Study The Mechanism Of Kangxian Yixin Decoction On Cardiomyocyte Apoptosis In Mice With Dilated Cardiomyopathy Based On The Mitochondrial Permeability Transition Pore

Objective:Kangxian Yixin Decoction is effective in the clinical treatment of dilated cardiomyopathy（DCM）.Early studies have found that it can inhibit cardiomyocyte apoptosis and its mechanism is not clear.In this study,we established the c Tn TR141W transgenic dilated cardiomyopathy mouse model to observe the effects of Kangxianyixin Recipe on cardiac function and cardiomyocyte apoptosis in mice with dilated cardiomyopathy;further use the cardiomyocyte hypertrophy model to explore the Kangxianyixin Recipe Mechanism of inhibiting cardiomyocyte apoptosis by regulating mitochondrial permeability transition pore.Methods:Animal experiment:Using the c Tn T^R141Wtransgenic DCM mouse model,through genetic identification,the successfully modeled mice were included in the experiment,and they were randomly divided into Model group,Kangxian Yixin Decoction group and Coenzyme Q10 Group,another C57BL/6J mouse was set as the normal group,and drug intervention was performed at the 10th week for 8 weeks of treatment.Detection:（1）Small animal echocardiography to detect the end-systolic diameter,end-diastolic diameter,short axis shortening rate and ejection fraction of mice;（2）HE and Masson staining to detect myocardial tissue morphology and myocardial fibrosis;（3）Transmission electron microscope to detect myocardial mitochondrial ultrasonography Microstructure;（4）Western blotting was used to detect the expression of Cyp-D,Cyt-C,and Caspase-3 protein in myocardial tissue.Cell experiment:Norepinephrine（NE）induced H9c2 cardiomyocyte hypertrophy model,divided into Control group,model group,Kangxian Yixin Decoction group（0.125 mg/m L）,cyclosporin A group（10μmol/L）.Detection:（1）RT-PCR detection of myocardial hypertrophy-related genes ANP and BNP m RNA expression;（2）flow cytometry to detect myocardial cell apoptosis rate and m PTP openness;（3）JC-1 to detect cell mitochondrial membrane potential changes;（4）RT-PCR to detect cardiomyocyte apoptosis The expression of apoptosis-related factors Cyp-D,Cyt-C,and Caspase-3 m RNA;（5）Western blotting was used to detect the protein expression of cardiomyocyte apoptosis-related factors Cyp-D,Cyt-C,and Caspase-3.Results:Animal experiments:（1）The results of cardiac ultrasound showed that compared with the normal group,the end-systolic and end-diastolic diameters of the model group increased（P<0.05）,and the short axis shortening rate and ejection fraction decreased（P<0.05）;compared with the model group,The Kangxian Yixin Decoction group and the coenzyme Q10 group reduced the end-systolic and end-diastolic diameters（P<0.05）,and the short axis shortening rate and ejection fraction increased（P<0.05）;（2）HE and Masson staining results showed that the myocardium of the model group Irregular arrangement of nuclei,pyknosis of some nuclei,disorder of myocardial fiber arrangement,collagen deposition,and local myocardial fibrosis;Compared with the model group,Kangxian Yixin Decoction and Coenzyme Q10 group,the arrangement of myocardial cell nuclei and muscle fibers were significantly improved,and myocardial fibrosis was reduced.（3）The electron microscope results showed that compared with the normal group,the mitochondrial ultrastructure of the model group was damaged,and some mitochondria were swollen.After the intervention of Kangxian Yixin Decoction,the mitochondrial ultrastructure was improved;（4）Western blotting results showed:compared with the normal group,the model group Cyp-D,Cyt-C,Caspase-3 protein expression increased（P<0.05）;Compared with the model group,Cyp-D,Cyt-C,Caspase-3 protein expression decreased after the intervention of Kangxian Yixin Fang（P<0.05）.Cell experiment:（1）RT-PCR detection of ANP and BNP m RNA results showed that norepinephrine（NE）had the best concentration at 200μmol/L（P<0.05）;（2）Flow cytometry results showed that compared with the normal group,The apoptosis rate of the model group was significantly increased.After the intervention of Kangxian Yixin Decoction,the apoptosis rate was significantly reduced,and it could inhibit the opening of m PTP（P<0.05）.（3）JC-1 test results show that Kangxian Yixin Decoctioncan increase mitochondrial membrane potential（P<0.05）;（4）Western blotting and RT-PCR results show:Compared with the normal group,the model group Cyp-D,Cyt-C,Caspase-3 protein And gene expression increased（P<0.05）,and Cyp-D,Cyt-C,Caspase-3 protein and gene expression decreased after the intervention of Kangxian Yixin Decoction（P<0.05）.Conclusion:1.Kangxian Yixin Decoction can improve cardiac function in DCM mice,reduce myocardial pathological damage and mitochondrial swelling,down-regulate the expression of apoptosis-related factors Cyp-D（m PTP regulator）,Cyt-C,and Caspase-3,and inhibit cardiomyocyte apoptosis The mechanism of death may be through inhibiting the openness of m PTP.2.Kangxian Yixin Decoction can reduce the expression of Cyp-D in the H9c2cardiomyocyte hypertrophy model,regulate the opening of m PTP,inhibit the expression of apoptotic factors Cyt-C,Caspase-3,and inhibit cardiomyocyte apoptosis.