Application of ginsenoside Rg3 in preparation of medicine for preventing and treating MC-induced heart injury

Abstract

The invention belongs to the technical field of medicine, and particularly relates to application of ginsenoside Rg3 in preparation of a medicine for preventing and treating MC-induced heart injury, experimental exploration shows that Rg3 improves the influence of MC on mouse cardiotoxicity, treatment of Rg3 reduces LDH and MDA levels of MC treated myocardial cells, cell apoptosis and oxidative stress are inhibited, and the effect of the Rg3 on preventing and treating the MC-induced heart injury is achieved. The miR-128-3p overexpression also finds that the protection effect of Rg3 on MC-induced cardiotoxicity is weakened, and the miR-128-3p negatively regulates and controls the expression of MDM4. In conclusion, by deeply researching the interaction relationship among the MiR-128-3p, the Rg3 and the MC, not only is a new molecular mechanism disclosed, but also a new thought and a new method are provided for reducing the cardiotoxicity of chemotherapeutic drugs, protecting myocardial cells and potentially treating tumors.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an application of ginsenoside Rg3 in preparing a medicine for preventing and treating MC-induced cardiac damage. Background Art

[0002] Cyanobacterial blooms frequently occur around the world, posing a serious threat to the life and health of animals and humans. Microcystins (MCs) are minor products of cyanobacterial metabolism that can induce cardiovascular toxicity by causing cardiac malformations, myocardial atrophy, and fibrosis. Exposure to high doses of MCs is associated with increased cardiotoxicity, leading to cytoskeletal changes and programmed cell death (e.g., necrosis and apoptosis) of cardiomyocytes. Previous evidence suggests that MCs have cytotoxic and genotoxic activities that can induce oxidative stress imbalance and toxicity in cardiomyocytes in a short period of time. However, few studies have reported MC-induced oxidative damage in myocardial tissue. Therefore, it is of great significance to better understand the mechanism of action of MC-induced myocardial oxidative damage and identify new MC antidotes.

[0003] Ginsenoside Rg3 extracted from ginseng has anti-angiogenic properties and has been widely used in traditional medicine. A previous study showed that Rg3 improved doxorubicin (ADM)-induced reductions in cardiac ejection fraction and left ventricular fractional shortening and improved left ventricular outflow. Rg3 also reduced aluminum-induced osteoporosis by regulating oxidative stress in rats and improved acetaminophen-induced hepatotoxicity in mice by inhibiting oxidative stress. In addition, Rg3 improved cardiac exercise compliance. At the same time, Rg3 improved myocardial glucose metabolism and insulin resistance by activating the AMP-activated protein kinase signaling pathway. However, the effect of Rg3 on MC-induced cardiotoxicity is still unclear. The role of ginsenoside Rg3 in MC cardiotoxicity and its potential mechanism are worth exploring. Summary of the invention

[0004] The purpose of the present invention is to provide an application of ginsenoside Rg3 in the preparation of a drug for preventing and treating MC-induced cardiac damage, which mainly prevents and treats MC-induced cardiac damage by regulating the expression of miR-128-3p.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention provides an application of ginsenoside Rg3 in preparing a medicine for preventing and treating MC-induced cardiac damage.

[0007] Furthermore, the ginsenoside Rg3 prevents and treats MC-induced cardiac injury by regulating the expression of miR-128-3p.

[0008] Furthermore, the ginsenoside Rg3 can protect cardiomyocytes by activating Keap-1 / Nrf2 / HO-1 signal transduction.

[0009] Furthermore, the ginsenoside Rg3 can induce the expression of Nrf2 / HO-1 in MC-stimulated muscle cells and inhibit MC-induced oxidative stress.

[0010] Furthermore, the dosage forms of the drug include tablets, capsules, oral preparations and injections.

[0011] Furthermore, the tablet comprises a coated tablet.

[0012] Furthermore, the oral preparation includes oral liquid, oral granules and oral powder.

[0013] Furthermore, the injection includes freeze-dried powder injection and injection emulsion.

[0014] The present invention also provides a medicine for preventing and treating MC-induced cardiac damage, wherein the medicine uses ginsenoside Rg3 as the only active ingredient.

