Application of pseudo-ginsenoside RT5 in preparation of anti-myocardial ischemia drugs

Abstract

The invention relates to application of pseudo-ginsenoside RT5 in preparation of an anti-myocardial ischemia medicine, and belongs to the field of medicines. According to the application of the pseudo-ginsenoside RT5 in preparation of the medicine for resisting myocardial ischemia, oxidative stress is relieved by regulating and controlling a cGMP-PKG channel, and therefore the effect of resisting myocardial ischemia is achieved. The pseudo-ginsenoside RT5 has the advantages that a new way is provided for preventing and treating myocardial ischemia, and the pseudo-ginsenoside RT5 medicine has the function of resisting myocardial ischemia by regulating and controlling a cGMP-PKG pathway and relieving oxidative stress. The preparation method is used for preparing the medicine with the active ingredients including the pseudo-ginsenoside RT5, the pseudo-ginsenoside RT5 and medically acceptable auxiliary materials are included, and the dosage form of the medicine can be any one of pills, tablets, capsules, granules, mixtures or oral liquid in any medicinal dosage form.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to the role of ginsenoside RT5 in resisting myocardial ischemia, and more specifically refers to ginsenoside RT5 exerting its anti-myocardial ischemia effect by regulating the cGMP-PKG pathway and reducing oxidative stress. Background Art

[0002] Myocardial ischemia (MI), also known as ischemic heart disease, refers to a pathological state in which the blood perfusion of the heart is reduced, resulting in reduced oxygen supply to the heart, abnormal myocardial energy metabolism, and inability to support the normal functioning of the heart. Long-term myocardial ischemia can cause irreversible damage to myocardial tissue, with the most common cause of death being myocardial infarction caused by thrombosis and ischemia-reperfusion injury. Currently, commonly used drugs for the treatment of MI in clinical practice include antiplatelet drugs, beta-blockers, statins, nitrates, RAS system blockers, etc. At present, ginsenoside RT5 has been semi-synthesized, and its related medical effects have been studied, and the mechanism needs to be further studied. Summary of the invention

[0003] The present invention provides an application of ginsenoside RT5 in the preparation of anti-myocardial ischemia drugs. Through biological research on ginsenoside RT5, it is revealed that it may reduce oxidative stress by regulating the cGMP-PKG pathway, thereby exerting an anti-myocardial ischemia effect, and can be used in the preparation of drugs for preventing and treating myocardial ischemia.

[0004] The technical solution adopted by the present invention is to use a pseudoginsenoside RT5 having the following structural formula in the preparation of an anti-myocardial ischemia drug,

[0005] .

[0006] The present invention reduces oxidative stress by regulating the cGMP-PKG pathway, thereby exerting an anti-myocardial ischemia effect.

[0007] Myocardial ischemia mentioned in the present invention specifically refers to reduced blood perfusion of the heart, resulting in reduced oxygen supply to the heart, abnormal myocardial energy metabolism, inability to support normal heart function, and tachycardia and bradycardia caused by abnormal cardiac autonomic rhythm or conduction disorder.

[0008] The advantage of the present invention is that it provides a new approach for preventing and treating myocardial ischemia. The pseudoginsenoside RT5 drug has the effect of reducing oxidative stress by regulating the cGMP-PKG pathway, thereby exerting an anti-myocardial ischemia effect. The present invention is used to prepare a drug whose active ingredient includes pseudoginsenoside RT5, including pseudoginsenoside RT5 and medically acceptable excipients. The dosage form of the drug is any one of pills, tablets, capsules, granules, mixtures or oral liquids in any pharmaceutically acceptable dosage form. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a graph showing the effect of H2O2 on the survival rate of H9c2 cardiomyocytes; Figure 2 This is a graph showing the effect of pseudoginsenoside RT5 on the survival rate of H9c2 cardiomyocytes; Figure 3 This is a graph showing the effect of ginsenoside RT5 on the survival rate of myocardial cells damaged by H2O2; Figure 4 This is a picture of the test results of the cell kit; Figure 5 This is a picture of the test results of the animal test kit; Figure 6 This is the effect diagram on myocardial infarction area; Figure 7 It is a diagram of the impact on myocardial tissue morphology; Figure 8 is the transcriptome analysis heat map; Fig. 9 It is the volcano map of differentially expressed genes; Fig.10 It is a Venn diagram of differentially expressed genes; Fig.11 This is the result diagram of differential gene enrichment. DETAILED DESCRIPTION

[0010] A use of pseudoginsenoside RT5 having the following structural formula in the preparation of a drug for preventing and treating myocardial ischemia, .

