrenone and CBX7 obtained through in vitro molecular interaction experiments; (b) Selecting the signal values at key time points before the end of the experiment and plotting the affinity fitting curve to deeply analyze the affinity characteristics between CBX7 and δ-Amyrenone);

[0018] Figure 6 Experimental results graph of the promoting effect of δ-Amyrenone on the viability and proliferation of AC16 cells ((a) After 24 hours of treatment, the CCK8 method was used to measure the effect of δ-Amyrenone at specified concentrations on the proliferation of AC16 cells; (b) After treating AC16 cells with 20 μM δ-Amyrenone for 24 hours, the cell survival status was evaluated by the trypan blue exclusion method and the survival ratio was calculated; (c) This part of the data is directly related to part (b), showing in detail the counting and calculation results of the survival ratio; (d) After treating cells with 20 μM δ-Amyrenone for 24 hours, the YO-PRO-1 fluorescence staining method was used to count the cell survival situation, image scale bar = 200 μm, and the shown images are representatives of three independent repeated experiments; All data are expressed in the form of Mean±SD; Significant differences are marked as: p<0.05， ***p < 0.0001);

[0019] Figure 7Experimental results of the inhibitory effect of δ-Amyrenone on the apoptosis of AC16 cells ((a) After AC16 cells were treated with 20 μM δ-Amyrenone for 24 hours, the apoptosis of cells was analyzed by flow cytometry; (b) Provided a statistical chart of the positive cell rate of the flow cytometry results in (a); (c) After AC16 cells were treated with 20 μM δ-Amyrenone for 24 hours, the apoptosis status of cells was observed using apoptosis and necrosis staining technology, image scale = 200 μm; (d) Through Western blot experiment, the changes in the expression levels of apoptosis-related proteins after δ-Amyrenone treatment were further explored; all the images shown were representatives of three independent repeated experiments; the data were presented in the form of Mean±SD, and the significant differences were marked as ****p<0.0001);

[0020] Figure 8 Experimental results of δ-Amyrenone promoting the proliferation of AC16 cells by regulating the cell cycle ((a) After AC16 cells were treated with 20 μM δ-Amyrenone for 24 h, the distribution of the cell cycle was analyzed by flow cytometry; (b) Showed a statistical chart of the positive cell proportion of the flow cytometry results in (a); (c) Through Western blot experiment, the changes in the expression levels of cell cycle-related proteins after δ-Amyrenone treatment were detected to further verify its effect on the cell cycle; all the images were representatives of three independent repeated experiments; the data were expressed as Mean±SD, and the significant differences were marked as *p<0.05, **p<0.01, ***p<0.001);

[0021] Figure 9 Experimental results of the effect of δ-Amyrenone on cell cycle-related proteins by regulating CBX7 protein ((a) After AC16 cells were treated with a specific concentration (20 μM) of δ-Amyrenone for 24 hours, the expression patterns of cell cycle-related protein genes were analyzed by qPCR technology; (b) Western blot detected the changes in cyclin-related markers in cells after δ-Amyrenone treatment; the data in the figure represented Mean±SD; significant difference: ****p<0.0001). Detailed implementation manners

[0022] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0023] The present invention provides an application of a CBX7 inhibitor in the preparation of a drug for preventing and / or treating ischemic heart disease, and the CBX7 inhibitor is δ-Amyrenone or its salt or isomer.

[0024] The CAS number of δ-Amyrenone is 20248-08-2, and the molecular formula is C 30 H 48 O, and the structural formula is as follows:

[0025] .

[0026] The inventors utilized the Discovery Studio software to implement a high-throughput screening strategy on the small molecule compound library provided by TargetMol, and successfully identified a variety of compounds that can effectively bind to the CBX7 protein. After screening, it was found that CBX7 has a strong binding affinity with δ-Amyrenone. Experiments have shown that δ-Amyrenone can act as an inhibitor of the CBX7 protein, thereby regulating the cell cycle, inhibiting apoptosis, promoting cardiomyocyte proliferation, and having good application prospects in preventing and / or treating ischemic heart disease.

[0027] In a preferred embodiment of the application 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.

[0028] In a preferred embodiment of the application of the present invention, the above-mentioned ischemic heart disease is myocardial infarction reperfusion injury.

[0029] In a preferred embodiment of the application of the present invention, the above-mentioned drug has at least one of the following uses:

[0030] (1) Promote the proliferation of cardiomyocytes;

[0031] (2) Inhibit the expression of apoptotic proteins;

[0032] (3) Slow down the apoptosis of cardiomyocytes treated with hypoxia reperfusion and / or ischemia reperfusion;

[0033] and (4) Improve the cell cycle abnormalities caused by hypoxia reperfusion and / or ischemia reperfusion.

[0034] In a preferred embodiment of the application of the present invention, the above-mentioned use (4) includes: upregulating the levels of at least one of the following genes and / or proteins: CDCA2, ARRB1, Sirt7, ALDH7A1, and FoxM1.

[0035] In a preferred embodiment of the application of the present invention, the above-mentioned drug has at least one of the following uses:

[0036] (1) Promote the repair of ischemic myocardial tissue;

[0037] (2) Reduce the area of myocardial infarction;

[0038] And (3) Reduce cardiac fibrosis.

[0039] In a preferred embodiment of the application of the present invention, the above-mentioned drug has at least one of the following uses:

[0040] (1) Reduce the overexpression of G1-phase related proteins in cardiomyocytes;

[0041] (2) Increase the expression of S-phase related proteins in cardiomyocytes;

[0042] (3) Promote the G1 / S-phase transition of cardiomyocytes;

[0043] (4) Promote the S / G2-phase transition of cardiomyocytes.

