Experimental Study On Apoptosis Of Myocardial Cells In The Intervention Of Wutou Chishizhi Pill Prescription

Objective:Acute myocardial infarction（AMI）is an acute and critical disease.In recent years,the morbidity and mortality of AMI in China have gradually increased.In modern medicine,thrombolytic therapy is often used in pre-hospital first aid for acutmyocardial infarction to gain time for in-hospital first aid.However,it is urgent to develop self-first aid drugs for patients with acute myocardial infarction in the ear-ly stage because of the failure of medical staff to take specific drugs for self-help in timebefore arrival and to gain effective time for pre-hospital first aid and in-hospital treatment.To investigate the mechanism of Wutou Chishizhi Pill inhibiting cardiomyoc yte apoptosis by inducing cardiomyocyte apoptosis with H₂O₂ and intervening with me dicated serum of Wutou Chishizhi Pill.Methods:Twenty rats were divided into normal group,Wutou Chishizhi pill grou pand positive control group（Shenfu Decoction）for routine feeding and administration.The state of rats was observed,and drug-containing serum was extracted 5 days later.H9C2 myocardial cell lines were cultured and passaged in groups.H9C2 myocardial cells were seeded into 96-well plate by cell counter,and the cell viability was tested after 24 hours.Four groups of time（1,2,4,8h）and four concentrations of H₂O₂（600,900,1200,1500μmol/L,respectively）were set.The optical density of each group was measured by enzyme labeling instrument,and the cell viability was calculated.The optimum modeling time（4 h）and concentration of H₂O₂（900μmol/L）were obtained.According to the concentration and time,the normal group,the H₂O₂ model grou the positive control group and the Wutou Chishizhi pill group were prepared,and the cell viability of the Wutou Chishizhi pill group was tested.Immunofluorescence technique was used to detect the expression of Bcl-2 and Bax in H9C2 myocardial cells.The expression levels of B-cell cymphoma-2（Bcl-2）and Bcl-2-Assaciated X protein（BAX）were detected by Western Blot（WB）and analyzed.Result:1.CCK-8 method was used to determine the viability of H9C2 myocardial cells injured by different concentrations of H₂O₂.The results showed that the mortality of H9C2 myocardial cells at 900μmol/L concentration for 4 hours was the closest to half,with statistical significance（p<0.01）.Compared with the model group,the activity of H9C2 myocardial cells in Wutou Chishizhi pill group increased significantly,which was close to the normal group,with statistical significance（p<0.05）.2.Fluorescence inversion microscopy showed that Bcl-2 was strongly positive in the c ytoplasm around the nucleus of myocardial cells in the normal group,while only a s mall amount was positive in the model group.The positive control group had relativel y high positive expression,while the Wutou Chishizhi pill group had a large number of positive expression,and the fluorescence intensity was higher than that of the mode l group,suggesting that Wutou Chishizhi pill could up-regulate the expression of Bcl-2in myocardial cells.The expression of BAX was not obvious in the normal group,b ut was high in the model group.The fluorescence expression in the positive control g roup was relatively low.The fluorescence intensity in the Wutou Chishizhi pill group was lower than that in the model group,suggesting that Wutou Chishizhi pill could d own-regulate the expression of BAX in myocardial cells.3.Western Blot assay showed that the expression of Bcl-2 protein in myocardial cells of Wutou Chishizhi pill group was significantly higher than that of model group（p<0.01）,with statistical significance;significantly higher than that of positive control g roup（p<0.01）,with statistical significance;close to normal group（p<0.01）,with s tatistical significance.The expression of BAX protein in myocardial cells of Wutou C hishizhi pill group was lower than that of model group（p<0.05）,with statistical sig nificance;slightly lower than that of positive control group,without statistical significa nce;higher than that of normal group（p<0.01）.Compared with the positive control group,the aconite Chishizhi pill group had no statistical significance.Conclusion:Wutou Chishizhi Pill can resist the damage of H9C2 myocardial cells caused by H₂O₂,up-regulate the expression of Bcl-2 in mitochondrial pathway and down-regulate the expression of BAX,thus inhibiting cardiomyocyte apoptosis.