[0011] The present invention reduces oxidative stress by regulating the cGMP-PKG pathway, thereby exerting an anti-myocardial ischemia effect.

[0012] Myocardial ischemia mentioned in the present invention specifically refers to reduced blood perfusion of the heart, resulting in reduced oxygen supply to the heart, abnormal myocardial energy metabolism, inability to support normal heart function, and tachycardia and bradycardia caused by abnormal cardiac autonomic rhythm or conduction disorder.

[0013] The present invention provides a new approach for preventing and treating myocardial ischemia, and the ginsenoside RT5 drug can reduce oxidative stress by regulating the cGMP-PKG pathway, thereby exerting an anti-myocardial ischemia effect.

[0014] The present invention is further illustrated by means of specific experimental examples.

[0015] Experiment 1: Effects of ginsenoside RT5 on H9c2 cardiomyocytes Objective: To observe the effects of different doses of ginsenoside RT5 on H9c2 cardiomyocytes under normal conditions and / or hypoxia-ischemia simulation fluid. The cell survival rate was determined by CCK-8 method, and various indices in the cell supernatant were detected by ELISA method.

[0016] 1. Materials 1. Cells H9c2 cardiomyocytes were obtained from Beina Biotechnology Co., Ltd.

[0017] 2. Instruments Inverted microscope (Olympus, Japan), constant temperature water bath (DR-HW-1, China), centrifuge (7LDZ5-2, China), and recording and analysis software (Patch Clamp 6.01, USA).

[0018] 3. Experimental drugs Ginsenoside RT5 (production batch number 20230726); enzyme-linked immunosorbent assay (ELISA) kits for lactate dehydrogenase (LDH), superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) were purchased from Jiangsu Feiya Biotechnology Co., Ltd. (Jiangsu, China). CCK-8 solution was purchased from Beijing Solebow Technology Co., Ltd.; H2O2 solution was purchased from Beijing Chemical Plant; all chemicals were analytically pure.

[0019] (II) Experimental methods 1. Cultivation of H9c2 cardiomyocytes Cell recovery Quickly take out the frozen H9c2 cardiomyocytes from the liquid nitrogen tank, immediately place them in a 37°C constant temperature water bath, gently shake them to thaw as quickly as possible, transfer them to a centrifuge tube, add 1 ml of culture medium to the centrifuge tube, centrifuge at 1000r for 5min, pour off the supernatant, and discard the excess liquid.

[0020] Cell passaging H9c2 cardiomyocytes were incubated in a carbon dioxide incubator at 5% CO2 and 37°C. The cell medium was changed every 1-2 days, and when the growth density reached about 80%, the cells were digested and passaged with 0.25% trypsin.

[0021] Cell cryopreservation Take H9c2 cardiomyocytes in the logarithmic growth phase, add an appropriate amount of 0.25% trypsin (containing 0.02% EDTA), digest and collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of cell freezing solution (FBS: DMSO = 9:1), gently pipette to mix, count, and adjust the cell concentration to (5-10) × 10 6pcs / mL, and dispense into cell cryopreservation tubes, 1 mL per tube. Tighten and seal the cryopreservation tubes, and label them. Place the cryopreservation tubes at -20 ℃ for several hours, quickly transfer to a -80 ℃ refrigerator overnight, and place in a liquid nitrogen tank for long-term storage.

