氧化应激介导的DNA损伤在大鼠心肌H9C2细胞缺氧/复氧损伤中的作用

Role of oxidative stress-mediated DNA damage in hypoxia/reoxygenation damage of rat myocardial H9C2 cells

目的探讨氧化应激介导的DNA损伤在大鼠心肌H9C2细胞缺氧/复氧损伤中的作用。方法采用大鼠心肌H9C2细胞制备缺氧/复氧损伤模型。实验分为对照组(Con组)、缺氧/复氧模型组(H/R组)、抗氧化剂NAC组(NAC组)、抗氧化剂+模型组(NAC+H/R组)。Western blot检测细胞凋亡相关蛋白(Bax、Bcl-2)表达;细胞免疫荧光法检测活性氧(ROS)水平,DNA损伤相关蛋白(p-γH2ax、8-OHdG)、细胞损伤指标[细胞色素c(Cytochrome c)]表达。结果与Con组比较,H/R组细胞内ROS水平升高,p-γH2ax、8-OHdG、Cytochrome c、Bax/Bcl-2表达增加,差异有统计学意义(P<0.05);与H/R组比较,NAC+H/R组ROS水平降低,p-γH2ax、8-OHdG、Cytochrome c、Bax/Bcl-2表达降低,差异有统计学意义(P<0.05)。结论抑制心肌细胞氧化应激,可明显改善心肌细胞DNA损伤和凋亡。

Objective To investigate the role of oxidative stress-mediated DNA damage in myocardial ischemia-reoxygenation injury in rat myocardial H9C2 cells.Methods The ischemia-reoxygenation injury model was prepared by using rat myocardial H9C2 cells.The experiment was divided into the control group(Con),hypoxia/reoxygenation model group(H/R),antioxidant NAC group(NAC)and antioxidant+model group(NAC+H/R).The expressions of apoptosis-related proteins Bax,Bcl-2 and cytochrome c were detected by Western blot and immunofluorescence assay.The level of ROS in the cells was detected by DCFH-DA staining.The expression of DNA damage-related proteins p-γH2ax and 8-OHdG were detected by immunofluorescence assay.Results Compared with the Con group,the intracellular oxidative stress ROS level in the H/R group was increased,and the expressions levels of p-γh2ax,8-OHdG and Cytochrome c were increased,Bax/Bcl-2 was increased,and the differences were statistically significant(P<0.05).Compared with the H/R group,the ROS level in the NAC+H/R group was significantly decreased,the expression levels of p-γH2ax,8-OHdG and Cytochrome c were significantly decreased,Bax/bcl-2 was decreased,and the differences were statistically significant(P<0.05).Conclusion Inhibiting the oxidative stress in cardiomyocytes can significantly improve the DNA damage and apoptosis of cardiomyocytes.