[0015] Beneficial effects:

[0016] The present invention has been found through experimental investigation that after MC treatment, MiR-128-3p expression is upregulated in vivo and in vitro, and downregulated after Rg3 treatment. Rg3 improves the effect of MC on mouse cardiotoxicity. In addition, Rg3 treatment reduces the levels of LDH and MDA in MC-treated cardiomyocytes, inhibits cell apoptosis and oxidative stress, and also finds that MiR-128-3p overexpression weakens the protective effect of Rg3 on MC-induced cardiotoxicity and MiR-128-3p negatively regulates the expression of MDM4. The present invention not only reveals new molecular mechanisms by deeply studying the interaction between MiR-128-3p, Rg3 and MC, but also provides new ideas and methods for reducing the cardiotoxicity of chemotherapeutic drugs, protecting cardiomyocytes and potential tumor treatment. These findings have important theoretical significance and clinical application value, and are expected to bring new breakthroughs in the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1The figure shows the effect of ginsenoside Rg3 of the present invention on the cardiac toxicity of mice caused by MC, wherein: (A) LVEF, LVSP and LVEDP were detected by ultrasound electrocardiogram; (B) H&E staining of myocardial tissue; (C) Mass on staining of myocardial tissue; (D) TUNEL staining of myocardial tissue; (EF) ELISA was used to detect the contents of LDH, cTnI, MDA, ROS and SOD in myocardial tissue; (G) WB was used to detect apoptotic proteins (cleaved caspase-3, Cyt-c and Bcl-2) and Nrf2 / HO-1 pathway (Nrf2 and HO-1) in myocardial tissue; (H) qRT-PCR was used to determine the expression of miR-128-3p in myocardial tissue; n=6*P<0.05, **P<0.01, ***P<0.001;

[0019] Figure 2 The figure shows the effect of ginsenoside Rg3 of the present invention on the cardiomyocyte cytotoxicity of H9C2 cells caused by MC, wherein (A) FCM was used to detect cell apoptosis; (BC) ELISA was used to determine the levels of serum LDH, MDA, ROS and SOD; (D) WB was used to detect the expression of cleaved caspase-3, Cyt-c, Bcl-2, Nrf2 and HO-1; (E) qRT-PCR was used to determine the expression of miR-128-3p; all experiments were repeated three times *P<0.05, **P<0.01, ***P<0.001;

[0020] Figure 3 Figure 1 is the effect of overexpression of miR-128-3p on Rg3 prevention and treatment of MC-induced cardiomyocyte toxicity, wherein (A) qRT-PCR was used to detect the expression of miR-128-3p; (B) flow cytometry was used to detect cell apoptosis; (C-D) ELISA was used to determine serum LDH, MDA, ROS and SOD levels; (E) WB was used to detect the expression of cleaved caspase-3, Cyt-c, Bcl-2, Nrf2 and HO-1; all experiments were repeated three times *P<0.05, **P<0.01, ***P<0.001;

[0021] Figure 4Figure 1 is the effect of MiR-128-3p on the expression of MDM4 of the present invention, wherein (A) Starbase software predicts the target binding site between miR-128-3p and MDM4; (B) the target binding relationship between miR-128-3p and MDM4 is detected by dual luciferase reporter gene analysis; (C) RIP assay is performed to confirm the binding between miR-128-3p and MDM4; (D) the mRNA expression of MDM4 is detected by qRT-PCR; (E) the expression of MDM4 protein is detected by WB method; all experiments were repeated three times *P<0.05, **P<0.01, ***P<0.001;

[0022] Figure 5 The figure shows the effect of knocking down MDM4 on miR-128-3p in MC-induced cardiomyocyte toxicity in the present invention, wherein (A) qRT-PCR was used to detect the expression of miR-128-3p and MDM4; (B) WB was used to detect the expression of MDM4; (C) FCM was used to detect cell apoptosis; (D-E) ELISA was used to determine the levels of serum LDH, MDA, ROS and SOD; (F) WB was used to detect the expression of cleaved caspase-3, Cyt-c, Bcl-2, Nrf2 and HO-1; all experiments were repeated three times *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field;

[0029] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0030] The abbreviations in the following text represent the following: MC, microcystin; SD, Sprague-Dawley; enzyme-linked immunosorbent assay; LVEF, left ventricular ejection fraction; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; PI, propidium iodide; qRT-PCR, real-time quantitative PCR; cTnI, cardiac troponin I; FCM, flow cytometry; DMEM, Dulbecco’s modified Eagle medium; reactive oxygen species; ADM, doxorubicin; miRNA, microRNA; ncRNA, noncoding RNA; DOX, doxorubicin; Nrf2, nuclear factor-erythroid 2-related factor; HO-1, heme oxygenase-1; H2O2, hydrogen peroxide; SOD, superoxide dismutase.

