Application of ginsenoside Rg5 in improvement of acute hypoxic myocardial injury

Abstract

The invention discloses application of ginsenoside Rg5 in improvement of acute hypoxic myocardial injury, and belongs to the field of medical preparations. One of the technical problems to be solved is how to prevent and / or treat hypoxic myocardial injury. In order to solve the technical problems, the invention provides application of ginsenoside Rg5 and pharmaceutically acceptable derivatives and / or compositions thereof in preparation of drugs for preventing and / or treating hypoxic myocardial cell injury, preventing and / or treating hypoxic myocardial injury and / or preventing and / or treating altitude stress. The invention discloses application of ginsenoside Rg5 in prevention and / or treatment of hypoxic myocardial cell injury, hypoxic myocardial injury and / or altitude stress for the first time. The medicinal value of the ginsenoside Rg5 is further developed, and meanwhile, a prevention and / or treatment scheme is provided for altitude stress and hypoxic myocardial injury.

Description

Technical Field

[0001] The present application belongs to the field of medical preparations, and specifically relates to the application of ginsenoside Rg5 in improving acute hypoxic myocardial injury. Background Art

[0002] With the development of transportation, people can quickly travel between low altitude and high altitude areas. However, the rapid change of environment will bring great challenges to the human body. When individuals rise rapidly from low altitude to high altitude, they often experience acute mountain sickness, such as headache, palpitations and shortness of breath. In high altitude environment, the heart will improve the efficiency of pumping blood by increasing the beating frequency and strengthening the contractility of the myocardium, so as to ensure that various tissues get as much oxygen and nutrients as possible. Hypoxia can cause irreversible damage to myocardial cells and even lead to myocardial cell death. Since myocardial cells cannot be regenerated, even if the oxygen supply is restored later, the damaged myocardial cells cannot be saved. Existing studies have shown that apoptosis is one of the main forms of myocardial cell damage under hypoxic conditions, and excessive oxidative stress and inflammatory response are important pathways for hypoxia-induced myocardial cell apoptosis.

[0003] As a long-standing medicinal plant, ginseng has excellent efficacy and a long history of use in the treatment of myocardial diseases. Its medicinal effects are mainly derived from its rich chemical components, including ginsenosides, polysaccharides, phenolic compounds, volatile oils, alkaloids and proteins. At present, the pharmacological research on ginseng is mainly focused on ginsenosides. Ginsenoside Rg5 is a unique saponin derivative in ginseng, which has attracted much attention due to its excellent biological activity. Rg5 has a wide range of therapeutic effects, including anti-cancer, anti-inflammatory, antioxidant and myocardial protection. Specifically, Rg5 can effectively reduce hypoxia-induced cardiomyocyte damage and improve the resistance of cardiomyocytes to ischemic damage by inhibiting cell apoptosis. In addition, it can improve mitochondrial function and further increase the survival rate of cardiomyocytes under ischemic conditions.

[0004] Signal transducer and activator of transcription 3 (STAT3) plays a key role in a variety of physiological and pathological processes. STAT3 is not only involved in basic biological processes such as cell proliferation, differentiation and survival, but also plays an important role in immune regulation, inflammatory response and tissue repair. In the heart, the high expression and active state of STAT3 make it a key molecule for maintaining cardiac function and protecting the myocardium from damage. The activation of STAT3 mainly depends on two key phosphorylation sites: tyrosine 705 (Y705) and serine 727 (S727). Y705 is particularly important for STAT3. When cytokines (such as IL-6) or growth factors (such as EGF) bind to their receptors, receptor-associated Janus kinases (JAKs) are activated, which in turn phosphorylate Y705 of STAT3. Phosphorylated STAT3 forms dimers, enters the cell nucleus, and binds to specific DNA sequences to regulate the expression of target genes. Phosphorylation of S727 can be mediated by a variety of kinases (such as MAPKs, PKC, etc.), and works together with phosphorylation of Y705 to regulate the activity of STAT3. This signaling pathway is crucial in a variety of physiological and pathological processes, especially in the heart, where activation of the JAK2 / STAT3 signaling pathway can reduce cardiomyocyte apoptosis and enhance cardiac function. Although the role of STAT3 in cardioprotection has been widely recognized, further research is needed to clarify its specific mechanism and potential clinical application in high-altitude myocardial injury. Summary of the invention

[0005] One of the technical problems to be solved by the present application is how to prevent and / or treat hypoxic myocardial damage caused by altitude sickness.

[0006] In order to solve the above technical problems, the present application provides the use of a substance in the preparation of a drug for preventing and / or treating hypoxic myocardial cell damage, preventing and / or treating hypoxic myocardial damage and / or preventing and / or treating altitude sickness, wherein the substance is A or a composition containing A, and the A is ginsenoside Rg5, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable salt hydrates or solvates, and its pharmaceutically acceptable modifications.

[0007] Furthermore, the altitude sickness includes producing hypoxic myocardial damage.

[0008] Furthermore, the hypoxic myocardial injury includes abnormal cardiac ejection fraction and / or abnormal cardiac minor axis shortening rate.

[0009] Furthermore, the abnormal cardiac ejection fraction may be a cardiac ejection fraction that is statistically significantly lower than that of a healthy subject under normal circumstances.

[0010] Furthermore, the abnormal cardiac minor axis shortening rate may be a cardiac minor axis shortening rate that is statistically significantly lower than that of healthy subjects under normal circumstances.

[0011] Furthermore, the myocardial injury also includes abnormal levels of myocardial injury markers, abnormal levels of cytokines, abnormal oxidative stress response and / or histological lesions.

[0012] Furthermore, the abnormal content of the myocardial injury marker may be statistically significantly higher than the content of the myocardial injury marker in healthy subjects under normal conditions.

[0013] Furthermore, the abnormal cytokine content may be a cytokine content that is statistically significantly higher than that of a healthy subject in a normal environment.

[0014] Furthermore, the abnormal oxidative stress response may be statistically significantly higher than the oxidative stress response of healthy subjects in a normal environment.

