Effects of Qishen Yiqi dripping pills-containing serum on KATP channel opening and PI3K/AKT signaling pathway in hypoxic/reoxygenated H9C2 cardiocytes

Gui-XinHe,Yu-Fei Feng,Wei-BinQin,Lin Lin, Meng-Xian Hu, Guo-Kun Zheng,Li-Yan Yu,Zi-Yong Jia,JuanWei, QiWen

1. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530022, China

2. Guangxi University of Chinese Medicine, Nanning 530299, China

ABSTRACT Objective: To investigate the effects of Qishen Yiqi dropping pills serum on KATP channel opening and PI3K/AKT signaling pathway of hypoxic/reoxygenated H9C2 cardiocytes.Methods: H9C2 cardiocytes cultured in vitro were randomly divided into five groups, A:H9C2 cell group B: H9C2 cells +H2O2 model group C:H9C2 cells +H2O2 model + Qishen Yiqi group D:H9C2 cells +H2O2 model + Qishen Yiqi +wort group E:H9C2 cells +H2O2 model + Qishenyiqi +5-HD group, the drug intervention is according to the corresponding conditions. CCK-8 method was used to detect the cell activity of each group; Western blot was used to detect the expression of AKT and P-Akt proteins in myocardial cells in each group. The current was recorded by the standard patch clamp whole cell recording method, and the current was collected and analyzed by Pclamp6.0 software. Results: CCK-8 test results showed that compared with group A, the activity of myocardial cells in group B was significantly decreased,and the difference was statistically significant (P＜0.01); compared with group B, the difference in group C was statistically significant (P＜0.01); compared with C, cardiomyocyte activity in D and E group were significantly decreased, and the difference was statistically significant(P＜0.05); WB results showed that compared with A, p-Akt protein expression in B, C, D and E groups were significantly decreased, and the difference was statistically significant (P＜ 0.01);compared with group B, p-Akt protein expression in C, D and E group were significantly increased, and the difference was statistically significant (P＜ 0.01),but there was no significant difference in AKT expression among groups (P＞0.05); The results of whole cell patch clamp experiment showed that the outward current of B was significantly increased compared with that of A, and the difference between groups was statistically significant (P＜0.01); compared with group B, cardiomyocytes in group C further increased the outward current, and the difference between groups was statistically significant (P＜0.01); compared with C, the current of D and E group were significantly decreased, with statistical significance between groups(P＜0.01). Conclusion: QishenYiqi dropping pills can protect cardiomyocytes by activating p-Akt protein expression and KATP channel opening in H9C2 cardiomyocytes.

Keywords:Qishen Yiqi dropping pills H9C2 Hypoxia/reoxygenation p-AKT mitoKATP?Corresponding author: HE Gui-xin, M.D., Chief Physician, Professor, Postgraduate Supervisor.E-mail: he_guixin@163.com.