[0022] 2. CCK-8 method to determine cell viability The CCK-8 kit is a rapid and highly sensitive detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) that is widely used in cell proliferation and cytotoxicity. WST-8 is an upgraded product of MTT. Its working principle is: in the presence of electron coupling reagents, it can be reduced by dehydrogenases in mitochondria to generate highly water-soluble orange-yellow formazan products. The depth of the color is proportional to the proliferation of cells and inversely proportional to the cytotoxicity. The absorbance value (OD) is measured at a wavelength of 450 nm using an enzyme reader, which indirectly reflects the number of living cells.

[0023] 3. Effect of H2O2 on the survival rate of H9c2 cardiomyocytes The culture medium of H9c2 cardiomyocytes in the logarithmic growth phase was discarded, and the cells were washed with PBS 1-2 times, digested with 0.25% trypsin (containing 0.02% EDTA), and centrifuged at 1000 rpm for 5 min. The cell density was adjusted to 5×10 4 / mL, inoculated in 96-well plates at 200 μL / well, set up normal control (Control) group and H2O2 group (nine concentration gradients: 100, 150, 200, 250, 300, 350, 400, 450, 500μM), and cultured in a carbon dioxide incubator at 5% CO2 and 37 ℃. When the cells adhered to the wall to 90%, the culture medium was discarded, washed twice with PBS, and cultured in a carbon dioxide incubator at 5% CO2 and 37 ℃ for 6 h to establish the injury model. The cells in the Control group were not treated with H2O2, and 6 replicates were set in each group. 10 μL CCK-8 solution was added to each well 2 h before the end of the culture, and the cells were placed in a carbon dioxide incubator for another 2 h. The OD value was measured at 450 nm using an enzyme reader, and the cell survival rate was calculated using the following formula: Cell survival rate (%) = (OD experimental group - OD blank) / (OD control group - OD blank) × 100%.

[0024] 4. Effect of ginsenoside RT5 on the survival rate of H9c2 cardiomyocytes H9c2 cardiomyocytes were seeded in 96-well plates. After 24 h of culture, ginsenoside RT5 was added to each well at a final concentration of 1, 5, 10, 25, 50, 100, 250, 500 and 1000 μM, 100 μL was added to each well for pretreatment for 24 h, and 6 replicates were set up in each group. 10 μL CCK-8 solution was added to each well 2 h before the end of culture, and the cells were placed in a CO2 incubator for another 2 h. The OD value was measured at 450 nm using an ELISA reader, and the cell survival rate was calculated using the following formula: Cell survival rate (%) = (OD experimental group - OD blank) / (OD control group - OD blank) × 100%.

[0025] 5. Effect of ginsenoside RT5 on the survival rate of myocardial cells injured by H2O2 H9c2 cardiomyocytes were seeded in 96-well plates. After culturing for 24 h, H9c2 cardiomyocytes in good growth state in the logarithmic growth phase were taken for the experiment and divided into the following groups: control group: cultured in normal culture medium; model group: cultured in 2% FBSDMEM culture medium for 24 h after normal culture for 24 h, and treated with H2O2 for 6 h; low-dose pseudoginsenoside RT5 group: cultured in 2% FBSDMEM culture medium (containing 1 μM pseudoginsenoside RT5) for 24 h after normal culture for 24 h, and treated with H2O2 for 6 h; medium-dose pseudoginsenoside RT5 group: cultured in 2% FBS DMEM culture medium (containing 5 μM pseudoginsenoside RT5) for 24 h after normal culture for 24 h, and treated with H2O2 for 6 h; high-dose pseudoginsenoside RT5 group: cultured in 2% FBS DMEM culture medium (containing 10 μM pseudoginsenoside RT5) for 24 h after normal culture for 24 h, and treated with H2O2 for 6 h h; Positive drug group: After normal culture for 24 h, the cells were cultured with 2% FBS DMEM medium (containing 5 μM diltiazem) for 24 h and treated with H2O2 for 6 h.

[0026] The cell survival rate was calculated by the formula: Cell survival rate (%) = (OD experimental group - OD blank) / (OD control group - OD blank) × 100%.