[0031] Example 1

[0032] 1. Experimental animals and research indicator determination

[0033] 1.1 Animals and treatment

[0034] A total of 30 male C57BL / 6 mice (6-8 weeks old, 22.9±0.1 g) were purchased from Slac Jinda Laboratory Animal Co., Ltd. (Hunan, China). The mice were given 0.4 mg / kg / day MC (Sigma, MO, USA) by oral gavage for 4 weeks, and the sham operation group was treated with the same amount of normal saline. The MC mice were then randomly divided into four groups (n=6) and injected intraperitoneally with 0, 10, 40 or 80 mg / kg / day of Rg3 (suspended in normal saline, MCE, New Jersey, USA) for 4 weeks. All mice were euthanized after 6 weeks of administration. Serum was once collected and stored at -80°C for post-hoc ELISA analysis. The heart was quickly removed, frozen in liquid nitrogen, and stored at -80°C for later studies. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Hunan University of Chinese Medicine.

[0035] 1.2 Research Indicators

[0036] 1.2.1 Echocardiographic analysis

[0037] All mice underwent blind echocardiography using NEMIOSSA-550A (Toshiba, Tokyo, Japan). Left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), and left ventricular end-diastolic pressure (LVEDP) were measured.

[0038] 1.2.2 Hematoxylin and eosin (H&E) staining

[0039] Heart tissues were fixed with 4% paraformaldehyde overnight at room temperature (RT), embedded in paraffin, and cut into 4 μm thick sections. Hematoxylin (Sigma-Aldrich, MO, USA) was used for staining for 5 minutes and eosin (Sigma, USA) was used for staining for 1 minute at RT. The slides were then observed under a microscope (Nikon, Japan).

[0040] 1.2.3 Masson staining

[0041] Treat the dehydrated tissue sections with xylene and then immerse them in potassium dichromate overnight. After staining in hematoxylin solution for 3 minutes, the sections are treated with hydrochloric acid to reverse the blue color. Then, the sections are treated in carmine and acid fuchsin dyes for 10 minutes, phosphomolybdic acid aqueous solution for 5 minutes, aniline blue dye for 10 minutes, and 1% glacial acetic acid for 3 minutes. Finally, the sections are observed under an optical microscope.

[0042] 1.2.4 TUNEL staining

[0043] The tissue sections were immersed in 0.01 M TBS containing 1:200 proteinase K at 37°C for 10 minutes. Then, 20 μL of labeling buffer containing 1 μL TdT and DIG-d-UTP was added to the slides at 37°C for 2 hours. After blocking with 50 μL of blocking buffer, the sections were incubated with hybridization buffer containing biotin-labeled anti-digoxigenin antibody at 37°C for 30 minutes (Roc he, Switzerland). Cell apoptosis was observed under an optical microscope (Olympus, Japan).

[0044] 2. Cell culture and determination of related indicators

[0045] 2.1 Cell culture and treatment

[0046] H9C2 cells were purchased from ATCC (VA, USA), cultured in DMEM (Hyclone, UT, USA) with 10% fetal bovine serum (Gibco, NY, USA), 1% GlutaMAX and 1% penicillin-streptomycin (Invitrogen, NY), and maintained in an incubator at 37°C and 5% CO2. To induce cardiotoxicity, cells were treated with 20 μM MC for 24 hours. Before MC, cells were treated with Rg3 at different concentrations (20, 60 and 100 μM) for 1 hour. The control group was treated with normal saline.

[0047] 2.2 Cell transfection

[0048] MDM4-shRNA was designed and synthesized by GenePharma (Shanghai, China). MiR-128-3p mimics and inhibitors were purchased from RiboBio (Guangzhou, China). Lipofectamine 2000 (Invitrogen, NY, USA) was used for transfection according to the supplier's specifications.