1. Introduction

Acute myocardial infarction (AMI) is one of the most common causes of morbidity, hospitalization and death worldwide. At present,coronary artery reconstruction procedures such as percutaneous coronary intervention, coronary artery bypass surgery, percutaneous puncture site selection and adjuvant antithrombotic therapy are the most effective strategies to improve the clinical survival rate of AMI.However, reperfusion therapy is a double-edged sword. The process of restoring blood flow to the ischemic myocardium may lead to myocardial reperfusion injury (MIRI), with the release of oxidative stress, cardiomyocyte apoptosis, autophagy and inflammatory cytokines Various pathological processes affect each other, which can further aggravate myocardial damage. Relevant animal model studies have shown that myocardial injury after acute myocardial infarction is caused by ischemia and reperfusion injury. The infarct area caused by reperfusion injury accounts for 50% of the final size of myocardial infarction[1]. Therefore, exploring the underlying molecular mechanism of myocardial ischemia-reperfusion injury and finding a treatment strategy that ultimately reduces the final infarct size is an urgent problem in the cardiovascular field. In recent years, many studies have confirmed that the activation of the mitoKATP channel plays a role in myocardial protection. On the one hand, the channel itself can prevent the driving force of reducing calcium uptake, prevent the formation of mitochondrial transition membrane pores (MPTP) and the generation of ROS to resist the myocardium ischemia[2], in addition, can affect endogenous active substances and downstream signaling pathways to play a protective role., so activating the mitoKATP channel is an important way for drugs to improve myocardial injury and protect myocardial cells[3].Studies have shown that the compound preparation Qishen Yiqi Dripping Pills has the following functions: anti-platelet aggregation,promoting angiogenesis, inhibiting inflammation, stabilizing plaque and inhibiting oxidative stress, thereby exerting its function of promoting blood circulation, removing blood stasis and dredging collaterals, and alleviating MIRI after myocardial injury. In the early stage, the team confirmed through animal experiments that Shenyiqi Dripping Pills can activate PI3K/AKT signal, regulate the expression of p-AKT protein, and improve the heart function of MIRI and protect myocardial cells[4-5]. In this experiment, on the basis of previous studies, serum pharmacology was used to prepare Qishen Yiqi Dropping Pills containing serum, and the protective effect and possible mechanism of the cardiomyocytes of Qishen Yiqi Dropping Pills containing serum were tested in vitro.

2. Materials and methods

2.1 Materials

Cell line Rat H9C2 cardiomyocyte cell line was purchased from Zhejiang Meisen Cell Technology Co., Ltd.

Experimental animals 20 SPF-grade male SD rats, 8-10 weeks old,weighing 220-250g, experimental animals were purchased from the Animal Experimental Center of Guangxi University of Traditional Chinese Medicine, animal license number: SCXK (Xiang) 2019-0014. Feeding conditions: Temperature: 22 ± 2℃; Humidity: 40%60%; Photoperiod: 12 /12h, ordinary feed, free eating and water;the experimental procedures strictly follow the "Guidelines for the Protection and Application of Laboratory Animals" issued by the US Health Agency "The experiment operation strictly abides by the relevant regulations of the Animal Experiment Management Committee of Guangxi University of Traditional Chinese Medicine.

2.2 Experimental drug

Qishen Yiqi Dripping Pills (Danshen, Panax notoginseng, Dalbergia odorifera, Astragalus, 0.5g/bag, Tianlishi Pharmaceutical Company,National Medicine Standard: Z20030319, batch number, 180615).

2.3 Main reagents

DMEM basic medium, 10% fetal bovine serum (Hyclone), blue chain double antibody (Shanghai Shenggong), H2O2 (analytical pure, Tianjin Jiangtian Chemical Reagent Factory), PI3K/AKT signaling pathway blocker wort (MCE, HY-10197), mitoKATP specific blocker 5-HD (abcam, ab141672), BCA protein quantitative kit (Biosharp, BL521A), anti-AKT antibody, anti-p-AKT antibody(American cell signaling technology company), Rabbit anti-GAPDH, goat rabbit secondary antibody IgG (American proteintech company), protein pre-stained Marker (American Thermo Scientific company), 30% acrylamide (29:1) (Amresco).

2.4 Main instruments

Cell incubator (Thermo Scientific 8000), optical microscope (XDS-1A), inverted camera microscope (OLYMPUS, IX71), centrifuge(Eppendorf, Germany), microplate reader (Thermo, MK3),refrigerated centrifuge (Effendorf), electrophoresis instrument (BIORAD, mini protean 3 cell), electrotransfer instrument (PS-9, Dalian Jingmai Technology Co., Ltd.), pipette (Thermo), water bath (Leica,HI1210), integrated chemistry Luminescence imager (ChemiScope 5300 Pro), patch clamp amplifier AXON200B, Pclam6.0 data acquisition software (AXON company).