[0015] Furthermore, the histological lesions include hypertrophy of myocardial endocardial cells, widening of intercellular spaces, and vacuoles in cells.

[0016] In the present application, the normal environment may be an environment with an altitude of 0-2500 meters. In some embodiments of the present application, the altitude of the normal environment is 50 meters.

[0017] In the present application, the myocardial injury markers include cardiac troponin I (cTnI), myoglobin (MYO), creatine kinase (CK) and / or creatine kinase mb (CKMB).

[0018] In the present application, the cytokines include IL-1β, IL-6 and / or TNF-α.

[0019] In the present application, the abnormal stress response includes increased malondialdehyde (MDA) content, increased superoxide dismutase (SOD) activity and / or increased reduced glutathione (GSH) content.

[0020] In the present application, the prevention and / or treatment of myocardial injury and / or the prevention and / or treatment of altitude sickness include administering an effective dose of a drug to a subject to achieve any of the following goals: improving abnormal cardiac ejection fraction and / or cardiac short axis shortening rate, reducing abnormal myocardial injury marker content, reducing abnormal cytokine content, reducing abnormal oxidative stress response and / or histological lesions. The improvement or reduction is carried out under comparable conditions. The "comparable conditions" refer to the same or similar environmental conditions. Environmental conditions include, for example, altitude, gas, temperature, humidity and / or light.

[0021] In this application, "subject" includes a person who is being treated for a disease or preventing a disease as a patient. The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, rodents (order Rodentia) mammals, such as mice and hamsters, and lagomorpha (order Logomorpha) mammals, such as rabbits. Mammals can be carnivores (order Carnivora), including felines (cats) and canines (dogs). Mammals can be even-toed ungulates (order Artiodactyla), including bovines (cows) and suids (pigs), or odd-toed ungulates (order Perssodactyla), including equines (horses). Mammals can be primates (order Primate), four-legged monkeys (order Ceboid) or monkeys (order Simoid) (monkeys) or humanoids.

[0022] The term "effective" as applied to dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0023] In the present application, the dosage is calculated based on the effective ingredient, which is ginsenoside Rg5, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable salt hydrates or solvates or its pharmaceutically acceptable modifications.

[0024] Furthermore, the effective dose of the drug may be 10 mg / Kg to 50 mg / Kg.

[0025] Furthermore, the effective dose of the drug may be 15 mg / Kg to 45 mg / Kg.

[0026] Furthermore, the effective dose of the drug may be 20 mg / Kg to 40 mg / Kg.

[0027] Furthermore, the effective dose of the drug may be 25 mg / Kg to 35 mg / Kg.

[0028] Furthermore, the effective dose of the drug may be 26 mg / Kg to 34 mg / Kg.

[0029] Furthermore, the effective dose of the drug may be 27 mg / Kg to 33 mg / Kg.

[0030] Furthermore, the effective dose of the drug may be 28 mg / Kg to 32 mg / Kg.

[0031] Furthermore, the effective dose of the drug may be 29 mg / Kg to 31 mg / Kg.

[0032] Furthermore, the effective dose of the drug may be 30 mg / Kg.

[0033] In the present application, the administration method may be oral administration.

[0034] The present application also provides a method for preventing hypoxic cardiomyocyte damage, which comprises the step of contacting the cardiomyocytes with the above-mentioned drug.

[0035] Furthermore, the cardiomyocytes are isolated cardiomyocytes.

[0036] In the present application, the myocardial cell injury includes: a decrease in the survival rate of myocardial cells and / or an increase in the content of myocardial injury markers. Further, the decrease in the survival rate of myocardial cells may be statistically significantly lower than the survival rate of normal myocardial cells in a normal environment. Further, the increase in the content of myocardial injury markers may be statistically significantly higher than the survival rate of normal myocardial cells in a normal environment.

[0037] In the present application, the prevention and / or treatment of myocardial cell injury includes administering an effective dose of a drug to myocardial cells to achieve any of the following goals: increasing the survival rate of myocardial cells and / or reducing the content of myocardial injury markers. The increase or reduction is performed under comparable conditions. The "comparable conditions" refer to the same or similar environmental conditions. Environmental conditions include, for example, temperature, humidity, light, altitude and / or gas.

[0038] Furthermore, the effective dose may be 0.25 to 1.00 μM.

[0039] Furthermore, the administration dosage may be 0.25 to 0.50 μM.

[0040] Furthermore, the effective dose may be 0.50 μM.

[0041] Another technical problem to be solved by the present application is: how to provide a STAT3 agonist. To solve this technical problem, the present application provides the use of a substance in the preparation of an agonist of signal transduction and transcription activator protein 3 (STAT3), wherein the substance is A or a composition containing A, wherein A is ginsenoside Rg5, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable salt hydrates or solvates, and its pharmaceutically acceptable modifications.

[0042] The UniProtKB number of mouse (MOUSE) signal transducer and activator of transcription protein 3 (STAT3) is P42227 (https: / / www.uniprot.org / uniprotkb / P42227 / entry). The UniProtKB number of rat (RAT) STAT3 is P52631 (https: / / www.uniprot.org / uniprotkb / P52631 / entry). The UniProtKB number of human (HUMAN) STAT3 is P40763 (https: / / www.uniprot.org / uniprotkb / P40763 / entry).

[0043] Furthermore, the agonist is used to promote the phosphorylation of tyrosine 705 of signal transduction and activator of transcription protein 3 (STAT3).

[0044] Furthermore, the tyrosine at position 705 of signal transduction and activator of transcription protein 3 (STAT3) includes but is not limited to: the tyrosine at position 705 of the amino acid sequence shown in UniProtKB number P42227, the tyrosine at position 705 of the amino acid sequence shown in UniProtKB number P52631 and / or the tyrosine at position 705 of the amino acid sequence shown in UniProtKB number P40763.

[0045] The activation of STAT3 mainly depends on two key phosphorylation sites: tyrosine at position 705 (Y705) and serine at position 727 (S727). Among them, phosphorylation of Y705 is particularly important for STAT3 activation.