[0049] 2.3 Flow cytometry

[0050] FCM was performed as previously reported. Briefly, H9C2 cells were collected with 0.25% trypsin and washed twice with phosphate-buffered saline (PBS), then resuspended in 400 μL binding buffer and mixed with 5 μL annexin VPE / 7-ADD and 5 μL propidium iodide (PI). Gently rotated, incubated at room temperature in the dark for 15 min, and then spotted with FCM (Thermo Scientific, USA).

[0051] 2.4 Reactive oxygen species (ROS) determination

[0052] Tissue and cell samples were treated with 10 M 2′,7′-dichlorofluorescein diacetate diluted in culture medium at 37° C. for 40 minutes. Then, the samples were washed three times with PBS. The multilabel counter (model: Wallac 1420; PerkinElmerLife) was used for determination.

[0053] 2.5 Enzyme-linked immunosorbent assay

[0054] Mouse serum and H9C2 cell culture supernatant were centrifuged to extract upper serum and supernatant, respectively. Follow-up lactate dehydrogenase (LDH), malondialdehyde (MDA), troponin I (cTnI) and superoxide dismutase (SOD) were evaluated according to the instructions provided by the kit (Shanghai Enzyme Biotechnology Co., Ltd., Shanghai, China). ciences, USA) recorded fluorescence intensity at 485nm and 535nm. Calculate relative ROS levels.

[0055] 2.6 Dual luciferase reporter gene assay

[0056] The feasible binding sites between miR-128-3p and MDM4 were predicted with the help of starbase (https: / / starbase.sysu.edu.cn / ). The 3'UTR sequence of MDM4 including the miR-128-3p binding site or mutant variants was compounded and inserted into the pmirGLO luciferase vector (Promega, Madison, WI) to establish the wild-type MDM4 reporter (MDM4-WT) and mutant MDM4 reporter (MDM4-8UT). MiR-128-3p mimics or NC mimics were co-transfected with MDM4-WT or MDM4-MUT into H9C2 cells with the help of Lipofectamine 2000. After incubation for 48 h, the luciferase activity was detected using a dual-luciferase assay device (Promega, USA).

[0057] 2.7 RNA immunoprecipitation

[0058] Imprint Immunoprecipitation kit (Sigma-Aldrich) was used for RIP assay. Cells were lysed in RIPA buffer and immunoprecipitated with 5 μg of mouse anti-IgG antibody or anti-Ago2 antibody (both from Abcam). After incubation at 4°C overnight, the immunoprecipitated RNA samples were isolated using GenEluteTM total RNA purification kit (Sigma-Aldrich). The enrichment of miR-128-3p and MDM4 was analyzed by real-time quantitative PCR (qRT-PCR).

[0059] 2.8 Quantitative real-time PCR (qRT-PCR)

[0060] Total RNA was extracted from mice or cells using Trizol reagent (Invitrogen, USA). cDNA was then synthesized using PrimeScript RT Master Mix (Takara, Dalian, China) according to the supplier's protocol. Real-time PCR evaluation was performed using PowerSY BR Green PCR Master Mix (Applied Biosystems, Irvine, CA, United States). The expression of miR-128-3p was normalized to U6, while the expression of others was normalized to GAPDH. The comparative Ct (threshold cycle) method (2-ΔΔCt) was used to determine the relative quantification of gene expression. The sequences of the primers are as follows:

[0061] miR-128-3p-F, 5'-GTCGTATCCAGTGCAGGTCCGAGGTATTCGCACTGGATACGAC AA AGAG-3',

[0062] miR-128-3p-R, 5′-CGTCACAGTGAACCGGT-3′;

[0063] MDM4-F,5'-gcgtgattttagcacggac-3',

[0064] MDM4-R, 5′-AAACTAAGAGCACCGCGGAA-3′;

[0065] U6-F, 5'-ctccttcggcagcaca-3',

[0066] U6-R, 5'-aacgccttcacgaattgcgt-3'.

[0067] GAPDH-F,5'-gcaagtcaacggcacag-3',

[0068] GAPDH-R, 5'-GCCAGTAGACTCCACGACAT-3'.