2.5 Methods

2.5.1 Preparation of medicated serum

20 SPF male SD rats were fed normally for 3 days and were divided into 2 groups according to the random number table method,namely the blank serum group and the medicated serum group. The dosage of Qishen Yiqi Dropping Pill is calculated by referring to the body surface area of "Methodology of Research on Pharmacology of Traditional Chinese Medicine"[6], the equivalent dose ratio of rats and humans (calculated as 70kg for human body weight) is 6.3, and the dosage of Qishen Yiqi Dropping Pills for adults is 1g/tid, and the daily dosage of Qishen Yiqi Dropping Pills is calculated according to the formula. It is 27mg/(100g·d). According to body weight, 27mg/100g Qishen Yiqi Dropping Pills in the medicated serum group were dissolved in 2ml/100g normal saline, and the blank serum group was given 2ml/100g normal saline, once a day in the morning and evening, for 7 consecutive days. Two hours after the last administration, anesthetized by intraperitoneal injection,blood was collected from the abdominal aorta, and allowed to stand at room temperature for 30 minutes, 3,000 r/min, centrifuged for 15 minutes, and the supernatant was taken. Inactivate in a water bath at 56℃ for 30 min, filter and sterilize with a 0.22μm microporous filter membrane, subpackage, and store at -20℃ for later use.

2.5.2 Cell culture and treatment

Take out the H9C2 cardiomyocytes cryopreserved in the liquid nitrogen tank, place them in a 37℃ water bath and shake them quickly to dissolve them, and suck the cell suspension in the lysing solution into a centrifuge tube, 1200r Centrifuge for 5 min at /min,discard the supernatant, and wash 1-2 times with DMEM medium.The cardiomyocytes were added to the complete medium and placed in a 37℃, 5% CO2 incubator for culture. The medium was changed every 1-2 days. The logarithmic growth period was used for subsequent experiments.

2.5.3 Determination of the concentration of drug-containing serum

After the cells grow up, adjust the cell concentration to 1×105 cells/mL, inoculate in a 96-well plate, 100μl per well, culture for 24h; randomly divide H9C2 cardiomyocytes into a blank serum group, 2.5% For the medicated serum group, 5% medicated serum group, 10% medicated serum group and 15% medicated serum group, the prepared medicated serum is 2.5%, 5%, 10%, 15% and DMEM high glucose respectively. The culture medium is mixed,the H9C2 cells are processed in groups, and the changes in cell morphology are observed under a microscope to confirm the optimal drug-containing serum concentration.

2.5.4 Preparation of hypoxia/reoxygenation model of cardiomyocytes and group dosing

According to reference [7] model preparation method, after the cells grow up, adjust the cell concentration to 1×105 cells/mL,inoculate it in a 96-well culture plate, 100μl per well, 37℃, 5% CO2 incubator for 24h; add H2O2 to DMEM Gaotang medium to make the final concentration of 62.5μM, 125μM, 250μM, 500μM, 1000μM respectively, treat H9C2 cells in groups, 3h, 8h, 16h after microscope Observe the changes in cell morphology to determine the optimal concentration and duration of H2O2.

2.6 Indicator detection

2.6.1 CCK-8 method to detect cell activity in each group

Inoculate cardiomyocytes with a concentration of 1×105cells/mL in a 96-well culture plate with 100μl per well to make the number of cells per well 1×104, then place the 96-well plate in a 37℃, 5%CO2 incubator for culture 24h; According to the operating instructions of the CCK-8 kit, add 10μl of CCK-8 solution to the hole by obliquely adhering to the wall of the hole after the treatment of cardiomyocytes, taking care to avoid air bubbles, and incubating the well plate in the incubator for 2 hours. Detect the OD value of each well at 450nm wavelength with a microplate reader, and calculate the survival rate of H9C2 cells in each group. Each group set up 6 multiple wells for experiment.