[0046] STAT3 phosphorylated at Y705 forms a dimer, enters the nucleus, and binds to specific DNA sequences to regulate the expression of target genes. Phosphorylation of S727 can be mediated by a variety of kinases (such as MAPKs, PKC, etc.), and works together with phosphorylation of Y705 to regulate the activity of STAT3. This signaling pathway is crucial in a variety of physiological and pathological processes, especially in the heart. Activation of the JAK2 / STAT3 signaling pathway can reduce cardiomyocyte apoptosis and enhance cardiac function.

[0047] The prior art has revealed that STAT3 agonists play a positive role in Alzheimer's disease, ischemic brain damage, increasing human platelet levels, inflammation such as atherosclerosis, bacterial infection, enhancing intestinal barrier function, intestinal immunity and tissue repair, etc. Therefore, the agonists provided in this application are also expected to be used to prevent and / or treat Alzheimer's disease, ischemic brain damage, inflammation such as atherosclerosis, bacterial infection and / or increase human platelet levels, enhance intestinal barrier function, intestinal immunity and tissue repair, etc.

[0048] The structural formula of ginsenoside Rg5 described in the present application is as shown in formula (I), and its CAS number is: 186763-78-0.

[0049]

[0050] The beneficial technical effects achieved by this application are as follows: 1. This application discloses for the first time the use of ginsenoside Rg5 in preventing and / or treating hypoxic myocardial cell damage, hypoxic myocardial damage and / or altitude sickness. It further develops the medicinal value of ginsenoside Rg5 and provides a prevention and / or treatment scheme for altitude sickness and hypoxic myocardial damage.

[0051] 2. The present application also reveals that the target of ginsenoside Rg5 in cardiomyocytes is STAT3, and its mechanism of action is to activate STAT3, that is, ginsenoside Rg5 can be used as an agonist of STAT3. At present, there are few agonists of STAT3 that have been excavated in the prior art, and the technical scheme provided in this application can be further used for the study of the mechanism and metabolic pathway of STAT3 activation. At the same time, ginsenoside Rg5 is expected to be used to prevent and / or treat related diseases that achieve prevention and / or treatment purposes through STAT3 activation, such as Alzheimer's disease, ischemic brain injury, increased human platelet levels, inflammation such as atherosclerosis, bacterial infection, enhanced intestinal barrier function, intestinal immunity and tissue repair, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the verification result that ginsenoside Rg5 improves myocardial damage caused by acute hypoxia; Figure 2 This is the verification result that ginsenoside Rg5 improves myocardial cell damage caused by acute hypoxia; Figure 3 This is the validation result that ginsenoside Rg5 alleviates acute hypoxia-induced cardiomyocyte oxidative stress and mitochondrial damage; Figure 4 This is the verification result that ginsenoside Rg5 alleviates myocardial cell apoptosis caused by acute hypoxia; Figure 5The results verify that STAT3 is the key target of Rg5 in alleviating acute hypoxic myocardial injury; Figure 6 Evidence that ginsenoside Rg5 targets STAT3; Figure 7 The results verify that Rg5 alleviates acute hypoxic myocardial injury by promoting STAT3 Tyr705 phosphorylation; Figure 8 The validation results show that STAT3 inhibitors aggravate acute hypoxic injury; Fig. 9 The results verify that Rg5 enhances the interaction between STAT3 and JAK2; Fig.10 These are the validation results that ginsenoside Rg5 improves acute hypoxic cardiac injury by targeting STAT3 at the animal level. DETAILED DESCRIPTION

[0053] Terms used in this application: Examples of resources describing many of the molecular biology-related terms used herein can be found in Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0054] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

[0055] To facilitate understanding of the present disclosure, several terms and abbreviations used herein are defined as follows: When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B, and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0056] The test methods involved in this application are as follows: 1 Animals and experimental protocols Male C57BL / 6 mice (20–22 g) were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All mice were 6 weeks old. All animal husbandry and experimental procedures in this study were approved by the Animal Ethics Committee of the Military Medical Research Institute. Mice were allowed to acclimate for 7 days before starting the in vivo experiments. Mice were placed on a 12:12 h light-dark cycle and provided with a standard rodent chow diet.

[0057] The mice were randomly divided into 3 groups: control group, model hypoxia group and hypoxia + ginsenoside Rg5 group (6 mice in each group). Ginsenoside Rg5 was dissolved in 2.5% sodium carboxymethyl cellulose solution and administered orally at a dose of 30 mg / kg of ginsenoside Rg5 for 7 days. After 7 days of administration, the mice in the hypoxia group were placed in a low-pressure hypoxia simulation chamber (Guizhou Fenglei Aviation Ordnance Co., Ltd., DYC-9070) for 3 days to simulate an altitude of 6500 meters. During this period, the mice could drink water and eat normally. After the simulation, the simulated altitude was lowered to 3500 meters, and the researchers entered the simulation chamber to collect mouse samples.

[0058] 2 Echocardiography Echocardiograms of mice were recorded using a 6 LAB animal ultrasound machine (Beijing Yiren Hengye Technology Co., Ltd.) equipped with a French VERMON high-frequency probe. M-mode scans were performed to evaluate changes in ejection fraction (EF, %) and fractional shortening (FS, %).

[0059] 3 Enzyme-linked immunosorbent assay (ELISA) The ELISA kits (catalog numbers: MM-0040M2, MM-0163M2, MM-0132M2, MM-0389M2, MM-0897M2, MM-0661M2, MM-0791M2, MM-0518M2, MM-0734M2, MM-43703M2) provided by Jiangsu Enzyme Absorption Biotechnology Co., Ltd. were used to measure the levels of serum interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), cardiac troponin I (cTnI), myoglobin (MYO), creatine kinase (CK) and creatine kinase isoenzyme MB (CK-MB).

[0060] 4. Cardiac Histological Examination After anesthetizing the animals, the heart tissues were removed from the chest cavity and fixed with 4% paraformaldehyde. The heart tissues were sliced ​​(5 mm thick), embedded in paraffin blocks, and stained with hematoxylin-eosin (HE) for morphological examination.

