miR-19b对H_2O_2诱导的大鼠心肌细胞H9c2氧化应激损伤的保护作用及机制研究

Protective Effects and Mechanisms of miR-19b on Oxidative Stress Injury Induced by Hydrogen Peroxide in H9c2 Myocardial Cells

目的研究microRNA(miR)-19b是否在H2O2诱导的心肌细胞的氧化应激损伤中具有保护作用。方法将大鼠H9c2心肌细胞株分为6组,空白对照组(L1),H2O2组(L2),H2O2+miR-19b mimic阴性对照(NC)组(L3),H2O2+miR-19b mimic组(L4),H2O2+miR-19b inhibitor组(L5),H2O2+miR-19b inhibitor NC组(L6)。NC组细胞转染随机合成的miRNA片段作为阴性对照。采用CCK8和流式细胞术检测细胞活性和凋亡情况,逆转录聚合酶链式反应(RT-PCR)法检测miR-19b的表达量,并利用试剂盒检测超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、丙二醛(MDA)和还原型谷胱甘肽(GSH)水平,Western blot法检测核因子相关因子2(Nrf2)、血红素氧合酶-1(HO-1)表达水平。结果与L1组比较,L2组和L3组中miR-19b表达量、H9c2细胞活性和细胞内SOD、GSH水平均显著降低(P<0.01),细胞凋亡率和细胞内LDH、MDA水平、Nrf2和HO-1表达水平均显著增加(P<0.01)。与L2组及L3组比较,L4组中miR-19b表达量、H9c2细胞活性和细胞内SOD、GSH水平、Nrf2和HO-1表达水平均显著增加(P<0.01),细胞凋亡率和细胞内LDH、MDA水平均显著降低(P<0.01),L5组中miR-19b表达量和H9c2细胞活性和细胞内SOD、GSH水平、Nrf2和HO-1表达水平均显著降低(P<0.01),细胞凋亡率和细胞内LDH、MDA水平均显著增加(P<0.01),L6组miR-19b表达量、H9c2细胞活性、细胞凋亡率、细胞内SOD、GSH、LDH、MDA水平以及Nrf2和HO-1表达水平均未见统计学差异(P>0.05)。结论miR-19b高表达可显著抑制H9c2细胞的氧化应激损伤,发挥心肌细胞保护作用。

Objective To investigate whether microRNA-19b(miR-19b)has a protective effect on H2O2-induced oxidative stress injury in rat myocardial cells.Methods Rat H9c2 myocardial cells were divided into 6 groups as follows:blank control(L1),H2O2(L2),H2O2+miR-19b mimic negative control(NC)(L3),H2O2+miR-19b mimic(L4),H2O2+miR-19b inhibitor(L5)and H2O2+miR-19b inhibitor NC(L6).Cells transfected with random miRNA fragments serve as NC.Cell activity and apoptosis were analyzed by CCK8 and flow cytometry.The expression of miR-19b was detected by RT-PCR.Superoxide dismutase(SOD),lactic dehydrogenase(LDH),malondialdehyde(MDA),glutathione(GSH)levels were tested by related kits and the expression of Nrf2 and HO-1 were decected by Western blot.Results Compared with L1 group,the expression of miR-19b,cell viability,SOD and GSH levels were significantly decreased(P<0.01)and cell apoptotic rate,LDH and MDA levels,Nrf2 and HO-1 proteins increased(P<0.01)in L2 and L3 group.Compared with L2 and L3 group,the expression of miR-19b,cell viability,SOD and GSH levels,Nrf2 and HO-1 proteins were markedly elevated(P<0.01)and cell apoptotic rate,LDH and MDA levels dropped(P<0.01)in L4 group.Meanwhile,the expression of miR-19b,cell viability,SOD and GSH levels,Nrf2 and HO-1 proteins were markedly declined(P<0.01)and cell apoptotic rate,LDH and MDA levels elevated(P<0.01)in L5 group,yet all of which showed no significant differences in L6 group(P>0.05).Conclusion MiR-19b could inhibits oxidative stress damage in H9c2 cells and play a protective role on myocardial cells.