[0027] Determination of oxidative stress markers LDH activity assay The cells cultured in 96-well plates were used for the experiment. The cells were randomly divided into 6 groups: normal control group, 300μM H2O2 injury group, pseudoginsenoside RT5 1μM group, pseudoginsenoside RT5 5μM group, pseudoginsenoside RT5 10μM group, and diltiazem 5μM group. Each medication group (pseudoginsenoside RT5 3 groups + diltiazem 1 group) was first treated with drugs for 24h. Then, H2O2 was added to the cell culture medium of the other groups except the normal control group to a final concentration of 300μM and incubated for 6h. The supernatant of the cell culture medium was taken, centrifuged at 1000 rpm for 5 min, and the supernatant was taken to determine the LDH content according to the reagent instructions.

[0028] MDA activity assay Cells cultured in 96-well plates were used for the experiment. The cells were randomly divided into 6 groups: normal control group, 300μM H2O2 injury group, pseudoginsenoside RT5 1μM group, pseudoginsenoside RT5 5μM group, pseudoginsenoside RT5 10μM group, and diltiazem 5μM group. Each medication group (pseudoginsenoside RT5 3 groups + diltiazem 1 group) was first treated with drugs for 24h. Then, H2O2 was added to the cell culture medium of the other groups except the normal control group to a final concentration of 300μM and incubated for 6h. The supernatant of the cell culture medium was taken, centrifuged at 1000 rpm for 5 min, and the supernatant was taken to determine the MDA content according to the reagent instructions.

[0029] SOD activity assay Cells cultured in 96-well plates were used for the experiment. The cells were randomly divided into 6 groups: normal control group, 300μM H2O2 injury group, pseudoginsenoside RT5 1μM group, pseudoginsenoside RT5 5μM group, pseudoginsenoside RT5 10μM group, and diltiazem 5μM group. Each medication group (pseudoginsenoside RT5 3 groups + diltiazem 1 group) was first treated with drugs for 24h. Then, H2O2 was added to the cell culture medium of the other groups except the normal control group to a final concentration of 300μM and incubated for 6h. The supernatant of the cell culture medium was taken, centrifuged at 1000 rpm for 5 min, and the supernatant was taken to determine the SOD content according to the reagent instructions.

[0030] ROS activity assay Cells cultured in 96-well plates were used for the experiment. The cells were randomly divided into 6 groups: normal control group, 300μM H2O2 injury group, pseudoginsenoside RT5 1μM group, pseudoginsenoside RT5 5μM group, pseudoginsenoside RT5 10μM group, and diltiazem 5μM group. Each medication group (pseudoginsenoside RT5 3 groups + diltiazem 1 group) was first treated with drugs for 24h. Then, H2O2 was added to the cell culture medium of the other groups except the normal control group to a final concentration of 300μM and incubated for 6h. The supernatant of the cell culture medium was taken, centrifuged at 1000 rpm for 5 min, and the supernatant was taken to determine the ROS content according to the reagent instructions.

[0031] (III) Experimental results 1. Effect of H2O2 on the survival rate of H9c2 cardiomyocytes The effects of different concentrations (100, 150, 200, 250, 300, 350, 400, 450, 500 μM) of ginsenoside RT5 on normal rat H9c2 cardiomyocytes are shown in the figure. As shown in the figure, compared with the control group, H2O2 at a concentration of 100-250 μM had no significant effect on the survival rate of normal H9c2 cardiomyocytes, while H2O2 at a concentration of 350-500 μM was too toxic to normal H9c2 cardiomyocytes. When the concentration of H2O2 was 300 μM, the survival rate of H9c2 cardiomyocytes was 67.6%, which was between 60% and 70%. Therefore, 300 μM H2O2 was selected as the modeling dose. Figure 1 .

[0032] 2. Effect of ginsenoside RT5 on the survival rate of H9c2 cardiomyocytes The effects of different concentrations (1, 5, 10, 25, 50, 100, 250, 500, 1000 μM) of ginsenoside RT5 on normal rat H9c2 cardiomyocytes are shown in Figure 2 As shown in the figure, compared with the control group, ginsenoside RT5 at a concentration of 25-1000 μM had a significant effect on the survival rate of normal H9c2 cardiomyocytes, while ginsenoside RT5 at a concentration of 1-10 μM had no significant effect on the survival rate of normal H9c2 cardiomyocytes, that is, ginsenoside RT5 at a concentration of 1-10 μM had no significant cytotoxic effect on normal H9c2 cardiomyocytes.