[0061] 5. Cell culture and treatment H9c2 cells (rat cardiomyocytes) were purchased from Wuhan Punosai Life Science Co., Ltd. (CL-0089). The cells were grown in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100 μg / ml streptomycin, and 100 μg / ml penicillin and cultured at 37°C in a humidified atmosphere with 5% CO2.

[0062] Before the experiment, the drugs were diluted in DMEM containing 2% fetal bovine serum (FBS). Then the cultured H9c2 cells were treated with hypoxia (5% CO2, 1% O2) for 24h. For the Rg5 group (drug treatment group), ginsenoside Rg5 was given at concentrations of 0.25, 0.5 and 1μM 24 hours before hypoxia exposure. For the Rg5+sta group, 5μM stattic was added under the treatment conditions of the Rg5 group. For the col group (colivelin): 1μM colivelin was given 24 hours before hypoxia exposure. For the sta group (stattic): 5μM stattic was given 24 hours before hypoxia exposure. No drugs were added to the culture medium of the model group (Mod) cells and the control group (Ctrl) cells. The difference is that the cells in the model group need to be treated with hypoxia, while the cells in the control group are cultured under normal conditions.

[0063] 6 CCK-8 cell proliferation assay H9c2 cells were seeded into 96-well plates at a density of 3 × 10^4 cells per well and cultured for 24 h at 37°C, 5% CO2. Cell proliferation was assessed using the CCK-8 kit (TargetMol, Shanghai) according to the manual. The absorbance was measured at 450 nm using a microplate reader.

[0064] 7. Cell apoptosis detection To distinguish apoptotic cells from viable or necrotic cells, Annexin V-FITC and PI (propidiumiodide) were used for co-staining (Wuhan Elaruite Biotechnology Co., Ltd.). To study the damage of H9c2 cells to hypoxia, the treated cells were trypsinized, washed with PBS, and resuspended in binding buffer. The cell suspension (1×10^5 cells / ml) was then incubated with FITC-labeled Annexin V and PI in the dark for 20 minutes. Cell counting was performed using BD FACSAria™ III (USA), and data were analyzed using FlowJo 10.8 software. 10,000 cells were recorded for each sample. The fluorescence spectra of Annexin V and PI were detected using 527 / 32 nm and 586 / 42 nm filters, respectively.

[0065] 8 JC-1 mitochondrial membrane potential detection Mitochondrial membrane potential was assessed using the JC-1 fluorescent probe (Wuhan Elaruite Biotechnology Co., Ltd.). Cells harvested during the experiment were trypsinized, washed with PBS and resuspended. Cell suspension (1×10^5 cells / mL) was prepared. The number of cells with green (JC-1 monomer) and red (JC-1 aggregate) fluorescence was determined using emission filters at 535 nm and 595 nm, respectively. Immunolabeled cells were analyzed using a BD FACSAria™ III flow cytometer.

[0066] 9 Reactive oxygen species (ROS) detection H9c2 cells were trypsinized and then treated with 500 nM DCFH-DA (Shanghai Biotech Co., Ltd.) probe at 37°C for 20 min, followed by two washes with PBS. The fluorescence intensity of DCFH-DA was measured at an excitation wavelength of 488 nm using a BD FACSAria™ III flow cytometer. FlowJo 10.8 software was used for analysis.

[0067] 10 Seahorse Analysis The oxygen consumption rate (OCR) of H9c2 cells was measured in real time using a Seahorse XFe96 analyzer (Agilent). Cells (3 × 10^4 cells per well) were seeded onto Seahorse 96-well cell culture plates. Cells were then treated with different concentrations of ginsenoside Rg5 for 24 h. After treatment, cells were subjected to hypoxia for 24 h. Subsequently, the culture medium was replaced with buffer-free, glucose-free DMEM supplemented with 2 mmol / L glutamine. The analysis was performed according to the Seahorse XF Cell Mitochondrial Stress Test Kit (103708-100, Agilent) and instrument instructions.

[0068] 11 Immunofluorescence staining After H9c2 cells were cultured on coverslips for 12 hours, the cells were washed three times with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. After fixation, the cells were rinsed with immunofluorescence washing buffer and blocked with immunofluorescence blocking buffer for 2 hours at 4°C. Subsequently, the coverslips were incubated with primary antibodies against phosphorylated signal transducer and activator of transcription 3 (STAT3) (Tyr705) (#9145, CST) at 4°C overnight. After that, the coverslips were incubated with fluorescent secondary antibodies for 1 hour. Finally, the coverslips were mounted with anti-fading mounting medium containing DAPI. Observation was performed using a confocal microscope.

[0069] 12 Cell thermal migration assay (CETSA) H9c2 cells were collected in PBS buffer, and whole-cell proteins were extracted by repeated freeze-thaw cycles. Then, the cell lysates were incubated with dimethyl sulfoxide (DMSO) (1%) or ginsenoside Rg5 (0.5 μM) on ice for 1 hour. The mixture samples were divided into eight equal parts and incubated in parallel at different temperatures from 42°C to 66°C for 3 minutes. After incubation, the samples were centrifuged, and the supernatant was used for Western blot analysis to detect the content of STAT3.

[0070] 13 Drug affinity reaction targeting stability H9c2 cells were collected and resuspended in PBS. Proteins were extracted by repeated freeze-thaw cycles and then centrifuged at 3500 rpm for 15 min at 4 °C. The lysate was divided into five equal parts and incubated with DMSO and different concentrations of ginsenoside Rg5 (0.25, 0.5, and 1 μM) on ice for 1 h. After incubation, protease (HY-B2228, MCE, China) (enzyme: cell protein = 1:500) was used for proteolysis on ice for 15 min. Finally, 5× loading buffer was added to denature the proteins, and the expression of STAT3 was detected by western blotting.