2.6.2 WB method to detect the expression levels of p-AKT and AKT in H9C2 cardiomyocytes of each group

Collect the cells that need to be extracted with an appropriate amount of pre-chilled RIPA lysis buffer with PMSF added, and then centrifuge at 12000r/min for 10 min after fully lysing, and collect the supernatant. The BCA protein quantification kit determines the protein concentration, and the sample amount is determined according to the sample concentration. Prepare protein samples and polyacrylamide gels, install the electrophoresis device, pour the electrophoresis buffer, inject the protein samples and the maker respectively, and perform SDS-PAGE electrophoresis. After the protein is separated, carefully transfer the gel into the transfer buffer, and transfer the membrane on the transfer device. Ponceau staining was used to test whether the transfer of the membrane was successful. The membrane was rinsed with TBST 3 times for 5 minutes each time, and then slowly shaken with 5% skimmed milk powder at 37℃ for 2 hours. According to the instructions, dilute the AKT and P-AKT antibody with blocking solution to a suitable concentration, and incubate overnight at 4℃. Wash the membrane of the incubation primary antibody with TBST, 3 times, 5 min each time. Then, according to the dosage, the HRP-labeled rabbit secondary antibody/mouse secondary antibody was diluted according to the instructions, and incubated with the membrane at 37℃ for 1 hour. After washing with TBST, the integrated chemiluminescence analyzer detects.

2.6.3 Whole-cell patch clamp technique to measure KATP channel current

2.6.3.1 Solution preparation

Preparation of extracellular fluid for recording KATP channel current (mmol/L): NaCl 137g, CaCl 1.2g, KCl 5.0g, MgSO4 1.2g,NaH2PO4 0.5g, glucose 10g, HEPES 10g (use NaOH to adjust pH to 7.35-7.40); Preparation of intracellular fluid for recording KATP channel current (mmol/L): NaCl 10g, CaCl 1g, KCl 125g, MgATP 1g, HEPES 10g, EGTA 14g (use KOH to adjust pH to 7.1)

2.6.3.2 Patch clamp whole cell recording

Drop the recalcified H9C2 cardiomyocyte suspension into the perfusion tank and place it on the workbench of the inverted microscope. After the cells adhere to the wall, use extracellular fluid perfusion to rinse. Under the microscope, select the cells to adhere firmly, with neat edges, and regular morphology. Cells with clear horizontal stripes and no spontaneous contraction are used for patch clamp experiment; the glass microelectrode is filled with the electrode fluid through a syringe to make the electrode resistance between 3-5Ω. Give proper negative pressure suction, adjust the focal length of the microscope and the operating instrument to keep the electrode and the cell surface close to the final full contact,forming a high-resistance seal, the resistance value reaches above GΩ,continue to increase the negative pressure to break the membrane,and compensate the capacitance and series resistance , At this time,a whole-cell recording mode is formed; each group of intervention drugs are added to the perfusion tank, and the current is recorded after 10-15 minutes of reaction. The current signal is guided by the Ag/AgCl electrode and amplified by the patch clamp AXON 200B amplifier. The current recording adopts the voltage clamp method,and the data acquisition and analysis are completed by the Pclamp6.0 software.

2.7 Statistical analysis

SPSS 25.0 statistical software was used for analysis. Measurement data were expressed as mean±standard deviation (±s). One-way analysis of variance (ANOVA) was used for comparison between groups if the variances were uniform, and the non-parametric rank sum test was used for uneven variances. The difference was statistically significant when P＜0.05.

3. Results

3.1 Determination of the best drug-containing serum concentration

The experimental results show that when the drug-containing serum is added at 2.5%, 5%, 10%, the cell condition is good, and when the concentration of the drug-containing serum is 15%, the cell condition is found to be worse, so in order to ensure the efficacy and balance the cell condition Therefore, 10% of the drug-containing serum concentration was selected for the experiment in the later stage, as shown in Figure 1.