[0044] In a preferred embodiment of the application of the present invention, the above-mentioned G1-phase related protein is Cyclin D1 protein.

[0045] In a preferred embodiment of the application of the present invention, the above-mentioned drug further comprises a pharmaceutically acceptable carrier.

[0046] In another embodiment, the drug can be in the form 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.

[0047] In one embodiment, the active ingredient (δ-Amyrenone) is formulated into a drug or a pharmaceutical composition such that the active ingredient is rapidly released, sustained released or delayed released after being administered 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 either be the carrier, excipient and diluent serving as the active ingredient or the carrier, excipient and diluent serving as the medium.

[0048] In another embodiment of the above-mentioned application, suitable carriers, excipients and diluents can be solid, semi-solid or liquid materials, such as: lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propyl parabens, talc, magnesium stearate or liquid paraffin.

[0049] In another embodiment of the drug or pharmaceutical composition in the above application, the drug or pharmaceutical composition may further include a lubricant, a wetting agent, an emulsifying and suspending agent, and a preservative.

[0050] In a preferred embodiment of the application of the present invention, the above drug is administered by injection.

[0051] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0052] Example 1

[0053] This example provides an experimental test on the expression of CBX7 protein in the infarcted area of the MI / R model in aged mice.

[0054] 1. Experimental animals

[0055] SPF-grade C57 mice provided by Chengdu Dashuo were used as experimental subjects, with no gender limitation, and continued to be cultured until 2 months of age. To ensure the hygienic conditions of the animals, the experimental mice were housed in an environment with a 12-hour light-dark cycle control, and had free access to clean drinking water and high-quality feed. Animal ethics standards: strictly follow the guiding principles of the Experimental Animal Ethics Review Committee of Sichuan Provincial People's Hospital.

[0056] 2. Experimental materials and reagents

[0057] Table 1 Main experimental materials and reagents

[0058]

[0059]

[0060] 3. Experimental methods

[0061] 3.1 Construction of the MI / R model

[0062] First, expose the pleura and open the chest between the second and third intercostal spaces on the left side of the chest. Then, open the chest between the second and third left intercostal spaces to expose the heart. Use 7-0 silk thread to tie a slipknot at the origin of the left anterior descending coronary artery and occlude the left anterior descending branch. Ensure that there is no obvious bleeding in the heart cavity during the operation, and keep the operation area clean at the same time. Finally, suture the operation area layer by layer to ensure that the tissue layers fit well. After 40 minutes of ischemia treatment, remove the coronary artery ligation thread. At the end of the experimental period, euthanize the animals with an overdose of isoflurane and remove the main organ tissues for further analysis.

[0063] 3.2 Tissue collection and real-time fluorescence quantitative PCR (RT-qPCR)

[0064] Table 2 Primers for different genes

[0065]

[0066] (1)Model construction and sample processing

[0067] Construct a mouse MI / R model according to 3.1. After 24 hours, anesthetize and humanely sacrifice the mice, and then remove the hearts for experiments.

[0068] (2)Heart region division and tissue sample collection

[0069] Divide the removed heart into three regions: infarct area, peripheral area, and non-infarcted normal area, and collect equal amounts of tissue from each area for subsequent experimental analysis.

[0070] (3)RNA extraction procedure

[0071] Extract RNA from the tissues of the three regions using the TRIzol reagent method, and purify the total RNA in combination with the SteadyPure Tissue & Cell Small RNA Extraction Kit.

[0072] (4)cDNA synthesis process

[0073] Use a reverse transcription kit to reverse transcribe the extracted RNA samples into cDNA.

[0074] (5)RT-qPCR detection

[0075] Perform RT-qPCR detection using a qPCR kit. Place the reaction system in a qPCR instrument, set the temperature gradient according to the instructions, and finally accurately determine the cycle threshold (Ct) of the PCR product.

[0076] 3.3 Tissue collection and Western blot

[0077] (1)Tissue sample acquisition and lysis treatment: Construct a mouse MI / R model according to 3.1. After 24 hours, anesthetize and humanely sacrifice the mice, and then remove the hearts for experiments. Obtain samples from the infarct area, marginal area, and normal area of the heart, add grinding beads and RIPA lysis buffer, and then use a grinding device to further refine the tissue, followed by lysis on ice bath. Centrifuge to separate and obtain the supernatant, and use the BCA method to determine the protein concentration therein. Subsequently, dilute the protein sample with 5× loading buffer and an appropriate amount of RIPA lysis buffer to 30 μg / μL, heat it in a 95°C metal bath for 10 minutes to denature it, and store it at -80°C after aliquoting for later use.

[0078] (2)Electrophoresis: Prepare the gel according to the kit instructions. Load the sample after the gel is stable, set the voltage to start electrophoresis, and end electrophoresis after all the bands are separated.

[0079] (3)Protein transfer and blocking steps: Transfer the protein onto a PVDF membrane in a transfer tank. After the transfer is completed, block the membrane with 5% non-fat milk powder for 1 - 2 hours, and then wash the membrane with PBST buffer on a shaker for 5 minutes each time, for a total of 6 times.

[0080] (4)Antibody binding reaction: Immerse the blocked membrane in the target primary antibody and incubate it overnight on a shaker at 4 °C. Recover the primary antibody, wash with PBST for 5 minutes, 6 times, and then place it in the corresponding secondary antibody of the same species and incubate at 37 °C for 1 - 2 hours, and then wash the membrane again.