[0071] 14 Immunoprecipitation (IP) The STAT3 plasmid with a Flag tag was transfected into H9c2 cells using a transfection reagent. After the drug experiment was completed, STAT3 and its interacting proteins were extracted from the transfected H9c2 cells using a Flag tag protein immunoprecipitation kit with agarose gel. The interaction was then confirmed by western blot analysis.

[0072] 15 Molecular docking The structural information of STAT3 was retrieved from the Protein Data Bank (RCSB). Modifications such as separation of the original ligand, removal of water molecules, addition of hydrogen atoms, and repair of amino acids were performed using PyMol software (version 1.8) and AutoDockTools software (version 1.5.6). Molecular docking was then performed using Autodock-vina (version 1.1.2). The docking results were visualized by PyMol software, and the docking interaction pattern was established.

[0073] 16 Western blot (WB) analysis Western blot (WB) analysis was performed according to conventional methods in the art. The corresponding proteins and primary antibodies were as follows: p-STAT3 (Tyr705) (#R381552, Zenbio), p-STAT3 (Ser727) (#9134, CST), STAT3 (#10253-2-ap, Proteintech), JAK2 (YT2426, Immunoway), cleaved caspase-3 (#9661, CST), cleaved caspase-9 (#9509, CST), Cytochrome c (#11940, CST), Bcl2 (YT0470, Immunoway), and Bax (#YT0449, Immunoway).

[0074] 17 Data Processing The quantitative tests in the following examples were repeated six times unless otherwise specified, and the results were averaged.

[0075] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used for multiple group comparisons, and t-test was used for two group comparisons. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a very significant difference, and P < 0.001 (***) indicates an extremely significant difference.

[0076] To study the effect of Rg5 on acute hypoxic myocardial injury, a hypobaric hypoxic environment simulation chamber (simulating an altitude of 6500 meters, such as Figure 1 The cells were placed in an anaerobic chamber (shown in (a)) and in a cell anaerobic chamber (1% oxygen concentration) to simulate the acute hypoxic state of mice and H9c2 cells.

[0077] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0078] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0079] Example 1: Verification of the effect of ginsenoside Rg5 on myocardial injury caused by hypoxia at animal level Eighteen 6-week-old male C57BL / 6J mice (weight 20-22 g) were randomly divided into 3 groups. They were provided with normal food and water during the treatment period. According to the different treatment methods, the groups were divided into the following groups: Rg5: Rg5 was continuously administered at a dose of 30 mg / kg, by oral administration, 0.3 mL / mouse, once a day; after 7 consecutive days of administration, the mice were placed in a hypobaric hypoxia simulation chamber and exposed to a simulated 6500m altitude environment for three days; after the treatment, the mice's cardiac ejection fraction and short-axis shortening rate, myocardial injury markers, and myocardial tissue microscopy were tested; Mod: The treatment method was the same as that of the Rg5 group, with the only difference being that Rg5 was replaced with the same volume of ddH2O; after 7 consecutive days of treatment, the mice were placed in a hypobaric hypoxia simulation chamber for three consecutive days; during this period, food and water were provided normally; Ctrl: The treatment method was the same as that of the Mod group, with the only difference being that after gavage with ddH2O for 7 consecutive days, the mice were placed in a normal environment (~50m above sea level) for another three days.

[0080] Three replicates were set for each treatment, and the results were averaged.

[0081] The detection methods are as follows: (1) The method for detecting the cardiac ejection fraction of mice is echocardiography. After the mice were anesthetized with isoflurane, the hair near the chest cavity of the mice was removed, and the ejection fraction of the heart of each group of mice was measured using a 6LAB portable small animal ultrasound instrument produced by Beijing Yiren Hengye Technology Co., Ltd.

[0082] (2) The detection and calculation method of the short-axis shortening rate of the mouse heart is the echocardiographic measurement method. The detection method refers to the ejection fraction of the mouse heart. The calculation formula of the short-axis shortening rate (FS%) is: FS%=[(LVEDD-LVESD) / LVEDD]×100%. Among them, LVEDD represents the left ventricular end-diastolic diameter, and LVESD represents the left ventricular end-systolic diameter.

[0083] (3) Detection of myocardial injury markers Myocardial injury markers include cardiac troponin I (cTnI), myoglobin (MYO), creatine kinase (CK) and creatine kinase mb (CKMB). The detection methods are all enzyme-linked immunosorbent assay (ELISA). The kits used for the above four indicators are from Jiangsu Enzyme-Free Industry Co., Ltd., with the item numbers: MM-0791M2, MM-0518M2, MM-0734M2, and MM-43703M2. Peripheral blood was collected and centrifuged to obtain the upper serum, and cardiac troponin I (cTnI), myoglobin (MYO), creatine kinase (CK) and creatine kinase mb were detected according to the operating methods in the instructions of the kit.

[0084] (4) Cytokine level detection: The cytokines IL-1β, IL-6 and TNF-α in cardiac tissue were detected by the following method: intact cardiac tissue was obtained, mechanically fragmented and centrifuged to extract cardiac lysate, and IL-1β, IL-6 and TNF-α were detected respectively according to the operating method in the instructions of the kit.

[0085] (5) Detection of oxidative stress response level The detection indicators of oxidative stress response level are: MDA, SOD, GSH. The specific detection method is: take intact heart tissue, mechanically fragment it and then centrifuge to extract heart lysate, and detect MDA, SOD, and GSH respectively according to the operating method in the instruction manual of the kit.

[0086] (6) The method for microscopic examination of myocardial tissue is hematoxylin-eosin (HE) staining, as follows: After the mouse is anesthetized, the heart tissue is removed from the chest cavity and fixed with 4% paraformaldehyde. A heart tissue section (5 mm thick) is taken and embedded in paraffin blocks, and finally morphological examination is performed under a microscope by hematoxylin-eosin staining.