Figure 1 Effects of different concentrations of drug-containing serum on cell morphology

3.2 Determination of the optimal H2O2 concentration and action time in the preparation of cardiomyocyte hypoxia/reoxygenation model

The experimental results show that: when H2O2 is only used for 3 hours, no matter what the concentration is, there is no significant oxidative damage to the cells; when the concentration is 250 μM for 8 hours, it has a significant oxidative damage to H9C2 cells.When the concentration increases or the time of action When it was extended to 16h, the cell condition was very poor and it was not suitable for later experiments, so 250μM H2O2 was selected for 8h for later experiments, as shown in Figure 2.

Figure 2 Oxidative damage of H2O2 at different concentrations and time of action on H9C cardiomyocytes

3.3 Changes in the activity of H9C2 cardiomyocytes in each group

CCK-8 results showed that compared with A, the activity of cardiomyocytes in group B was significantly decreased, and the difference was statistically significant (p＜0.01); compared with cardiomyocytes of group B, the activity of cardiomyocytes in group C added with Qishen Yiqi Drop Pills Increase, the difference is statistically significant (p＜0.01); compared with group C, the cardiomyocyte activity of group D and E is significantly reduced,and the difference is statistically significant (p＜0.05), see Table 1 for details.

The WB results showed that there was no statistically significant difference in AKT protein expression between the groups (P＞0.05);compared with A, the p-AKT protein expression in groups B, C,D, and E was significantly reduced, and the differences between the groups were statistically significant. Significance (P＜0.01);Compared with group B, the expression of p-AKT protein in groups C, D and E was significantly increased, and the difference between the groups was statistically significant (P＜0.01). See Table 2 and Figure 3 for details.

Table 2 Comparison of the relative expression levels of AKT and P-Akt protein in cardiomyocytes of each group

Figure 3 P-Akt protein electrophoresis of myocardial cells in each group

3.4 Whole-cell patch clamp experiment

Under normal conditions, the KATP channel of H9C2 cardiomyocytes is not open. Therefore, the cell membrane potential is clamped to -40 mV, and a ramp stimulation from -80 to 60 mV is given to induce background current. Under the test voltage of 0mV, compared with A cardiomyocytes, the outgoing current of cardiomyocytes in group B increased significantly, and the difference between groups was statistically significant (P＜0.01); compared with group B, the current of group C had a further increasing trend.The difference was statistically significant (P＜0.01), indicating that oxidatively damaged cardiomyocytes showed a significantly enhanced outward current after Qishen Yiqi intervention. Compared with group C, the current in group D and group E was significantly reduced, which was statistically significant between the groups(P＜0.01). It is suggested that PI3K/AKT specific blocker and mitoKATP specific blocker can block this part of the current to varying degrees. See Table 3 for details.

Plotting current versus voltage can get the current-voltage relationship curve (I-V) of IKATP. In the test voltage range of 0-20 mV, the outward current is significantly enhanced, and the latter part of the current curve of each group is raised. Compared with the background current, the higher part is the open KATP channel current. See Figure 4 for details.

Table 3 mitoKATP channel current of myocardial cells in each group at 0mV voltage

Figure 4 KATP channel currents of cardiomyocytes in each group

4. Discussion

At present, reperfusion has been identified as a feasible treatment strategy for ischemic heart disease. However, the recovery of blood flow after insufficient blood supply to myocardial tissue usually leads to further aggravation of myocardial damage.More seriously, myocardial I/R may have serious pathological effects, such as excessive production of reactive oxygen species and nitrogen substances, increased release and recruitment of inflammatory mediators, activation of immune cells, apoptosis or necrosis of myocardial cells, and ultimately myocardial damage and Dysfunction[8-9]. As a common and inevitable pathophysiological change after interventional operation, MIRI can lead to arrhythmia and enlargement of infarct size. It is a disease with high morbidity and high mortality, and its morbidity is on the rise among young people. Therefore, how to reduce the damage caused by MIRI has attracted the attention of scholars in the cardiovascular field.Although previous studies have partially revealed the molecular mechanism of MIRI, the specific mechanism of MIRI is still not clear enough, and effective prevention and treatment is imminent.