[0081] (5)Chemiluminescence: Prepare the developing solution according to the instructions of the developing solution. Place the PVDF membrane in an exposure machine, add the developing solution, and record and analyze the developing results.

[0082] 3.4 Tissue collection and immunofluorescence

[0083] (1)Procedure for obtaining, fixing and dehydrating heart samples: Construct an MI / R model in aged mice according to 3.1. After 24 hours, anesthetize and humanely sacrifice the mice, and then remove the hearts for experiments. The heart tissues need to be immersed in a 4% paraformaldehyde solution for 24 hours of fixation to ensure the integrity of the tissue structure is maintained. Next, dehydrate the heart tissues using a fully automatic tissue dehydrator.

[0084] (2)Paraffin embedding and section preparation of heart tissues: The dehydrated heart tissues need to be further paraffin-embedded for subsequent sectioning. When sectioning, ensure that the thickness of the heart tissue sections is 5 μm to meet the specific requirements of the experiment.

[0085] (3)Deparaffinization and hydration: Place the paraffin sections in a fully automatic staining machine for baking and deparaffinization, and complete the hydration.

[0086] (4)Antigen retrieval: Place the sections in a steam pressure cooker containing sodium citrate buffer (pH 6.0) for high-temperature and high-pressure retrieval for 5 - 10 minutes. After natural cooling to room temperature, rinse the sections with PBS 3 times, 5 minutes each time.

[0087] (5)Permeabilization and blocking: Treat the sections with a 0.5% Triton X-100 solution to enhance the penetration ability of the antibody in the tissue. Subsequently, block the sections with goat serum at room temperature for 30 minutes to reduce the interference of non-specific binding and ensure the accuracy of the experimental results.

[0088] (6)Primary antibody incubation: Add the primary antibody according to the ratio in the instructions, cover the section tissues, and incubate overnight in a wet box at 4 °C. Subsequently, wash the sections with PBS 3 times, 5 minutes each time.

[0089] (7)Secondary antibody incubation: Then add the fluorescently labeled secondary antibody (select the corresponding secondary antibody according to the host of the primary antibody, and the dilution ratio is shown in the instruction manual), incubate at 37 °C for 1 hour, and wash again with PBS 3 times, 5 minutes each time.

[0090] (8)DAPI staining and mounting: Under the condition of 37 °C, stain with DAPI for 20 minutes to label the cell nuclei. Wash with PBS 3 times, 5 minutes each time, to remove the excess staining solution. Drop an appropriate amount of mounting medium, cover the section with a coverslip, and gently flatten it to prevent air bubbles.

[0091] (9)Microscopic observation and photographing: Observe the section using a fluorescence microscope, and sequentially capture the target fluorescence signal and nuclear staining signal using an appropriate excitation wavelength, and save the images for subsequent analysis.

[0092] 3.5 TTC staining

[0093] (1)Sample collection: Anesthetize the mice and then sacrifice them, and then remove the hearts for experimental use.

[0094] (2)Prepare the TTC solution: Weigh 0.5 g of TTC powder and dissolve it in 50 mL of distilled water to prepare a 1% (mass fraction) TTC solution.

[0095] (3)Staining: Wash the heart and cut it into sections with a thickness of 1 to 2 mm from the apex to the base of the heart. Place the obtained sections in the TTC solution to ensure that they are submerged, and then incubate at 37 °C for 30 minutes.

[0096] (4)Observation and photographing: Take out the sections and blot the liquid dry, arrange the sections in order and take pictures with a camera.

[0097] To explore the expression changes of CBX7 after MI / R, this study established a model using aged mice, collected heart tissues 24 h after infarction, and compared the electrocardiograms before infarction and after infarction in mice. The ST segment elevation of the electrocardiogram of the infarction model indicated that the infarction model was successfully established ( Figure 1 in a). The TTC results of the mouse heart sections 1 day later also proved this point ( Figure 1 in b). The expression of CBX7 was analyzed by qPCR, Western Blot and immunofluorescence detection. First, 1 day after the successful establishment of the model, myocardial tissues from the infarcted zone (IZ), margin zone (MZ) and normal zone (NZ) were taken for RT-qPCR detection respectively. The experimental results showed that compared with the normal myocardial tissue area, the myocardial tissue in the infarcted area showed a significant upward trend in the mRNA expression level of CBX7, specifically manifested as a P value of 0.0013 ( Figure 1In c), the differences at the gene transcription level were highlighted. To further explore this change, the Western Blot technique was further adopted to verify the situation of CBX7 at the protein expression level. The results showed that the expression level of CBX7 protein in the infarct area was significantly higher than that in the normal area ( Figure 1 In e). In addition, the immunofluorescence technique was also used to observe the localization and expression intensity of CBX7, and it was found that the fluorescence signal of CBX7 in the infarct area was significantly enhanced, as shown in Figure 1 d in, which was consistent with the aforementioned mRNA and protein expression results.

[0098] In summary, after MI / R, the expression of CBX7 in the myocardium of old mice changed significantly. This finding suggests that CBX7 may become a potential therapeutic target in the process of myocardial infarction repair.

[0099] Example 2

[0100] In this example, the information of CBX7 protein was obtained from the Uniport database, and a small molecule compound library was obtained from TargetMol. The compound with the most potential specific binding ability was screened out through the Discovery Studio software. Subsequently, through biological experiments, it was verified and evaluated whether this small molecule compound could regulate the cell cycle of cardiomyocytes by inhibiting CBX7, thereby promoting the repair process of cardiomyocytes and inhibiting apoptosis, providing an experimental basis for subsequent related research.

