参附益心方对血管紧张素Ⅱ诱导H9c2心肌细胞凋亡及线粒体含量、膜电位的影响

Effects of Shenfu Yixin Formula(参附益心方) on Angiotensin Ⅱ-Induced H9c2 Cardiomyocytes Apoptosis,Mitochondrial Content and Membrane Potential

目的探讨参附益心方对血管紧张素Ⅱ(AngⅡ)诱导的H9c2心肌细胞损伤的干预作用及相关机制。方法采用CCK-8法筛选参附益心方、辅酶Q10、氯沙坦钾实验药物浓度,MTT法筛选AngⅡ实验药物浓度。以H9c2心肌细胞为研究对象,以AngⅡ诱导H9c2心肌细胞损伤为模型,将细胞分为正常对照组、模型组、辅酶Q10组、氯沙坦组和参附益心方低、高剂量组。正常对照组正常培养24 h;模型组用含筛选浓度的AngⅡ的培养基培养24 h;参附益心方低、高剂量组及氯沙坦组、辅酶Q10组用含筛选出相应药物浓度及AngⅡ的培养基培养24 h。检测各组H9c2心肌细胞活性、线粒体含量及膜电位、活性氧簇(ROS)含量、细胞凋亡率,凋亡相关因子Caspase-3、Caspase-8、Caspase-9、Bax、Bcl-2基因表达,Caspase-3、Caspase-8、Caspase-9蛋白活性。结果最终选择参附益心方低、高浓度为0.25、0.5 mg/ml,辅酶Q10研究浓度为1×10^-4mol/L,氯沙坦钾研究浓度为1×10^-4mol/L,AngⅡ建立实验模型浓度为1×10^-5mol/L。与正常对照组比较,模型组大鼠心肌细胞活性、线粒体数量及膜电位、Bcl-2基因表达降低,ROS含量、细胞凋亡率及Caspase-3、Caspase-8、Caspase-9、Bax基因表达升高,Caspase-3、Caspase-8、Caspase-9蛋白活性亦升高(P<0.05);与模型组比较,参附益心方高剂量组和氯沙坦组、辅酶Q10组细胞活性、线粒体数量及膜电位升高,ROS含量、细胞凋亡率、Caspase-3、Caspase-9、Bax基因表达降低,Caspase-3、Caspase-9蛋白活性亦降低(P<0.05)。结论参附益心方可能通过改善心肌细胞线粒体功能,降低ROS含量,从而减少AngⅡ诱导的H9c2心肌细胞发生凋亡。

Objective To investigate the intervention effect and related mechanism of Shenfu Yixin Formula(参附益心方)on angiotensinⅡ(AngⅡ)-induced H9c2 cardiomyocyte injury.Methods CCK-8 method was used to screen the concentration of Shenfu Yixin Formula,Coenzyme Q10 and Losartan potassium,and the concentration of AngⅡ was screened by MTT method.H9c2 cardiomyocytes were used as the research object,and the injury of H9c2 cardiomyocytes induced by AngⅡ was used as a model.The cells were divided into a normal control group,a model group,a coenzyme Q10 group,a losartan group and Shenfu Yixin Formula low and high dose groups.The normal control group was normally cultured for 24 hours.The model group was cultured for 24 hours with the culture medium containing the screening concentration of AngⅡ.The low-dose and high-dose groups of Shenfu Yixin Formula,the losartan group and the coenzyme Q10 group were cultured with the culture medium which were screened for the corresponding drug concentration and AngⅡ for 24 hours.The activity of H9c2 cardiomyocytes,mitochondrial content and membrane potential,reactive oxygen species(ROS)content,apoptosis rate,expression of apoptosis-related factors Caspase-3,Caspase-8,Caspase-9,Bax,Bcl-2,Caspase-3,Caspase-8 and Caspase-9 protein activity were detected.Results Finally,the low-concentration and high-concentration of Shenfu Yixin Formula was 0.25 and 0.5 mg/ml respectively,the concentration of coenzyme Q10 was 1×10^-4mol/L,and the concentration of losartan potassium was 1×10^-4mol/L.The experiment of AngⅡ model was established with the concentration of 1×10^-5mol/L.Compared with the normal control group,myocardial cell activity,mitochondrial number,membrane potential,Bcl-2 gene expression decreased in the model group.ROS content,apoptosis rate and expression of Caspase-3,Caspase-8,Caspase-9 and Bax genes increased.The activities of Caspase-3,Caspase-8 and Caspase-9 also increased(P<0.05).Compared with the model group,the cell activity,mitochondrial number and membrane potential of Shenfu Yixin Formula high-dose group,losartan group and coenzyme Q10 group increased,ROS content,apoptosis rate,gene expression of Caspase-3,Caspase-9,Bax were decreased,and the activities of Caspase-3 and Caspase-9 also decreased(P<0.05).Conclusion Shenfu Yixin Formula may reduce the mitochondrial function of cardiomyocytes and reduce the ROS content,thereby reducing the apoptosis of H9c2 cardiomyocytes induced by AngⅡ.