There is no description of ischemia-reperfusion injury in Chinese ancient books. It can be considered that MIRI is a product of the development of modern medicine. According to the clinical manifestations of a series of myocardial injury after reperfusion therapy, it can be summarized into the categories of "chest pain" and"true heartache". At present, there is no unified expert consensus regarding the etiology, pathogenesis, syndrome differentiation and treatment of MIRI. Li Hui [10] on the basis of phlegm, blood stasis and arthralgia blocking the heart, the key is the decay of yang and the prosperous yin, the endogenous turbid toxin, and the damage of the body and collaterals of the heart. Weng Jinlong[11] believe that reperfusion therapy can reopen blood vessels, mainly to promote blood circulation to remove blood stasis and dredge collaterals.It should be a method of "eliminating evil" to treat symptoms.However, the symptoms of "intrinsic deficiency" such as shortness of breath, fatigue, dizziness, etc. Still exists. Cao Jiao [12] emphasizes that MIRI is based on deficiency of Yang Qi, phlegm turbidity and blood stasis as the standard, warming Yang to transform qi, and treating phlegm and blood stasis at the same time can restore Yang Qi and unobstructed pulse. Qishen Yiqi Dropping Pills are composed of Huangqi, Danshen, Panax notoginseng and Dalbergia odorifera. It is a traditional Chinese medicine compound preparation for treating chest obstruction of Qi deficiency and blood stasis type. It is widely used in clinical treatment of cardiovascular diseases such as ischemic heart failure and angina pectoris, as well as myocardial diseases.Secondary prevention of infarction[13-14]. Animal experiments have shown that Qishen Yiqi Dropping Pills have significant myocardial protection in CHF rats, which may enhance the degree of myocardial fibrosis by inhibiting the TGF-β1/Smads pathway; at the same time,inhibit the caspase-3 signaling pathway and improve myocardial cell apoptosis, Thus play a role in protecting the myocardium[15] .In addition, Qishen Yiqi Drop Pills can partially activate the PI3K/Akt signaling pathway, thereby protecting H9c2 cells from a series of injuries induced by high glucose[16]. Clinical experiments have confirmed that Qishen Yiqi Dropping Pills can effectively protect the vascular endothelial function after PCI after acute myocardial infarction, reduce the body's inflammatory response, help improve heart function, and have high safety[17]. As a multi-channel, multitargeted traditional Chinese medicine compound preparation, Qishen Yiqi Dropping Pills contain 53 kinds of chemical components,including organic acids, flavonoids, quinones, saponins, amino acids and other compounds, and various types of drug activity.The ingredients can work together to protect ischemic/reperfused cardiomyocytes through a synergistic effect.

In this study, Qishen Yiqi Dropping Pills were used as a representative prescription, and serum pharmacology experimental methods were used to further explore the effect of Chinese herbal compound on MIRI and its possible mechanism. In the experiment,SD rats were taken orally with Qishen Yiqi Dropping Pills to prepare different concentrations of medicated serum, and the optimal concentration was determined by observing the changes in the morphology of cardiomyocytes. The results showed that 10% was the optimal concentration of medicated serum. In addition, taking H9C2 cardiomyocytes cultured in vitro as the experimental object,according to the principle of enhanced oxidation reaction of the heart in the process of oxidative damage, H2O2 was selected to induce cell damage to simulate the hypoxia/reoxygenation pathological model. The observation results of this experiment suggest that after pretreatment with 250μM H2O2 for 8 hours, the damage effect is the most significant, and it can be used for later cell experiments.