[0101] 1. Experimental materials

[0102] (1) Experimental cell line: The AC16 human cardiomyocyte cell line is derived from ATCC (American Type Culture Collection) in the United States.

[0103] (2) Experimental software:

[0104] Table 3 Experimental software

[0105]

[0106] (3) Experimental reagents

[0107] Table 4 Main experimental materials and reagents

[0108]

[0109] Preparation of common reagents

[0110] (1) Cell culture medium: 5.6 ml of double antibody and 56 ml of FBS were added to every 500 ml of the basal medium DMEM / F-12.

[0111] (2) Escherichia coli culture medium: Add 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride to 1000 ml of double-distilled water, and then adjust the pH value of the solution to 7.2.

[0112] 2. Experimental methods

[0113] 2.1 High-throughput screening with Discovery Studio software and verification process with AutoDock

[0114] (1) Obtain the CBX7 protein structure information: By accessing the Uniprot database (website: https: / / www.uniprot.org / ), the three-dimensional structure file of the CBX7 protein was downloaded and then imported into the Discovery Studio software. Subsequently, necessary preprocessing of the protein structure was performed, including removing irrelevant ligands and redundant parts, adding hydrogen atoms, and performing an energy minimization optimization step. Finally, the active site of the protein was determined, and the preprocessing of the structure was completed.

[0115] (2) Integration and preparation of the small molecule compound library: After introducing the small molecule compound library from TargetMol, a series of preprocessing processes were performed on it, covering adding hydrogen atoms, removing salt ions, adjusting the charge distribution, etc., and energy minimization calculations were performed by applying the molecular force field to generate the three-dimensional structure of small molecules suitable for molecular docking analysis.

[0116] (3) Perform simulated docking: When the small molecule and the protein are ready, click libdock in the Discovery Studio software for docking.

[0117] (4) Screening and comprehensive evaluation of candidate molecules: Based on criteria such as the rationality of the binding site, the minimum value of the binding free energy, and the LibDockScore, the small molecule compounds were screened to identify candidate molecules with the best binding potential, providing valuable alternatives for the subsequent experimental verification stage.

[0118] (5) AutoDock verification: To further verify the binding mode and energy stability of the screened candidate compounds, molecular docking analysis was performed using AutoDock4. The protein and small molecule structure files were processed into the PDBQT format, water molecules and impurities were removed, and polar hydrogen atoms were added. The active site was defined as a cube grid of 126 Å × 126 Å × 126 Å, and the grid point spacing was set to 0.05 nm. The docking operation was run under the conditions of Lamarckian Genetic Algorithm (LGA) to obtain the binding poses 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.

[0119] 2.2 Visualization of Protein-Small Molecule Compounds with PyMOL Software

[0120] (1)Import the structure file: Import the best protein-small molecule complex structure file (in PDB format) obtained by AutoDock4 docking into PyMOL software for visualization analysis.

[0121] (2)Display the structures of the protein and small molecule: Load the protein structure and small molecule compound in PyMOL, and adjust the colors and display modes of the protein backbone, small molecule compound, and related ligands respectively. Common display modes include wireframe (line), stick (sticks), and surface (surface) modes to highlight the interaction between the binding position of the small molecule and the protein active site.

[0122] (3)Analysis of the binding site: Use the measurement tools in PyMOL to analyze the key interactions between the small molecule compound and the protein active site, including hydrogen bonds, hydrophobic interactions, π-π stacking, etc. By labeling and highlighting these key interactions, it provides an intuitive basis for analyzing the binding stability.

[0123] (4)Optimized display of secondary structure and binding mode: Display the secondary structure of the protein (such as α-helix and β-sheet) in cartoon mode, and display the binding region of the small molecule compound and the protein in stick or surface mode to ensure that the overall structure is clear and hierarchical.

[0124] (5)Labeling of key interacting residues: Label the key residues and their numbers that bind to the small molecule, and at the same time display the distance data of hydrogen bonds or non-bonded interactions to provide support for explaining the binding mechanism of the small molecule.

[0125] (6)Export of pictures and videos: Take screenshots of the three-dimensional structure of the key binding site from multiple angles and save them as high-resolution pictures for reports and publications; or generate a rotating animation to show the dynamic interaction effect of the protein-small molecule complex. Based on the visualization results, summarize the binding characteristics and interaction modes of the small molecule and the protein to provide a basis for subsequent molecular optimization and function verification.

[0126] 2.3 Recombinant Protein Expression and Purification

[0127] (1)Expression of CBX7 protein: Obtain the base sequence of human CBX7 protein from the database, add His tag and Flag tag to the head and tail respectively, clone it into a plasmid and load it into Escherichia coli, and then carry out bacterial culture. When the OD600 value of every 200 ml of bacterial liquid grows to the appropriate range of 0.6 to 0.8, different concentrations of IPTG from 0.1 mM to 1.0 mM are used to induce protein expression at 16 °C for 12 hours.

[0128] (2)Verification process of protein expression effect: After the induction expression is completed, collect the bacterial precipitate and perform ultrasonic lysis treatment under ice bath conditions to obtain the lysate containing the target protein. Subsequently, add 20 μL of 5× loading buffer to 100 μL of the lysate and heat it in a 95 °C metal bath for 10 minutes to denature the protein. Separate the protein by SDS-PAGE electrophoresis technology and use Coomassie Brilliant Blue staining method to detect the induction effect at different IPTG concentrations, so as to determine the optimal IPTG concentration. At the same time, the induction expression temperature and time were also optimized by the same method.