[0033] 3. Effect of ginsenoside RT5 on the survival rate of cardiomyocytes injured by H2O2 300 μM H2O2 was used to model H9c2 cardiomyocytes, and different concentrations (1, 5, 10 μM) of pseudoginsenoside RT5 were administered to normal rat H9c2 cardiomyocytes. Figure 3 As shown, compared with the model group, after administration, the H9c2 cell growth inhibition caused by H2O2 was effectively reversed, showing a significant antioxidant effect in a dose-dependent manner.

[0034] 4. Effects of ginsenoside RT5 on the activities of LDH, SOD, MDA and ROS in the supernatant of H9c2 cardiomyocytes damaged by H2O2 like Figure 4As shown, compared with the normal group, the activities of LDH, MDA and ROS in the supernatant of cardiomyocytes in the model group were increased, and the activity of SOD was decreased; compared with the model group, the activities of LDH, MDA and ROS in the supernatant of cardiomyocytes in the high and medium doses of ginsenoside RT5 and positive drug diltiazem groups were decreased, and the activity of SOD was increased; there was no significant change in the activities of LDH, SOD, MDA and ROS in the supernatant of cardiomyocytes in the low dose group.

[0035] 4. Discussion Different concentrations of H2O2 were set for modeling, and the modeling dosage was finally determined to be 300μM. If the dose was too high, the cell survival rate was low, and if the dose was too low, the modeling failed. At the same time, we set a series of RT5 dosing concentrations to observe whether different concentrations of RT5 would have toxic side effects on cells, and finally determined the dosing concentrations to be 1, 5, and 10μM. The effects of pseudo-ginsenoside RT5 on the activities of LDH, SOD, MDA, and ROS in the supernatant of H9c2 cardiomyocytes damaged by H2O2 were detected. It was finally found that compared with the control group, the LDH level of the model group cells was significantly increased after being treated with H2O2, the MDA level was significantly increased, the SOD level was significantly decreased, and the ROS level was significantly increased; after the intervention of medium, high, and low concentrations of RT5, the LDH level in the cells was significantly decreased, and the MDA level was significantly decreased; after the intervention of medium and high concentrations of RT5, the SOD level was significantly increased, and the ROS level was decreased.

[0036] (V) Summary The modeling dose of H2O2 was determined to be 300 μM; the dosage of ginsenoside RT5 was 1, 5, and 10 μM; ELISA detection revealed that ginsenoside RT5 had an antioxidant stress effect.

[0037] Experiment 2: Study on the effect of ginsenoside RT5 on myocardial ischemia model in rats 1. Materials 1. Animals Wistar rats, weighing about 220-250 g, clean grade, were provided by the Experimental Animal Center of the School of Basic Medical Sciences of Jilin University, with the use license number: SYXK(Jilin)2024-0005.

[0038] 2. Experimental Instruments Inverted microscope (Olympus, Japan) Power Lab data recording and analysis system (AD Instruments, Australia) MICROM HM340E Pathological Tissue Slicer (Microm, Germany) FDU-1200 Freeze Drying Instrument (EYELA Tokyo Rika Instruments Co., Ltd.) Constant temperature water bath (DR-HW-1 model, China) Centrifuge (7LDZ5-2, China) Recording and analysis software (Patch Clamp 6.01, USA) 3. Experimental drugs Sodium carboxymethyl cellulose, NaCl solution, and TTC stain were purchased from Sigma.

