白藜芦醇对CoCl_2诱导H9C2心肌细胞损伤的保护作用研究

Study on the protective effect of Resveratrol on H9C2 myocardial cell injury induced by CoCl_2

目的探讨白藜芦醇(Resveratrol,RES)对二氯化钴(CoCl_2)诱导H9C2心肌细胞损伤保护作用的机制。方法 500μmol/L CoCl_2处理H9C2心肌细胞24h以建立H9C2心肌细胞缺氧损伤模型。实验分组:空白对照组(control);模型损伤组(0.1%DMSO培养液预处理H9C2细胞24h后,500μmol/L CoCl_2继续处理H9C2细胞24h);低剂量RES组(12.5μmol/L RES预处理H9C2细胞24h后,500μmol/L CoCl_2继续处理H9C2细胞24h);中剂量RES组(25μmol/L RES预处理H9C2细胞24h后,500μmol/L CoCl_2继续处理H9C2细胞24h);高剂量RES组(50μmol/L RES预处理H9C2细胞24h后,500μmol/L CoCl_2继续处理H9C2细胞24h)。采用MTT法评价H9C2心肌细胞活力。Hoeschst 33342染色观察H9C2细胞核形态的变化。采用流式细胞技术检测H9C2细胞线粒体膜电位的变化、细胞内活性氧的产生及细胞凋亡的情况。Western blot检测H9C2细胞中凋亡相关蛋白的变化。结果 MTT结果显示,RES浓度依赖性抑制CoCl_2诱导的H9C2细胞活力的降低;Hoeschst 33342染色观察显示,RES浓度依赖性抑制CoCl_2引起的H9C2细胞凋亡形态的出现;流式细胞术显示,RES浓度依赖地抑制CoCl_2诱导的H9C2细胞内活性氧的产生、线粒体膜电位的降低、细胞凋亡的增加;Western blot检测结果显示,RES浓度依赖性抑制CoCl_2引起的H9C2细胞中促凋亡蛋白(Bax、cleaved-caspase-3、cleaved-caspase-9)的增加和抑凋亡蛋白(Bcl-2)的降低。结论 RES对CoCl_2引起的H9C2心肌细胞损伤具有保护作用,其机制可能是通过抑制CoCl_2引起的活性氧增加、线粒体膜电位降低进而阻碍H9C2细胞线粒体凋亡的发生而实现的。

Objective To study the protective mechanism of resveratrol（RES）on H9C2 cells injury induced by two cobalt chloride（CoCl2）.Methods CoCl2（500μmol/L）was used to treat H9C2 cardiac muscle cell for 24 hin order to establish H9C2 myocardial cell hypoxia injury model.The experiment was divided into five groups：blank control group（control）;model injury group（H9C2 cells were pretreated with 0.1% DMSO culture medium for 24 h,and then they were continued to treat by 500μmol/L CoCl2 for 24 h）;low dose RES group（H9C2 cells were pretreated with 12.5μmol/L RES for 24 h,and then they were continued to treat by 500μmol/L CoCl2 for 24 h）;medium dose RES group（H9C2 cells were pretreated with 25μmol/L RES for24 h,and then they were continued to treat by 500μmol/L CoCl2 for 24 h）;high dose RES group（H9C2 cells were pretreated with 50μmol/L RES for 24 h,and then they were continued to treat by 500μmol/L CoCl2 for 24 h）.MTT method was used to evaluate the viability of H9C2 myocardial cell.Morphological changes of H9C2 nucleus were observed by Hoeschst 33342 staining.The changes of mitochondrial membrane potential,intracellular reactive oxygen species（ROS）production and cell apoptosis were detected by flow cytometry in H9C2 cells.Western Blot was used to investigate the changes of apoptosis related proteins in H9C2 cells.ResultsMTT results showed that RES concentration dependently inhibited the decrease of H9C2 cell viability induced by CoCl2.Hoeschst 33342 staining showed that RES concentration dependently inhibited the appearance of H9C2 cells apoptosis induced by CoCl2.Flow cytometry revealed that RES concentration dependently inhibited ROS production,decreased mitochondrial membrane potential,and increased apoptosis in H9C2 cells induced by CoCl2.Western blot showed that RES concentration dependently inhibited the increase of pro-apoptotic proteins（Bax,cleaved-caspase-3,cleaved-caspase-9）and decreased expression of apoptotic protein（Bcl-2）in H9C2 cells induced by CoCl2.Conclusion RES has the protective effect on H9C2 myocardial cell injury induced by CoCl2,the mechanism may be related with inhibiting the increase of ROS induced by CoCl2 and decreasing the mitochondrial membrane potential,thus hindering the occurrence of mitochondrial apoptosis in H9C2 cells.