[0069] 2.9 Western Blotting (WB)

[0070] Proteins from tissues or cells were extracted using a RIPA kit (Beyotime, China). Protein samples were quantified by a BCA protein assay kit (Jiangsu Bitian, China). Equal amounts of protein were loaded onto 10% SDS-PAGE. And transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk for 2 h at room temperature and incubated with the primary antibody overnight at 4 °C. Cleaved caspase-3 (1:1000, CST#9661), Bcl-2 (1:2000, ab196495), Cyt-c (1:5000, CST#4280), Nrf2 (1:1000, ab92946), HO-1 (1:10000, ab68477), MDM4 (1:10000, SAB4501852), and GAPDH (1:2000, ab8245) antibodies were used in this study. After washing, the protein bands were incubated with appropriate secondary HRP antibodies (1:5000) for 2 h at room temperature. Proteins were examined using ECL Western blotting substrate (Thermo Scientific). ImageJ software (Bethesda, MD, USA) was used to quantitatively analyze the band signal intensities.

[0071] 3. Statistical Analysis

[0072] All statistical data are presented as mean ± standard deviation (SD). Differences between the two companies were analyzed using Student's t-test, and multiple group comparisons were analyzed using one-way analysis of variance (ANOVA), and post hoc comparisons were performed using Tukey's test. In the past, each set of files was repeated at least three times. The applicant used IBM SPSS Statistics 20.0 (Chicago, USA) for analysis. P < 0.05 was considered statistically significant.

[0073] 4. Results

[0074] 4.1 Ginsenoside Rg3 improves the cardiotoxicity of MC in mice

[0075] Rg3 can reduce inflammation in a mouse myocardial infarction model. To explore the effect of Rg3 on MC-induced cardiotoxicity, mice were given MC by oral gavage and treated with different concentrations of Rg3 (10, 40, or 80 mg / kg). After MC injection, the LV EF and LVSP of mice decreased, and the LVEDP increased. After injection of ginsenoside Rg3, LVEF and LV SP increased, and LVEDP decreased with increasing Rg3 concentrations, as shown in Figure 2. Figure 1As shown in A. H&E staining results showed that myocardial tissue damage was aggravated after MC treatment, manifested as disordered arrangement of myocardial cells, irregular arrangement of myocardial fibers, enlarged cell nuclei, and strange shapes. The above damage was alleviated after injection of ginsenoside Rg3, as shown in Figure 1 As shown in B. Masson staining showed that the collagen fibers of the control animals were arranged normally, while the cardiomyocytes of the MC-treated group were arranged in disorder and the collagen fibers were reduced, as shown in Figure 1 As shown in C. TUNEL staining showed that MC induced apoptosis of mouse cardiomyocytes. Figure 1 As shown in D. However, injection of ginsenoside Rg3 alleviated MC-induced myocardial tissue pathological changes and cell apoptosis. At the same time, the best therapeutic effect was observed at a concentration of 40 mg / kg. Therefore, 40 mg / kg was selected for subsequent experiments. After MC treatment, LDH activity in mouse serum increased, cTnI, MDA and ROS levels increased, and SOD level decreased, as shown in Figure 1 These effects were reversed after treatment with ginsenoside Rg3, as shown in Figure 1 As shown in Figure EF. After MC treatment, the expression of cleaved caspase-3 and Cyt-c proteins in myocardial tissue homogenate was upregulated, and the expression of Bcl-2 protein was downregulated. At the same time, the levels of Nrf2 and HO-1 were downregulated, indicating that the activation of the Nrf2 / HO-1 pathway was inhibited, as shown in Figure EF. Figure 1 These results were reversed after treatment with Rg3, as shown in Figure 1 As shown in G. Ginsenoside Rg3 has been shown to interact with miRNAs in the pathogenesis of diseases. Here, the applicants detected the expression of miR-200a-3p, miR-4732-3p, iR-320a-3p, miR-128-3p, miR-146a-3p, and miR-495-3p in MC mice and found that the levels of miR-200a-2p, miR-128-3, and miR-146a-3p were significantly increased in MC mice, among which miR-128-3p had the highest expression level. However, the expression of miR-128-3p was downregulated after Rg3 treatment, as shown in Figure 1 As shown in H. The above data showed that ginsenoside Rg3 improved the effect of MC on cardiotoxicity in mice.