[0039] 2. Experimental methods 1. Establishment of acute myocardial ischemia model in rats The rats were first anesthetized, and then fixed on the operating table in the supine position and connected to the PowerLab data recording and analysis system for ECG monitoring. The biological signal collector was used to record the II lead ECG of the rats, and the rats were intubated through the mouth and connected to the small animal ventilator (the parameters were set as follows: tidal volume of 20-30 ml / kg, respiratory rate of 70 times / min, respiratory ratio of 1:1, and inspired oxygen concentration of 21%). The chest was dehaired, disinfected with 75% alcohol, and the skin was cut longitudinally on the left side of the middle for about 2 cm. At the most obvious cardiac pulsation between the 3rd and 4th ribs, the muscle layer was bluntly separated with hemostatic forceps. After the thoracotomy, the venous retractor was used to expand the chest, and the pericardium was removed to expose the heart. At the junction of the left atrial appendage and the pulmonary artery cone, that is, the left anterior descending branch of the coronary artery, a 5-0 non-traumatic silk thread was inserted for ligation. After ligation, the left ventricular anterior wall was observed to be pale and the pulsation was weakened. The heart was quickly reset and the chest cavity was closed. The whole thoracotomy time was controlled within 30 s, and the gas and blood in the thoracic cavity were discharged by external chest compression, spontaneous breathing was restored, and the heart wall was sutured. The II lead electrocardiogram was recorded, and the ST segment deviation amplitude was observed. The ST segment elevation of 0.2 mv was defined as a successful model. The sham operation group used the same surgical method, except that the surgical line was placed under the left coronary artery without ligation.

[0040] 2. Experimental Grouping and Drug Administration The rats with normal electrocardiogram were tested by standard lead II. The rats were selected as experimental subjects. The rats were randomly divided into 6 groups: sham operation group (Sham group), model group (Model group), positive drug group, RT5 low-dose group (L-RT5 group), RT5 medium-dose group (M-RT5 group), and RT5 high-dose group (H-RT5 group). The gavage volume was 10 mL / kg. The number and frequency of administration were twice a day for 3 consecutive days. The Sham group and the Model group were gavaged with CMC-Na, and the pseudo-ginsenoside RT5 groups were gavaged with RT5 solution, and the DIL group was gavaged with 2 mg / mL DIL solution.

[0041] 3. Selection of anti-myocardial ischemia evaluation indexes Myocardial enzyme activity After the experiment, about 3 ml of blood was collected from the abdominal aorta of the rats and centrifuged at 4000 rpm for 15 min to prepare serum. The activities of lactate dehydrogenase (LDH), creatine kinase (CK) and aspartate aminotransferase (AST) were determined according to the instructions of each kit to evaluate the protective effect of RT5 on the heart during myocardial injury.

[0042] Oxidative stress markers Rat serum and were taken, and the activities of superoxide dismutase (SOD), reactive oxygen species (ROS), malondialdehyde (MDA), cardiac troponin T (cTnT), creatine kinase isoenzyme (CK-MB) and glutathione peroxidase (GSH-Px) were determined according to the instructions of the kit to further verify the anti-myocardial ischemia activity of RT5.

[0043] Determination of myocardial infarction area After blood collection, the heart was immediately removed and washed with saline. After being stored at -20°C overnight, each heart was cut into 5 pieces with a thickness of about 0.1 cm, and then placed in a 1% TTC solution and incubated in a 37°C water bath for 5 minutes. As a result, normal tissue was stained red, while the infarcted myocardium was white. The myocardial infarction was analyzed using BI-2000 image analysis software, and the myocardial infarction area was calculated.

[0044] The percentage of myocardial infarction area = the sum of unstained myocardial area / total myocardial section area × 100%.

[0045] Histomorphological examination The heart tissue was separated as described above, and quickly fixed in 10% formalin (24-48h), dehydrated in 75%-85%-95%-100% ethanol gradient, transparent in xylene, and embedded in paraffin. The embedded wax block was fixed on a paraffin slicer, sliced ​​to a thickness of 4μm, mounted and dried. Before staining, the paraffin in the slice was removed with the help of xylene, dehydrated with alcohol from high to low concentrations, rinsed with distilled water, and stained with hematoxylin-eosin (HE), and myocardial tissue damage was observed under an optical microscope.

