抗纤益心方干预线粒体通透性转换孔抑制心肌细胞凋亡的机制

Kangxian Yixin Prescription Interferes with Mitochondrial Permeability Transition Pore to Inhibit Cardiomyocyte Apoptosis

目的:探讨抗纤益心方通过调控线粒体通透性转换孔(mPTP)对心肌细胞凋亡的影响及相关作用机制。方法:H9c2心肌细胞常规培养,饥饿8 h后分为正常组,模型组,抗纤益心方(0.25 g·L^(-1))组,环孢素A(10μmol·L^(-1))组,药物作用24 h用于后续实验。利用去甲肾上腺素(NE)诱导H9c2心肌细胞肥大模型,通过实时荧光定量聚合酶链式反应(Real-time PCR)检测NE的最佳造模浓度,流式细胞仪检测mPTP的开放程度,同时检测mPTP开放后引起的凋亡相关因子Cyp-D,Cyt-C,Caspase-3 mRNA和蛋白的表达水平,和线粒体膜电位的水平。结果:NE浓度在200μmol·L^(-1)时心房钠尿肽(ANP),脑钠肽(BNP)mRNA的表达水平最高,此浓度作为诱导H9c2心机细胞肥大模型的最佳浓度,与正常组比较,模型组mPTP过度开放,线粒体内相对荧光强度减弱和线粒体膜电位降低(P<0.05,P<0.01);凋亡相关因子Cyp-D,Cyt-C,Caspase-3 mRNA和蛋白表达升高(P<0.05)。与模型组比较,抗纤益心方组和环孢素A组mPTP开放受到抑制,线粒体相对荧光强度和线粒体膜电位升高(P<0.05,P<0.01);Cyp-D,Cy-tC,Caspase-3 mRNA和蛋白表达降低(P<0.05)。结论:抗纤益心方能够抑制心肌细胞凋亡,其作用机制可能是通过调控mPTP的开放,抑制相关凋亡因子Cyp-D,Cyt-C,Caspase-3的表达有关。

Objective:To explore the mechanism of Kangxian Yixin prescription in regulating mitochondrial permeability transition pore(mPTP)and inhibiting cardiomyocyte apoptosis.Method:H9 c2 cardiomyocytes were cultured routinely.After 8 h of starvation,the cells were divided into the normal group,model group,Kangxian Yixin prescription(0.25 g·L^(-1))group,and cyclosporin A(CsA,10μmol·L^(-1))group and treated with the corresponding drugs for 24 h for follow-up experiments.The H9 c2 cardiomyocyte hypertrophy model was induced by norepinephrine(NE),whose optimal concentration was determined by real-time polymerase chain reaction(Real-time PCR).The degree of mPTP opening was detected by flow cytometry,followed by the measurement of mRNA and protein expression levels of apoptosis-related factors cyclophilin D(Cyp-D),cytochrome C(Cyt-C),and cysteine aspartate-specific protease-3(Caspase-3)after mPTP opening and the quantification of mitochondrial membrane potential.Result:When the concentration of NE was200μmol·L^(-1),the mRNA expression levels of atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)were the highest,implying that it was the optimal concentration to induce H9 c2 cell hypertrophy.Compared with the normal group,the model group exhibited excessive opening of mPTP,weakened relative fluorescence intensity in mitochondria,decreased mitochondrial membrane potential(P<0.05,P<0.01),and elevated mRNA and protein expression of Cyp-D,Cyt-C,and Caspase-3(P<0.05).Compared with the model group,both Kangxian Yixin prescription and CsA inhibited mPTP opening,enhanced the relative fluorescence intensity of mitochondria,increased mitochondrial membrane potential(P<0.05,P<0.01),and lowered the mRNA and protein expression of Cyp-D,Cyt-C,and Caspase-3(P<0.05).Conclusion:Kangxian Yixin prescription inhibits cardiomyocyte apoptosis possibly by regulating mPTP opening and inhibiting the expression of apoptosis-related factors Cyp-D,Cyt-C,and Caspase-3.