[0087] Results Figure 1 Compared with the control group (Ctrl group) that was not exposed to hypoxia, the cardiac ejection fraction and short-axis shortening rate of the model group (Mod group) mice were significantly reduced. However, administration of Rg5 was able to restore myocardial function during hypoxia ( Figure 1b, c). The results of myocardial enzyme and myocardial injury marker tests showed that pre-administration of Rg5 could effectively improve the relevant indicators ( Figure 1 (d in the middle), indicating that Rg5 can reduce myocardial damage caused by hypoxia. From the observed indicators, hypoxia leads to increased inflammatory response and oxidative stress in the body, and pre-intervention with Rg5 can reduce inflammation and oxidative stress damage in mice ( Figure 1 In addition, pathological changes in myocardial tissue were detected under an optical microscope by hematoxylin-eosin (H&E) staining. No obvious abnormalities were observed in the control group, while hypertrophy of myocardial endothelial cells, widening of intercellular spaces, and vacuoles in some cells were observed in the model group. Rg5 treatment significantly improved these pathological injuries ( Figure 1 Middle g).

[0088] Example 2: Verification at the cell level that ginsenoside Rg5 alleviates myocardial damage caused by hypoxia H9c2 cells (rat cardiomyocytes) were purchased from Wuhan Punosai Life Science Co., Ltd. (CL-0089). The culture medium was DMEM supplemented with 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 μg / mL penicillin; the culture conditions were 37°C, 20% O2, 5% CO2 in a humidified gas incubator. The collected and cultured activated H9c2 cells were divided into the following two groups according to the treatment method: Rg5: Rg5 was diluted with DMEM containing 2% fetal bovine serum (FBS) to obtain cell culture media with final Rg5 concentrations of 0.25µM, 0.5µM, and 1µM, respectively. Activated H9c2 cells were added to a 96-well plate at a concentration of 3×10^4 / well and cultured under normal conditions (37°C, 20% O2, 5% CO2 humidified incubator) for 24 hours before hypoxia treatment (5% CO2, 1% O2). Hypoxia treatment was maintained for 24 hours.

[0089] Mod group: The treatment method is similar to that of Rg5 group, with the only difference being that Rg5 is replaced with the same volume of ddH2O; Ctrl: The treatment method was the same as that of the Mod group, with the only difference being that the cells were not exposed to hypoxia and were still cultured under normal conditions: 37°C, 20% O2, 5% CO2 in a humidified gas incubator.

[0090] Three replicates were set up for each treatment group.

[0091] 2.1. Cell activity and cell damage detection After the hypoxia treatment, the contents of myocardial enzymes (creatine kinase (CK) and creatine kinase mb (CKMB)) in the supernatant of the cell culture medium were detected, and the results were expressed as the ratio of creatine kinase mb (CKMB) to creatine kinase (CK); the cell viability was determined using the CCK-8 kit according to the kit instructions; The results of cell activity test showed that Rg5 (0.25µM, 0.5µM, 1µM) could improve the cell survival rate under hypoxic conditions ( Figure 2 The results of the detection of cardiac enzymes in the supernatant of H9c2 cell culture also showed that the degree of cell damage in the Rg5 group was milder than that in the model group ( Figure 2 This result is consistent with the above findings, indicating that Rg5 has a protective effect on the myocardium during hypoxia exposure.

[0092] 2.2. Rg5 reduces oxidative stress and mitochondrial damage in cardiomyocytes induced by acute hypoxia The heart consumes a lot of oxygen and is easily affected by hypoxia, which can lead to excessive oxidative stress. Next, we used reactive oxygen species detection and Seahorse analysis to explore whether Rg5 can alleviate myocardial oxidative stress by improving mitochondrial function.

[0093] 2.2.1. Reactive oxygen detection After hypoxia treatment, H9c2 cells were trypsinized and then treated with 500 nm DCFH-DA (Shanghai Biotech Co., Ltd.) probe at 37°C for 20 min, followed by two washes with PBS. The fluorescence intensity of DCFH-DA was measured at an excitation wavelength of 488 nm using a BD FACSAria™ III flow cytometer. FlowJo 10.8 software was used for analysis.

[0094] Flow cytometry results showed that Rg5 reduced the level of reactive oxygen species (ROS) in H9c2 cells after hypoxia exposure ( Figure 3 In order to further investigate the generation of mitochondrial ROS in H9c2 cells after Rg5 treatment, MitoSOX dye was used, which enables us to observe the distribution of ROS in mitochondria. The results showed that, similar to the overall ROS level, the generation of mitochondrial ROS in H9c2 cells after Rg5 treatment was significantly reduced ( Figure 3 (b) ROS generation is often associated with mitochondrial damage.

[0095] 2.2.2 Seahorse Analysis The oxygen consumption rate (OCR) of H9c2 cells was measured in real time using a Seahorse Xfe96 analyzer (Agilent). Cells (3 × 10^4 cells per well) were seeded onto Seahorse 96-well cell culture plates. Cells were then treated with different concentrations of ginsenoside Rg5 for 24 h. After treatment, cells were subjected to hypoxia for 24 h. Subsequently, the culture medium was replaced with buffer-free, glucose-free DMEM supplemented with 2 mmol / L glutamine. The analysis was performed according to the Seahorse XF Cell Mitochondrial Stress Test Kit (103708-100, Agilent) and instrument instructions.

[0096] The results showed that after hypoxia exposure, the basal respiration rate and ATP production rate of cells were significantly reduced, and Rg5 treatment was able to improve these symptoms ( Figure 3 In summary, Rg5 protected mitochondrial function and reduced ROS generation.

[0097] 2.3. Rg5 alleviates myocardial cell apoptosis induced by acute hypoxia 2.3.1. Cell apoptosis detection To distinguish apoptotic cells from viable or necrotic cells, Annexin V-FITC and PI co-staining (Wuhan Elaruite Biotechnology Co., Ltd.) was used. To study the damage of H9c2 cells to hypoxia, the cells after hypoxia treatment were digested with trypsin, washed with PBS, and resuspended in binding buffer. The cell suspension (1×10^5 cells / ml) was then incubated with FITC-labeled Annexin V and PI in the dark for 20 minutes. Cell counting was performed using BD FACSAria™ III (USA), and data were analyzed using FlowJo 10.8 software. 10,000 cells were recorded for each sample. The fluorescence spectra of Annexin V and PI were detected using 527 / 32 nm and 586 / 42 nm filters, respectively.