[0129] (3)Purification process of CBX7 protein: Under the determined optimal induction conditions, collect the bacterial precipitate again and perform ultrasonic lysis treatment, and then obtain the supernatant by centrifugation. The Ni-NTA affinity chromatography column can capture the His-tagged CBX7 protein. Wash out the impurity proteins with 6 times of Tris, and then use different concentrations of imidazole elution to recover CBX7 protein. Detect the protein content in each concentration of imidazole by gel electrophoresis to determine the optimal imidazole concentration.

[0130] (4)Collection and purification improvement of CBX7 protein: After collecting the eluate containing CBX7 protein, use an ultrafiltration tube for concentration treatment. In order to further improve the protein purity, load the concentrated solution onto an SDS-PAGE gel, determine the band position by electrophoresis separation and Coomassie staining, cut off the band, and use a kit to recover the protein to extract high-purity CBX7 protein. Finally, a purified CBX7 protein sample is obtained.

[0131] 2.4 Surface plasmon resonance molecular interaction experiment

[0132] (1)Determine the protein concentration: Use the BCA method to measure the concentration of the obtained CBX7 protein.

[0133] (2)Biotinylation labeling: Biotinylate the CBX7 protein according to the kit instructions for immobilization on the sensor chip.

[0134] (3)Binding of CBX7 to the sensor: Contact the SSA sensor with the protein in (2) and solidify it according to the instructions.

[0135] (4)Sample injection treatment: Small molecule solutions with different concentrations and blank controls were injected successively (0 - 100 nM).

[0136] (5)Recording and analysis: Parameters were set according to the standard procedure, and corresponding programs were set to monitor the signal changes during the baseline, binding, and dissociation processes. The data was processed using analysis software to calculate the affinity constant between the two, thus providing strong data support for subsequent research.

[0137] 2.5 Construction of myocardial cell ischemia-reperfusion / hypoxia model

[0138] (1)Cell culture: Human myocardial cells AC16 were selected and cultured in a suitable culture medium environment to ensure that the cells maintained a good growth state.

[0139] (2)Simulated ischemia treatment: To simulate ischemic conditions, the culture medium of AC16 cells was replaced with serum-free medium, and the cells were placed in a triple-gas incubator. By using the mixed-gas culture method and introducing nitrogen to evacuate other gases in the incubator, an anaerobic environment was created. For the hypoxia group, complete medium was used for culture, and the gas conditions were the same as those in the ischemia group.

[0140] (3)Collection and analysis of cell samples: When the oxygen concentration in the incubator was monitored to be lower than 0.5%, timing started. After 2 hours, the culture conditions were restored to normal conditions, which was reperfusion. After reperfusion for more than 24 hours, the cells were collected.

[0141] 2.6 Determination of cell viability by CCK-8 method, trypan blue staining, and YP-1 staining

[0142] (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, the 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 values were processed and analyzed using GraphPad software to quantify cell viability.

[0143] (2)Trypan blue staining: Myocardial cells were seeded into 6-well plates, and the reperfusion model was constructed. The small molecule drug was added according to the optimal concentration determined by the CCK8 method. After 24 hours, the cells were labeled by trypan blue staining method. Observation and recording were carried out under a microscope, and the stained images were analyzed using ImageJ software to distinguish live cells from dead cells. Furthermore, the quantitative data of cell survival rate was statistically analyzed using GraphPad software.

[0144] (3)YO-PRO-1 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 h, cells were labeled with YO-PRO-1 staining. Subsequently, fluorescence microscopy was used to observe and capture images to visually display the apoptosis of cells.

[0145] 2.7 Flow cytometry for cell cycle detection

[0146] (1)Preparation process of cardiomyocyte samples: The ischemia-reperfusion model of cardiomyocytes was constructed according to the method shown in 2.5, and small molecule drugs were introduced for intervention. After 24 h, cells were digested and collected, centrifuged after washing with PBS to remove excess liquid, fixed with 70% ethanol for more than half an hour, and then the cells were resuspended.

[0147] (2)Staining treatment steps of samples: An appropriate amount of propidium iodide staining solution was added to each prepared cell sample. After thorough mixing, it was incubated at 37 °C in the dark for 30 minutes to ensure sufficient and uniform staining.

[0148] (3)Flow cytometry detection and data analysis method: Using a flow cytometer, the fluorescence signal and light scattering characteristics of cells were detected under 488 nm excitation light. The collected data were deeply analyzed using FlowJo software to reveal the changes in the cell cycle.

[0149] 2.8 Flow cytometry for apoptosis detection

[0150] (1)Preparation process of cardiomyocyte samples: The ischemia-reperfusion model of cardiomyocytes was constructed according to the method shown in 2.5, and small molecule drugs were applied for treatment. After 24 hours of treatment, the cells were digested with trypsin, resuspended after washing with PBS, counted, and 100,000 cells were collected into a 1.5 mL centrifuge tube. After centrifugation to remove the supernatant, 195 μL of Annexin V-FITC binding solution was added, and the cells were gently resuspended.

[0151] (2)Apoptosis staining steps: 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution were added to the sample in (1) according to the instructions. The mixture was incubated at room temperature (20 - 25 °C) in the dark for 10 to 20 minutes, and during this period, it could be wrapped with aluminum foil to fully block light. After incubation, the sample was transferred to ice to maintain a low temperature state and reduce false positives caused by continued staining.

[0152] (3)Flow cytometry detection and data: The FITC and PI wavelength data of the sample were detected using a flow cytometer. The downloaded data were analyzed using FlowJo software to quantify apoptosis.