[0046] 4. Statistical processing of data The data were expressed as mean ± standard deviation, and the numerical comparisons among the groups were statistically analyzed using the sample mean t-test.

[0047] (III) Experimental results 1. Effects on the activity of three myocardial enzymes like Figure 5As shown in the figure, the results of serum LDH, CK and AST levels in rats of each group showed that compared with the Sham group, myocardial ischemia injury caused a significant increase in the levels of LDH, CK and AST in the Model group (P<0.01). Compared with the Model group, the levels of LDH, CK and AST in the serum of rats in the DIL group were significantly decreased (P<0.01, P<0.01 and P<0.05). The RT5 group had similar changes to the DIL group. The high-dose and medium-dose RT5 groups significantly inhibited the levels of LDH, AST and CK (P<0.01), and the low-dose RT5 group had no significant effect.

[0048] 2. Effects on oxidative stress indicators The results of the activity detection of SOD, ROS, MDA, GSH-Px, CK-MB and cTnT in myocardial tissue showed that myocardial ischemia caused the activities of MDA, ROS, GSH-Px, CK-MB and cTnT in the Model group to be significantly higher than those in the Sham group (P<0.01). On the contrary, compared with the Sham group, the activity of SOD in the Model group was significantly reduced (P<0.01). Compared with the Model group, the DIL group increased the level of SOD and significantly reduced the contents of MDA, ROS, GSH-Px, CK-MB and cTnT (P<0.05; P<0.01). Figure 5 As shown in the results, compared with the Model group, the M-RT5 and H-RT5 groups significantly reduced the activity of MDA and significantly increased the activity of SOD (P<0.05; P<0.01). At the same time, after treatment with RT5, the three dose groups of low, medium and high effectively inhibited the increase of cTnT levels caused by myocardial injury (P<0.05; P<0.01). The experimental results showed that RT5 therapeutic intervention had a great effect on SOD, MDA and cTnT, significantly increased the level of SOD, and significantly reduced the content of MDA and cTnT, indicating that RT5 therapeutic intervention can reduce oxidative stress damage and has significant anti-myocardial ischemia activity.

[0049] 3. Effect on myocardial infarction area Compared with the heart of the Sham group, the myocardial tissue of the Model group was pale, and obvious ischemic and infarcted areas were visible. The RT5 treatment group and the DIL group alleviated the symptoms of myocardial infarction to varying degrees. The heart images of the H-RT5 group and the DIL group were closer to those of the Sham group, such as Figure 6As shown. The comparison of myocardial infarction area in rats of each group showed that the M-RT5, H-RT5 and DIL groups significantly reduced the myocardial infarction area compared with the Model group (P<0.05, P<0.01), among which the change in the H-RT5 group was the most significant. The results of the effect on myocardial infarction area showed that medium and high doses of RT5 can significantly reduce myocardial cell death, reduce myocardial infarction area, and have a good anti-myocardial ischemia effect.

[0050] 4. Effects on myocardial tissue morphology The results of myocardial tissue morphological changes showed that the myocardial tissue structure and morphology of the Sham group were normal, with tight and regular arrangement. The myocardial cells of the Model group were swollen and deformed, with disordered arrangement, edema and more inflammatory cell infiltration. The distance between myocardial fibers in the L-RT5 group was widened, and there were more inflammatory cells infiltrating in the necrotic area. Most of the myocardial tissue structures in the DIL group, M-RT5 group and H-RT5 group were normal, with a small amount of interstitial edema, and the lesion range was limited. The degree of lesions was significantly milder than that in the Model group. Figure 7 The experimental results showed that RT5 preventive intervention showed a significant improvement in the tissue characteristics of cardiomyocytes and had significant anti-myocardial ischemic activity.

[0051] 4. Discussion We established a model in rats by ligating the left anterior descending branch of the coronary artery, and detected various indicators of myocardial three enzymes and oxidative stress. We found that medium and high doses of ginsenoside RT5 have antioxidant effects and a protective effect on the myocardium. We then performed TTC staining and HE staining on the heart tissue to further confirm that RT5 has a protective effect against myocardial ischemia.