[0076] 4.2 Ginsenoside Rg3 improves MC cardiomyocyte cytotoxicity in H9C2 cells

[0077] To further explore the role of Rg3 in MC-induced cardiotoxicity, the applicants conducted relevant studies on MC-treated H9C2 cells, and these results were reversed after treatment with Rg3, such as Figure 2 As shown in A. After MC treatment of H9C2 cells, the levels of LDH, MDA and ROS increased significantly, while the level of SOD decreased. Figure 2 Ginsenoside Rg3 reversed the above results, as shown in Figure BC. Figure 2 As shown in BC. After MC treatment, the expression of cleaved caspase-3 and Cyt-c was upregulated, and the expression of Bcl-2 was downregulated. At the same time, the activation of the Nrf2 / HO-1 pathway was inhibited, and the above results were reversed after Rg3 treatment. Finally, it was found that the expression of miR-128-3p was upregulated after MC treatment and downregulated after Rg3 treatment, as shown in Figure 2 Likewise, these results suggest that ginsenoside Rg3 can reduce the cytotoxicity of MC to cardiomyocytes.

[0078] Overexpression of miR-128-3p weakens the protective effect of ginsenoside Rg3 against MC-induced cardiomyocyte toxicity

[0079] To further explore the mechanism of Rg3 in MC-induced cardiotoxicity, miR-128-3p was overexpressed in Rg3-treated cells, as Figure 3 As shown in A. Overexpression of miR-128-3p in Rg3-treated cells significantly promoted cell apoptosis, which reversed the therapeutic effect of Rg3. Figure 3 As shown in Figure B. When Rg3 was treated alone, the levels of LDH and MDA decreased, while the level of SOD increased. Compared with Rg3 treatment alone, miR-128-3p overexpression significantly increased the levels of LDH, MDA, and RO S, while the level of SOD decreased, as shown in Figure 2 Figure 3 As shown in Figures C and D. Compared with Rg3 treatment alone, after miR-128-3p overexpression, the expression of cleaved caspase-3 and Cyt-c was upregulated, while the expression of Bcl-2 was downregulated. After miR-128-3p overexpression, the expression of Nrf2 and HO-1 was downregulated, as shown in Figures C and D. Figure 3 As shown in E. In conclusion, overexpression of miR-128-3p impaired the protective effect of ginsenoside Rg3 against MC-induced cardiomyocyte toxicity.

[0080] 4.4MiR-128-3p negatively regulates the expression of MDM4.

[0081] To further discover the regulatory mechanism of miR-128-3p, starbase predicted that there are binding sites between miR-128-3p and MDM4, such as Figure 4 As shown in A. In the MDM4-WT group with miR-128-3p mimics, the luciferase reporter gene activity was significantly reduced, but there was no effect in the MDM4-MUT group with miR.128-3p mimics, as shown in Figure 4 As shown in Figure B. RIP assay further confirmed the binding between miR-128-3p and MDM4. Figure 4As shown in C. In addition, after miR-128-3p overexpression, the expression of MDM4 was significantly decreased, as shown in Figure 4 In conclusion, miR-128-3p negatively regulates the expression of MDM4.

[0082] 4.5 Knockdown of MDM4 reverses the effects of miR-128-3p knockdown in MC-induced cardiomyocyte toxicity

[0083] To discover whether miR-128-3p plays a role in MC-induced cardiotoxicity by regulating MDM4, we knocked down the expression of miR-128-3p alone in MC-induced cells and set up a purposeful rescue experiment: MC+mi R-128-3p inhibitor+sh-NC, MC+miR-128-3p inhibitor+sh-MDM4. After MC treatment, miR-128-3p expression increased and MDM4 expression was downregulated. After miR-128-3p knockdown, MDM4 expression was upregulated. However, after knocking down miR-128-3p and MDM4 at the same time, the expression of MDM4 was significantly decreased, and the expression of miR-128-3p did not change, as shown in Figure 5. Figure 5 As shown in Figure AB. After knocking down miR-128-3p alone, the apoptosis rate was significantly reduced, while after knocking down miR-138-3p and MDM4 expression at the same time, the above results were reversed, as shown in Figure AB. Figure 5 As shown in C. In addition, after knocking down miR-128-3p alone, the levels of LDH, MDA, and ROS in the cells were significantly reduced, while the level of SOD was increased. However, after knocking down miR-128-3p and MDM4 expression at the same time, the above results were reversed, as shown in Figure 5 As shown in Figure 5. After knocking down miR-128-3p alone, the expression of cleaved caspase-3 and Cyt-c in cardiomyocytes was reduced, Bcl-2 was increased, and the Nrf2 / HO-1 pathway was activated. However, this was reversed when both miR-128-3p and MDM4 were knocked down, as shown in Figure 5. Figure 5 As shown in F. Therefore, MDM4 knockdown can reverse the effect of miR-128-3p knockdown on MC-induced cardiomyocyte toxicity.