[0098] Flow cytometry analysis showed that the number of apoptotic cells in H9c2 cells exposed to hypoxia showed an increasing trend. However, in H9c2 cells pre-treated with Rg5 before hypoxia exposure, the number of apoptotic cells was significantly reduced ( Figure 4 a, b).

[0099] 2.3.2. JC-1 mitochondrial membrane potential detection Mitochondrial membrane potential was assessed using the JC-1 fluorescent probe (Wuhan Elaruite Biotechnology Co., Ltd.). The cells harvested after group treatment were trypsinized, washed with PBS and resuspended. Cell suspension (1×10^5 cells / mL) was prepared. The number of cells with green (JC-1 monomer) and red (JC-1 aggregate) fluorescence was determined using emission filters at 535 nm and 595 nm, respectively. Immunolabeled cells were analyzed using a BD FACSAria™ III flow cytometer.

[0100] Changes in mitochondrial membrane potential are also a signal of apoptosis. The flow cytometry results of membrane potential detection are consistent with the apoptosis results, indicating that Rg5 can reduce the occurrence of membrane potential changes ( Figure 4 (c, d).

[0101] 2.3.3 Western blot (WB) analysis Subsequently, we evaluated the activation of the Bcl2 family in hypoxia-treated H9c2 cells by Western blot (WB) analysis ( Figure 4 e). After Rg5 treatment, pro-apoptotic proteins such as Bax (Bax is the English name for Bcl-2 homologous antagonist / killer, referred to as Bax or Bak) ( Figure 4 f), cytochrome C ( Figure 4 middle g), caspase-9 ( Figure 4 h) and caspase-3 ( Figure 4 The anti-apoptotic protein Bcl2 ( Figure 4 These results indicate that Rg5 can inhibit the apoptosis of H9c2 cells under hypoxia.

[0102] Example 3: Rg5 is an agonist of STAT3 3.1. In vitro screening to verify that STAT3 is the target of Rg5 3.1.1. STAT3 is the key target of Rg5 in alleviating acute hypoxic myocardial apoptosis In order to clarify the specific mechanism by which Rg5 reduces acute hypoxic myocardial apoptosis, we first used thermal proteomics technology to screen the potential target proteins of Rg5 ( Figure 5 Among the potential target proteins, signal transducer and activator of transcription 3 (STAT3) attracted our attention. The results of thermoproteomics showed that the peptide content of STAT3, such as AILSTKPPGTFLLR, was significantly upregulated after Rg5 treatment ( Figure 6b), and the spectrum of the peptide also shows a good match ( Figure 6 In addition, we also performed molecular docking to evaluate the binding ability of Rg5 to STAT3, and the results showed that the affinity reached -8.1 kcal / mol ( Figure 6 Therefore, the preliminary screening results showed that there was a strong interaction between Rg5 and STAT3. Furthermore, cell thermal migration assay (CETSA) showed that Rg5 could significantly slow down the degradation of STAT3 caused by temperature increase ( Figure 5 The drug affinity response target stability (DARTS) experiment also showed that Rg5 could delay the degradation of STAT3 ( Figure 5 The above results prove that Rg5 can target STAT3.

[0103] Next, we demonstrated that Rg5 functionally interacts with STAT3. Figure 5 The cells in each treatment group shown in Figure d were subjected to reactive oxygen species detection and cell apoptosis detection. The results showed that the STAT3 inhibitor stattic could significantly reverse the decrease of mitochondrial ROS levels in H9c2 cardiomyocytes induced by Rg5 ( Figure 5 In addition, compared with the Rg5 group, flow cytometry showed that the overall ROS level in cells was significantly increased after stattic treatment ( Figure 5 e, f). At the same time, we found that the STAT3 agonist colivelin has the effect of reducing the intracellular ROS content, which is the same as Rg5. In terms of apoptosis, the inhibitor stattic significantly increased the occurrence of apoptosis compared with the Rg5 group. In contrast, compared with the model group (Mod), colivelin can significantly reduce the occurrence of apoptosis ( Figure 5 These results suggest that Rg5 not only physically interacts with STAT3 but also functionally promotes the activity of STAT3.

[0104] 3.1.2 Rg5 alleviates hypoxia-induced myocardial apoptosis by promoting Tyr705 phosphorylation of STAT3 STAT3 is an important signal transduction protein, whose activity is mainly regulated by the phosphorylation levels of tyrosine 705 (tyrosine (Y) at position 705 of the amino acid sequence shown in UniProtKB: P52631) and serine 727 (serine (S) at position 727 of the amino acid sequence shown in UniProtKB: P52631). WB results showed that Rg5 significantly increased the phosphorylation level of STAT3 Tyr705 in H9c2 cardiomyocytes after hypoxia exposure ( Figure 7a, b), but had no effect on the protein content and phosphorylation of Ser727 of STAT3. Phosphorylation of STAT3 at Try705 can promote its translocation to the cell nucleus. By isolating the cell nucleus, it was found that after administration of Rg5, the phosphorylation of STAT3 at Tyr705 in the cell nucleus increased significantly ( Figure 7 At the same time, we detected the downstream target proteins of STAT3, Bcl2 and Mcl-1. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) results showed that Rg5 promoted Bcl2 ( Figure 7 f) and Mcl-1 ( Figure 7 These results suggest that Rg5 exerts its pharmacological effects by activating the phosphorylation of Tyr705 of STAT3.

[0105] In addition, we found that stattic significantly increased ROS levels in hypoxia-treated H9c2 cardiomyocytes ( Figure 8 Correspondingly, compared with the hypoxia exposure, stattic further promoted the apoptosis of H9c2 cardiomyocytes ( Figure 8 This result was also confirmed by the significant changes in mitochondrial membrane potential ( Figure 8 In addition, compared with the model group (Mod), the level of STAT3 phosphorylated at Tyr705 in the nucleus was further reduced after stattic treatment ( Figure 8 At the same time, the level of STAT3 phosphorylated at Tyr705 in the whole cell was also significantly reduced ( Figure 8 (middle f).

