敲低EphB1基因对过氧化氢诱导的大鼠心肌细胞氧化损伤的影响

Effect of knockdown of EphB1 gene on oxidative damage of cardiomyocytes induced by hydrogen peroxide in rats

目的:探讨敲低EphB1基因对过氧化氢(H 2O 2)诱导的大鼠心肌H9c2细胞氧化损伤的影响,为EphB1基因治疗心血管疾病提供依据。方法:体外培养大鼠心肌H9c2细胞,分为空白组(不做任何处理)、H 2O 2组(H 2O 2细胞损伤)、阴性对照组(转染siRNA control)和转染si-EphB1组(转染si-EphB1后H 2O 2细胞损伤)。实时荧光定量聚合酶链反应(qRT-PCR)检测各组H9c2细胞中EphB1基因表达水平,噻唑蓝(MTT)法检测各组H9c2细胞存活率,流式细胞术检测各组细胞凋亡情况,DCFH-DA探针法检测各组细胞内活性氧(ROS)水平,分光光度计法检测各组细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)水平。结果:与空白组比较,H 2O 2组H9c2细胞中EphB1 mRNA表达水平明显升高(P<0.05),阴性对照组H9c2细胞中EphB1 mRNA表达水平无明显变化(P>0.05);与H 2O 2组比较,转染si-EphB1组H9c2细胞中EphB1 mRNA表达水平明显降低(P<0.05)。与空白组比较,H 2O 2组细胞存活率降低(P<0.05),细胞凋亡率升高(P<0.05),ROS和MDA水平升高(P<0.05),SOD水平降低(P<0.05),而阴性对照组细胞存活率、凋亡率、ROS、MDA和SOD水平差异无统计学意义(P>0.05);与H 2O 2组比较,转染si-EphB1组细胞存活率升高(P<0.05),细胞凋亡率降低(P<0.05),ROS和MDA水平降低(P<0.05),SOD水平升高(P<0.05)。结论:敲低EphB1基因能够抑制H 2O 2诱导的大鼠心肌细胞氧化损伤,起到心肌保护作用,其机制与提高心肌细胞存活率、抑制细胞凋亡、改善抗氧化酶活性和提高细胞内氧化应激产物清除能力有关。

Objective:To investigate the effect of knockdown of EphB1 gene on the oxidative damage of cardiomyocytes (H9c2) induced by hydrogen peroxide (H 2O 2) in the rats,and to provide the evidence for EphB1 gene in the treatment of cardiovascular diseases.Methods: The rat cardiomyocytes H9c2 were cultivated in vitro and divided into blank group (no treatment),H 2O 2 group (H 2O 2-induced cell damage),negative control group (transfected with siRNA control) and si-EphB1 transfection group (transfected with si-EphB1 after H 2O 2-induced cell damage).The expression levels of EphB1 mRNA in the H9c2 cells in various groups were detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR),the survival rates of the H9c2 cells in various groups were tested by MTT assay,the apoptosis of H9c2 cells were measured by flow cytometry,the ROS levels in the H9c2 cells in various groups were determined by DCFH-DA,and the levels of MDA and SOD in the H9c2 cells in various groups were determined by spectrophotometer.Results:Compared with blank group,the expression level of EphB1 mRNA in the H9c2 cells in H 2O 2 group were significantly increased(P <0.05);the expression level of EphB1 mRNA in the H9c2 cells in negative control group had no change(P >0.05);compared with H 2O 2 group,the expression level of EphB1 mRNA in the H9c2 cells in si-EphB1 transfection group was significantly decreased(P <0.05).Compared with blank group,the survival rate of H9c2 cells in H 2O 2 group was decreased(P <0.05),the apoptotic rate was increased(P <0.05),the ROS and MDA levels were increased(P <0.05),and the SOD level was decreased(P <0.05);but the differences in the survival rate,apoptotic rate,ROS,MDA and SOD levels in negative control group were not significant(P >0.05).Compared with H 2O 2 group,the survival rate of H9c2 cells in si-EphB1 transfection group was increased(P <0.05),the apoptotic rate was decreased(P <0.05),the ROS and MDA levels were decreased(P <0.05),and the SOD level was increased (P <0.05).Conclusion: Knockdown of EphB1 gene can significantly inhibit the H 2O 2-induced oxidative damage of the rat cardiomyocytes and protect against myocardial injury;its mechanism is related to improving the survival rate of cardiomyocytes,inhibiting the apoptosis,improving the antioxidant enzyme activity,and increasing the scavenging abilities of oxidative stress products in the cells.