[0084] In summary, MC is positively correlated with cardiotoxicity and therefore poses a risk to human cardiovascular health. Ginsenoside Rg3 plays an indispensable role in scavenging free radicals and protecting the heart. It has been used to induce healing and inhibit scar hyperplasia. In the present invention, the applicant proposes that ginsenoside Rg3 can improve MC-induced cardiotoxicity and protect cardiomyocytes both in vitro and in vivo. The prior art has clearly shown that ginsenoside Rg3 regulates the progression of different diseases, such as ischemic stroke recovery and septic liver injury, through an integrated miRNA mRNA regulatory network. Downregulation of miR-128 alleviates angiotensin II-induced cardiac dysfunction, hypertrophy, fibrotic cell apoptosis and oxidative stress damage in C57BL / 6 mice. The applicant observed that the expression of miR-128-3p in cardiomyocytes was enhanced after MC treatment, while the expression of miR-128-3p was reduced after Rg3 treatment. Overexpression of miR-128-3p also significantly promoted apoptosis and elevated LDH levels in Rg3-treated cells, indicating severe myocardial damage. SOD is a common antioxidant enzyme that catalyzes the conversion of superoxide radicals into H2O2. Oxidative stress causes damage to cardiomyocytes. Overexpression of miR-128-3p in Rg3-treated cells promoted the increase in MC-induced SOD levels. In summary, ginsenoside Rg3 may play a protective role in MC-induced cardiomyocyte toxicity by regulating the expression of miR-128-3p. The applicant also found that miR-128-3p aggravated MC-induced cardiomyocyte toxicity by targeting MDM4. The transcription factor Nrf2 is a major regulator of the adaptive response to oxidative damage and is considered to be a key component in the pathogenesis of cardiac injury in mammals. It has been reported that excessive production of free radicals can regulate the Nrf2 / HO-1 signaling pathway and protect the myocardium from oxidative stress-induced damage by enhancing oxidation. In addition, Rg3 protects cardiomyocytes from damage by activating Keap-1 / Nrf2 / HO-1 signaling. In the above experiments, Rg3 can induce the expression of Nrf2 / HO-1 in MC-stimulated muscle cells and inhibit MC-induced oxidative stress. In summary, the applicant first confirmed that Rg3 attenuated MC-induced cardiotoxicity through the miR-128-3p / MDM4 / Nrf2 / HO-1 axis. The above research results of the applicant indicate that Rg3 is a therapeutic agent for MC-induced cardiotoxicity and miR-128-3p is the target of Rg3 for the treatment of MC-induced cardiotoxicity.

[0085] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An application of ginsenoside Rg3 in the preparation of a drug for preventing and treating MC-induced cardiac damage.

2. The use according to claim 1, characterized in that The ginsenoside Rg3 prevents and treats MC-induced cardiac injury by regulating the expression of miR-128-3p.

3. The use according to claim 2, characterized in that The ginsenoside Rg3 can also protect cardiomyocytes by activating Keap-1 / Nrf2 / HO-1 signaling.

4. The use according to claim 3, characterized in that The ginsenoside Rg3 induces the expression of Nrf2 / HO-1 in MC-stimulated muscle cells and inhibits MC-induced oxidative stress.

5. The use according to claim 1, characterized in that The dosage forms of the drug include tablets, capsules, oral preparations and injections.

6. The use according to claim 5, characterized in that: The tablets include coated tablets.

7. The use according to claim 5, characterized in that: The oral preparation includes oral liquid preparation, oral granule preparation and oral powder preparation.

8. The use according to claim 5, characterized in that: The injection includes freeze-dried powder injection and injection emulsion.

9. A drug for preventing and treating MC-induced cardiac damage, characterized in that: The drug contains ginsenoside Rg3 as the only active ingredient.

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