[0153] 2.9 Fluorescence microscopy for detecting cell apoptosis

[0154] (1) Preparation process of cell samples: Cardiomyocytes were seeded in 96-well plates at a density of 3000 cells per well and cultured. Subsequently, a cardiomyocyte ischemia-reperfusion model was constructed according to the method shown in 2.5, and small molecule drugs were introduced for intervention. After 24 hours of treatment, the 96-well plates were centrifuged for 5 minutes to prepare for the subsequent staining steps.

[0155] (2) Cell apoptosis staining process: Staining was performed according to step (2) shown in 2.8.

[0156] (3) Observation under fluorescence microscope: After staining, the cells were observed using a fluorescence microscope, and corresponding fluorescence images were taken to record the staining of cell apoptosis.

[0157] 2.10 RT-qPCR

[0158] (1) Sample treatment: AC16 cells were seeded in 6-well plates. According to the method of constructing the reperfusion model shown in 2.5, after adding small molecules for 24 hours, the cells were collected.

[0159] (2) Total RNA extraction: Total RNA of the cells was extracted according to the kit instructions, and the concentration and purity of the obtained RNA were measured.

[0160] (3) The subsequent steps were carried out with reference to the steps in 3.2 of Example 1.

[0161] Table 5 Primers for different genes

[0162]

[0163] 2.11 Protein extraction and Western blotting

[0164] (1) Construction of cell model and pretreatment: Cardiomyocytes were seeded in 10-cm cell culture dishes. Following the steps shown in 2.5, an ischemia-reperfusion model was constructed. After adding small molecule drugs for 24 hours, cell samples were prepared for subsequent protein analysis experiments.

[0165] (2) Protein extraction: The cells in (1) were washed three times with PBS. Subsequently, the PBS was blotted dry, lysis buffer was added, and the cells were scraped repeatedly to obtain a homogenate, which was transferred to a centrifuge tube. After standing on ice for 30 minutes, ultrasonic treatment was performed to break the cells. After centrifugation at 12000 g for 20 minutes, the supernatant was collected without touching the tube wall. Then, the concentration was determined by the BCA method, and loading buffer and lysis buffer were added to adjust the total protein concentration, and it was denatured under the conditions of 95 °C for 10 minutes in a metal bath.

[0166] (3) The subsequent steps refer to 3.3 of Example 1.

[0167] 2.12 Statistical analysis

[0168] The data obtained from the experiment were expressed in the form of 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. Among them, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001, all of which are used to indicate significant differences in statistical significance.

[0169] 3. Experimental results

[0170] 3.1 Screening of CBX7 inhibitors

[0171] Using Discovery Studio software, a high-throughput screening strategy was implemented on the small molecule compound library provided by TargetMol, and a variety of compounds that could effectively bind to the CBX7 protein were successfully identified. They could all form a stable three-dimensional binding with the target protein. Based on LibdockScore, the most promising small molecule δ-Amyrenone was selected for subsequent verification. Table 6 lists some information of the top 10 small molecule compounds in terms of docking scores, Figure 2 showing the corresponding chemical structures. The binding mode and energy stability of the candidate compound δ-Amyrenone were verified by AutoDock software. Among them, δ-Amyrnone and CBX7 had a binding energy of -11.61 kcal / mol, and the ligand efficiency was -0.37 kcal / mol, indicating very stable binding.

[0172] Table 6 Top 10 small molecule compounds in terms of docking scores after high-throughput screening

[0173]

[0174] 3.2 Molecular docking analysis of δ-Amyrenone and CBX7 protein

[0175] To more deeply verify the molecular docking between δ-Amyrenone and CBX7, PyMOL software was used to visually display the relevant structures. After careful analysis, the results showed that CBX7 tightly wrapped and bound δ-Amyrenone (see specifically Figure 3 ). In Figure 3It can be clearly observed that there is an efficient binding interaction between δ-Amyrenone and CBX7. 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 the interaction forces, δ-Amyrenone forms stable bindings 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 establishes interactions with HIS through Pi-Alkyl and Pi-Sigma interaction forces. These interactions play a key role in the binding stability between δ-Amyrenone and the target protein, indicating that these amino acid residues have important effects 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 respectively. This docking analysis reveals the binding mode between δ-Amyrenone and CBX7, providing reference information and theoretical basis for the subsequent biological verification experiments.

[0176] 3.3 Systematic optimization of the expression and purification conditions of CBX7 protein

[0177] To explore the effects of IPTG concentration, induction time, and culture temperature on the induced expression of CBX7 protein, the method of controlling variables was adopted, and a series of experiments were carefully designed, including different IPTG concentration gradients (0 to 1 mM), time gradients (0 to 12 hours), and temperature gradients (16 °C to 37 °C). Subsequently, Coomassie Brilliant Blue staining technology was used to verify the experimental results. The experimental data clearly show (see Figure 4 ), under the conditions of an IPTG concentration of 0.2 mM, an induction temperature of 22 °C, and a duration of 10 hours, the expression level of CBX7 protein reaches the peak (see a-c in Figure 4 for the specific images).

[0178] The Ni-NTA purification column was used to extract CBX7 protein, and the target protein was isolated from the bacterial lysate. The optimal number of Tris washes was determined to be 5 times by Coomassie staining (see specifically in Figure 4In step d), because the impurity protein components had been removed at 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 eluent containing CBX7 protein, it was concentrated using an ultrafiltration tube. To further improve the protein purity, the concentrated solution was loaded onto an SDS-PAGE gel. After electrophoresis separation, high-purity CBX7 protein was extracted by cutting the gel and recovery, and finally a purified CBX7 protein sample was successfully obtained (see Figure 4 in step e).