MIRI is a complex pathophysiological process involving multiple factors, involving multiple mechanisms and signaling pathways.Among them, cell apoptosis is a key factor in myocardial injury and determines cardiac function and prognosis. In this study, the CCK-8 method was used to detect the survival rate of cardiomyocytes. The results suggest that Qishen Yiqi Dropping Pills containing serum can significantly increase the survival rate of H9C2 cardiomyocytes after oxidative injury, and have a certain protective effect on cardiomyocytes. PI3K and its downstream target serine/threonine kinase AKT belong to a family of conserved signal transduction enzymes, involved in a variety of cell biological processes[18]. The PI3K/AKT signaling pathway is considered to be an endogenous negative feedback regulation mechanism that promotes cell survival in response to harmful external stimuli and plays a key role in the pathological progression of MIRI [19]. Among them, the activated form of AKT, that is, p-AKT can regulate a large number of apoptosis-related mediators. For example, when p-AKT is activated,p-AKT acts on native cells through Bcl2 family members (Bcl2,Bclxl, Bax and Bad) Death pathway, and cell death pathway that acts on the outside through caspase family members (cleaved caspase 3, caspase 8 and caspase 9) [20]. The results of this study suggest that the serum containing Qishen Yiqi Dripping Pills significantly activates the expression of p-AKT protein in oxidatively damaged cardiomyocytes, while in cardiomyocytes with PI3K/AKT inhibitors,the expression of p-AKT protein decreases, and the cardiomyocytes The activity of the drug is also relatively reduced, indicating that Qishen Yiqi Dripping Pills can interfere with the expression of p-AKT protein in the PI3K/AKT signaling pathway and increase the activity of cardiomyocytes at the same time.

ATP-sensitive potassium channel (KATP channel) is an inwardly rectifying potassium ion channel regulated by the intracellular ATP concentration [21]. Tinker’s [22] studies have found that KATP channels are abundantly present in heart tissues such as the atria,ventricles, and atrioventricular conduction systems. They can not only improve the ischemic preconditioning of cardiomyocytes,but also play a role in the signal transduction pathway of cardiomyocytes, and play a role in the resistance to MIRI. It plays a very important role in the process. Costa[23] believed that activating KATP channels can reduce the damage and apoptosis of cardiomyocytes under various stress states. The vitality of KATP channels is determined by the number and opening degree of KATP channels in the cell. Its activity is closely related to cell metabolism and is regulated by the intracellular ATP concentration: under physiological conditions, ATP has a higher concentration in the cell.KATP channels are inhibited and closed; and when cardiomyocytes are subject to pathological stimuli such as ischemia, reperfusion injury and apoptosis, the intracellular ATP will be consumed and the concentration will decrease. At this time, KATP channels are no longer inhibited, resulting in ATP channels Open, potassium ions outflow, the resting potential of the cell membrane is reduced,the action potential is shortened, the influx of calcium ions is reduced, and the calcium overload of myocardial cells is reduced[24], thereby reducing the energy consumption of myocardial cells,increasing their stress tolerance, and protecting the myocardium[25].This study found that when the cells were oxidized damage, the cell outgoing current increased significantly, resulting in stress myocardial protection; after the intervention of Qishen Yiqi on the basis of oxidative damage, the myocardial cell current increased significantly, which was similar to the model group. The ratio is statistically significant. When the mitoKATP specific blocker 5-HD is added, the current is partially blocked, indicating that the generation of the current is closely related to the activation of mitoKATP channel. It is speculated that Qishen Yiqi Dropping Pills may have a KATP channel opener-like effect. In addition, the activity of cardiomyocytes added with blocker was significantly lower than that of Qishenyiqi dripping pill intervention group, indicating that the opening of mitoKATP channel may have a protective effect on cardiomyocytes.In summary, this experiment confirms that Qishen Yiqi Dropping Pills can protect cardiomyocytes by activating the expression of p-AKT protein and the opening of KATP channels in H9C2 cardiomyocytes. However, the specific mechanism of Qishen Yiqi Dripping Pills and related reactions have not yet been explored, and further experiments are needed to clarify the mechanism.