[0052] summary The mechanism of action of pseudoginsenoside RT5 against MI may be related to anti-oxidative stress; TTC staining showed that the ischemic area of ​​the heart was significantly reduced after administration of RT5; HE staining showed that RT5 had a significant improvement on the characteristics of cardiac tissue.

[0053] Transcriptomics Since RT5 can significantly improve ventricular function, reduce myocardial enzyme activity, enhance antioxidant stress capacity, reduce oxidative stress damage, and reduce myocardial infarction area in rats with myocardial ischemia, metabolomics and transcriptomics techniques and methods were used to analyze the serum and heart tissue of rats after RT5 intervention to explore the anti-myocardial ischemia mechanism of RT5. Rat heart tissues were collected, and differential metabolites were detected and screened by UPLC-Q-TOF / MS; high-resolution and high-sensitivity sequencing platforms were used to fully reflect gene expression levels. In order to clarify the molecular mechanism and potential targets of RT5-mediated MI improvement, transcriptomics analysis was performed on the heart tissues of rats in the sham operation group, model group, and RT5-H group.

[0054] The heat map shows the expression pattern of each group of samples (Model, RT5, Sham) in terms of features, with the color gradient ranging from blue (low expression) to red (high expression). The rows and columns reflect the similarities between features and samples through cluster analysis, revealing the differences in expression features among the Model group, RT5 group, and Sham group ( Figure 8 ). Compared with the expression levels of the Sham group, the Model group resulted in a significant upregulation of the expression of 278 genes and a significant downregulation of the expression of 179 genes. Compared with the Model group, the RT5-H group significantly downregulated the expression of 328 genes and upregulated the expression of 346 genes ( Fig. 9 ). Among the 179 genes that were significantly downregulated in the Model group, 70 genes were significantly restored after RT5 treatment. Among the 278 genes that were significantly upregulated in the Model group, 81 genes were significantly downregulated after RT5 treatment. These 151 genes were considered to be DEGs regulated by RT5 on MI ( Fig.10 Subsequently, GO enrichment and KEGG pathway analysis were performed on DEGs to explore the potential mechanism of ginsenoside RT5 against MI ( Fig.11 ). The enrichment results showed that among the top ten enriched pathways, the activation of the cGMP-PKG pathway was widely reported to be associated with reducing myocardial damage and improving cardiac function. Therefore, activation of the cGMP-PKG pathway may be the potential mechanism of ginsenoside RT5 in resisting MI. Further analysis of the genes enriched in this pathway revealed that compared with the Sham group, the Model group significantly downregulated the expression levels of Nppb and Myh7, while RT5 adjusted the expression of Nppb and Myh7. In addition, there are literature reports that upregulating Nppb and Myh7 has a protective effect on myocardial ischemia. Therefore, Nppb and Myh7 and their downstream signaling pathways may be potential targets for ginsenoside RT5 in resisting MI.

Claims

1. Use of a pseudoginsenoside RT5 having the following structural formula in the preparation of an anti-myocardial ischemia drug, 。 2. The use as claimed in claim 1, wherein the pseudoginsenoside RT5 reduces oxidative stress and exerts an anti-myocardial ischemia effect by regulating the cGMP-PKG pathway.

3. The use as claimed in claims 1 and 2, wherein the myocardial ischemia refers to a decrease in blood perfusion to the heart, resulting in a decrease in oxygen supply to the heart, abnormal myocardial energy metabolism, inability to support normal heart function, and tachycardia or bradycardia caused by abnormal cardiac autonomic rhythm or conduction disorder.

4. A drug, characterized in that: The active ingredient comprises the pseudoginsenoside RT5 described in any one of claims 1-2.

5. The drug according to claim 4, characterized in that: The invention comprises the pseudoginsenoside RT5 and medically acceptable auxiliary materials.

6. The drug according to claim 5, characterized in that: The dosage form of the drug is any one of pills, tablets, capsules, granules, mixtures or oral liquids in any pharmaceutically acceptable dosage form.

Images