[0179] 3.4 In vitro molecular interaction between CBX7 and δ-Amyrenone

[0180] The interaction mechanism between CBX7 and δ-Amyrenone was discovered through in vitro molecular interaction experiments. The experimental results showed that the two exhibited kinetic characteristics of rapid binding and rapid dissociation, and with the increase in the concentration of δ-Amyrenone, the binding strength also increased significantly (see Figure 5 in step a). Through detailed data analysis, the dissociation constant Kd value of CBX7 and δ-Amyrenone was calculated to be 49.65 nM, which strongly demonstrated the strong binding affinity between them (see Figure 5 in step b).

[0181] Although the current experimental results revealed their high affinity characteristics, to comprehensively and deeply analyze their potential pharmacological effects, more extensive and in-depth research work needs to be carried out.

[0182] 3.5 δ-Amyrenone promotes the proliferation of AC16 cells in vitro

[0183] To explore the effect of δ-Amyrenone on the proliferation of cardiomyocytes, the CCK-8 experimental method was used. The experimental results showed (see Figure 6 in step a) that different concentrations of δ-Amyrenone had different promoting effects on the activity of cardiomyocytes after ischemia-reperfusion treatment, and 20 μM was its optimal concentration. Compared with the ischemia-reperfusion group, after treatment with 20 μM δ-Amyrenone, the viability of cardiomyocytes increased significantly by 13.87% ± 7.87%. Based on this finding, 20 μM was selected as the working concentration of δ-Amyrenone in subsequent experiments.

[0184] In addition, to further evaluate the proliferative effect of δ-Amyrenone, CCK-8 assays were performed on the other nine small molecule compounds with the top docking scores in Table 6 at the same concentration (20 μM). The results showed that among all candidate molecules, δ-Amyrenone still exhibited the best proliferative activity (see Figure 6 b) in

[0185] To further verify the proliferative effect at this concentration, trypan blue and YO-PRO-1 staining assays were carried out. The trypan blue staining results showed that the proliferation of cardiomyocytes in the 20 μM δ-Amyrenone treatment group was obvious (see Figure 6 c) in Figure 6 . Meanwhile, a significant decrease in fluorescence intensity was observed in the YO-PRO-1 staining assay (see Figure 6 d) in

[0186] These results suggest a change in the cell state in the δ-Amyrenone treatment group, which may be related to the enhanced proliferative activity. These experimental results further strengthen the potential role of δ-Amyrenone in promoting cardiomyocyte proliferation.

[0186] 3.6 Analysis of δ-Amyrenone regulating cardiomyocyte apoptosis

[0187] Apoptosis, as a core mechanism regulating cell fate, plays an indispensable role in maintaining tissue and organ homeostasis. To deeply explore the specific effect of δ-Amyrenone on cardiomyocyte proliferation, flow cytometry was carried out to evaluate cell apoptosis. The experimental data revealed that the apoptosis rate of the normal (control) group remained at a relatively low level of 5.15% ± 0.13%. However, in the cells subjected to hypoxia-reoxygenation treatment and ischemia-hypoxia-reoxygenation treatment, the apoptosis rates increased to 6.60% ± 0.24% and 15.91% ± 0.77% respectively. However, in the cells of the ischemia-hypoxia-reoxygenation group after δ-Amyrenone intervention, the apoptosis rate was significantly inhibited, decreasing to 10.42% ± 0.25% (see specifically Figure 7 a-b) in Figure 7 . In addition, the fluorescence image results ( Figure 7 c) in

[0188] also confirmed this result.

[0188] Changes in apoptosis are usually related to apoptosis-related proteins. Western blotting (WB) was used to explore the changes in the expression patterns of related proteins. In the established ischemia-reperfusion injury model, significant upregulation of the expression levels of two key apoptosis proteins, c-caspase-3 and c-PARP, was observed compared with the normal group. However, in the δ-Amyrenone treatment group, the expression levels of both proteins showed an obvious 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 treatment group also showed a decreasing trend (see specificallyFigure 7 In d). This series of experimental results strongly indicate that δ-Amyrenone significantly slows down the process of apoptosis by effectively inhibiting the expression of apoptosis-related proteins, thereby playing a positive protective role.

[0189] 3.7 Regulation of cardiomyocyte cell cycle analysis by δ-Amyrenone

[0190] Studies have shown that the CBX7 protein plays an inhibitory role in cardiomyocyte proliferation by regulating the cell cycle process. The effects of the small molecule δ-Amyrenone on the cell cycle of human cardiomyocytes were explored by flow cytometry experiments, and the results are shown in Figure 8 a and b in the figure. The proportions of each stage of the cell cycle of normal cardiomyocytes were: G1 phase 42.57% ± 0.94%, S phase 36.77% ± 0.78%, and G2 phase 16.73% ± 0.58%. In contrast, hypoxia-reoxygenation treatment led to cell arrest mainly in the G1 phase, and its proportion increased to 45.4% ± 0.67%. For cardiomyocytes intervened with the small molecule δ-Amyrenone, the proportion of the G1 phase in the cell 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-reoxygenation, reduce cell arrest in the G1 phase, and may promote the smooth transition of cells from the G1 phase to the S phase and the G2 phase, thus contributing to the normal progression of the cell cycle. Based on these findings, it is concluded that δ-Amyrenone regulates the cell cycle by acting on the CBX7 protein, alleviates the functional damage of cardiomyocytes under hypoxia-reoxygenation conditions, and thus promotes the proliferation of AC16 cells.