[0106] 3.1.3 Rg5 enhances the interaction between STAT3 and JAK2 Subsequently, we further verified the regulatory effect of Rg5 on STAT3 Tyr705 phosphorylation using STAT3 agonists and inhibitors. First, immunofluorescence results showed that compared with the Rg5 group, the inhibitor stattic did significantly reduce the accumulation of STAT3 phosphorylated at Tyr705 in the nucleus ( Fig. 9 (a). Compared with the model group (Mod), the STAT3 agonist colivelin significantly promoted the accumulation of STAT3 in the nucleus. The WB results of the isolated nuclear components also showed that the content of STAT3 phosphorylated at Tyr705 was significantly reduced after stattic administration compared with the Rg5 group ( Fig. 9 b, c), while colivelin significantly increased the content of STAT3 phosphorylated at Tyr705 in the nucleus. The downstream anti-apoptotic protein Bcl2 also showed the expected effect. Compared with the Rg5 group, its content was significantly reduced after stattic administration ( Fig. 9Finally, immunoprecipitation experiments confirmed that the binding ability of STAT3 to its kinase JAK2 was significantly enhanced after Rg5 treatment ( Fig. 9 These results indicate that Rg5 can enhance the interaction between STAT3 and JAK2 and further promote the phosphorylation of STAT3 Tyr705.

[0107] 3.2 In vivo experiments confirm that STAT3 is the target of Rg5 After clarifying the relationship between Rg5 and STAT3 in vitro, we tried to explore whether stattic could reverse the pharmacological effects of Rg5 in vivo. Three groups were set up in the experiment: model group (Mod), Rg5 group and Rg5+stattic group. The Rg5 dosage was 30 mg / kg, and the operation steps were referred to Example 1. All mice were exposed to hypoxia ( Fig.10 First, the echocardiographic results showed that the ejection fraction and short-axis shortening rate of the Rg5+stattic group mice were significantly reduced compared with the Rg5 group ( Fig.10 Compared with the Rg5 group, the levels of myocardial enzymes (including creatine kinase CK, troponin cTnI and myoglobin Myo) in the serum of mice in the Rg5+stattic group were significantly increased ( Fig.10 d), while the content of creatine kinase isoenzyme CKMB also showed an upward trend, but due to large fluctuations, no statistical difference was achieved. At the same time, the hearts of mice in the Rg5+stattic group also showed an aggravated inflammatory response ( Fig.10 In addition, HE staining results showed that although Rg5 could reduce the edema of hypoxic cardiomyocytes, this protective effect was significantly weakened after stattic administration, and the mouse heart showed obvious edema similar to that of the model group ( Fig.10 f). Finally, we confirmed again through WB experiments of mouse heart tissue that Rg5 promoted the phosphorylation of STAT3 at tyrosine 705 (tyrosine (Y) at position 705 of the amino acid sequence shown in UniProtKB number P42227), while stattic reversed this increase. Compared with the Rg5 group, the levels of apoptosis-related proteins were also significantly reversed after stattic administration ( Fig.10 These results indicate that the pharmacological effect of Rg5 in vivo is also achieved by promoting STAT3 phosphorylation.

[0108] In summary, this study revealed that acute hypoxia exposure can lead to impaired cardiac function, manifested as increased cardiomyocyte apoptosis, aggravated inflammatory response, and elevated oxidative stress levels. Rg5, a natural product in ginseng, can improve cardiac function and reduce myocardial apoptosis in mice exposed to hypoxia. The specific mechanism is that Rg5 promotes the interaction between JAK2 and STAT3 by targeting STAT3, specifically inducing phosphorylation of STAT3 tyrosine 705, thereby alleviating apoptosis and enhancing the survival of cardiomyocytes. This study clarifies the pharmacological effects and molecular mechanisms of Rg5 in alleviating acute hypoxic myocardial injury, providing a potential therapeutic option for hypoxia-induced myocardial injury.

[0109] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. Use of a substance in the preparation of a drug for preventing and / or treating hypoxic myocardial cell damage, preventing and / or treating hypoxic myocardial damage and / or preventing and / or treating altitude sickness, characterized in that: The substance is A or a composition containing A, wherein A is ginsenoside Rg5, its stereoisomer, its solvate, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt hydrate or solvate, or its pharmaceutically acceptable modification.

2. The use according to claim 1, characterized in that: The altitude sickness includes hypoxic myocardial damage; the hypoxic myocardial damage includes abnormal cardiac ejection fraction and / or abnormal cardiac minor axis shortening rate.

3. The use according to claim 1 or 2, characterized in that: The hypoxic myocardial injury also includes abnormal levels of myocardial injury markers, abnormal levels of cytokines, abnormal oxidative stress response and / or histological lesions.

4. The use according to claim 3, characterized in that: The myocardial injury markers include cardiac troponin I, myoglobin, creatine kinase and / or creatine kinase mb.

5. The use according to claim 3, characterized in that: The cytokines include IL-1β, IL-6 and / or TNF-α.

6. The use according to claim 3, characterized in that: The abnormal stress response includes increased malondialdehyde content, increased superoxide dismutase activity and / or increased reduced glutathione content.

7. A method for preventing hypoxic myocardial cell damage, characterized in that: The method comprises the step of contacting the cardiomyocytes with the drug of claim 1.

8. The method according to claim 7, characterized in that: The cardiomyocytes are isolated cardiomyocytes.

9. Use of a substance for preparing an agonist of signal transduction and transcription activator protein 3, characterized in that: The substance is A or a composition containing A, wherein A is ginsenoside Rg5, its stereoisomer, its solvate, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt hydrate or solvate, or its pharmaceutically acceptable modification.

10. The use according to claim 9, characterized in that The agonist is used to promote the phosphorylation of tyrosine 705 of signal transduction and transcription activator protein 3.

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