[0191] To further verify the regulatory effect of δ-Amyrenone on the CBX7 protein, Western blot experiments were conducted (see specifically in Figure 8In c). The experimental results showed that, compared with normal cardiomyocytes, the expression levels of Cyclin D1 protein (a protein related to the G1 phase) were significantly increased in both the hypoxia-reperfusion group and the ischemia-hypoxia-reperfusion group. However, in the ischemia-reperfusion group after δ-Amyrenone treatment, the expression level of Cyclin D1 showed an obvious decrease, which suggested that δ-Amyrenone could reduce the overexpression of this protein. The expression of CDK2 protein (a protein related to the 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 was restored and increased. It is worth noting that the expression level of Cyclin A2 protein (a protein related to the G2 phase) in the δ-Amyrenone treatment group was the highest among the four groups of experiments. These protein blotting results were consistent with the conclusion obtained from the previous flow cytometry analysis (i.e., promoting the smooth transition of cardiomyocytes from the G1 phase to the S phase and the G2 phase), all indicating that the small molecule δ-Amyrenone could inhibit the function of CBX7 protein, thereby regulating the cell cycle and promoting the proliferation of ischemia-reperfusion cardiomyocytes.

[0192] 3.8 δ-Amyrenone regulates cardiomyocyte proliferation through SIRT7 / ALDH7A1 / FOXM1 proteins

[0193] After investigating the effect of δ-Amyrenone on CBX7 protein, the expression changes of related genes were further verified to reveal its potential regulatory mechanism on cardiomyocyte proliferation. The results showed that after δ-Amyrenone treatment, the expressions of genes such as CDCA2, ARRB1, FOXM1, SIRT7, and ALDH7A1 all changed significantly ( Figure 9). Specifically, after the action of δ-Amyrenone, the gene expressions of CDCA2, ARRB1, FOXM1, SIRT7, and ALDH7A1 were all up-regulated. Further literature analysis showed that CDCA2 could promote the G1 / S phase transition by up-regulating and activating CCND1 / CDK4 / 6 and CCNE1 / CDK2, thereby promoting the proliferation of hepatoma cells and enhancing their colony formation ability. At the same time, CDCA2 inhibited apoptosis in a p53 / p21-dependent manner by inhibiting the p38 MAPK pathway and activating the JNK / c-Jun pathway, and promoted the migration of p53-mutated Huh7 cells through promoting epithelial-mesenchymal transition. Experimental findings showed that δ-Amyrenone could up-regulate the expression of CDCA2, thus promoting cardiomyocyte proliferation. Another study showed that ARRB1 drives the G1-S phase transition of the cell cycle in liver cancer. In other words, δ-Amyrenone has the ability to up-regulate the expression of ARRB1, thereby promoting the transition of cells from the G1 phase to the S phase, and this mechanism of action may contribute to promoting the growth and repair of damaged myocardium. The Sirt7 protein plays a role in the progression of non-small cell lung cancer, promoting tumor progression by facilitating the G1 / S phase transition, epithelial-mesenchymal transition, and activating the AKT and ERK1 / 2 signaling pathways. After cardiomyocyte injury, the expression of Sirt7 decreased, but after treatment with δ-Amyrenone, the expression of Sirt7 returned to normal levels, which may be one of the mechanisms by which it promotes cardiomyocyte proliferation. After myocardial ischemia-reperfusion, the expression of ALDH7A1 was down-regulated, and ALDH7A1 has been confirmed to be able to promote the G1-S phase transition. Treatment with δ-Amyrenone contributed to the increase in the expression of ALDH7A1. FoxM1 is an important cell cycle regulator that controls the expression of genes required for the G1 / S phase and S / G2 phase transitions. After treatment with δ-Amyrenone, the expression of FoxM1 protein was up-regulated, further promoting the proliferation of damaged cardiomyocytes. Overall, δ-Amyrenone regulates the cell cycle through multiple signaling pathways to promote cardiomyocyte proliferation. In addition to the signaling pathway mediated by the CBX7 protein, δ-Amyrenone also promotes the G1 / S and G2 phase transitions by up-regulating genes / proteins such as CDCA2, ARRB1, Sirt7, ALDH7A1, and FoxM1, thereby promoting cardiomyocyte proliferation. This indicates that δ-Amyrenone promotes the repair and regeneration of damaged myocardium through the synergistic action of multiple mechanisms.

[0194] 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 targets the CBX7 protein. Computer simulation revealed the binding mode and binding site between the two, while in vitro molecular interaction experiments verified the direct interaction between δ-Amyrenone and CBX7 from a practical perspective.

[0195] The screened small molecule δ-Amyrenone can promote the proliferation and inhibit the apoptosis of cardiomyocytes: the results of cell viability detection experiments and staining experiments both show that the screened inhibitor has a significant effect on promoting proliferation. At the same time, flow cytometry analysis and Western blot experiments also revealed its significant effect on inhibiting cardiomyocyte apoptosis.

[0196] Through flow cytometry analysis, protein and gene levels, it was found that the small molecule inhibitor δ-Amyrenone has a regulatory effect on the cell cycle of ischemic reperfusion cardiomyocytes.

[0197] In summary, δ-Amyrenone can act as an inhibitor of the CBX7 protein, thereby regulating the cell cycle, inhibiting cell apoptosis, promoting cardiomyocyte proliferation, and having good application prospects in the prevention and / or treatment of ischemic heart disease.

[0198] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within 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 myocardial infarction reperfusion injury, characterized in that: The CBX7 inhibitor is δ-Amyrenone or a salt thereof.

2. The use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that: The drug is administered by injection.

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