NgR介导的氧化应激反应在葡萄糖诱导RGC凋亡中的作用

Role of NgR mediated oxidative stress in glucose induced RGC apoptosis

目的:探讨NgR介导的氧化应激在葡萄糖诱导视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡中的作用。方法:RGC-5细胞株分3组培养:正常对照组(DMEM高糖培养基+100m L/L胎牛血清),高糖组(DMEM高糖培养基+100m L/L胎牛血清+30mmol/L葡萄糖)、NEP1-40组(DMEM高糖培养基+100m L/L胎牛血清+30mmol/L葡萄糖+1μmol/L NEP1-40)。各组RGC-5细胞培养3d后检测各指标:显微镜观察细胞生长状态,cell counting KIT-8(CCK-8)试剂盒检测细胞活力,Annexin V/PI双染流式细胞仪(flow cytometry,FCM)检测RGC细胞凋亡率,试剂盒检测线粒体内ROS含量的变化、胞内MDA水平及SOD活力,Western-blot检测凋亡蛋白Bcl-2和Bax的表达。结果:与正常对照组相比,高糖组细胞生长状态差、活力下降、凋亡率显著增加,ROS及MDA水平显著升高,SOD活力下降,抗凋亡蛋白Bcl-2表达降低,促凋亡蛋白Bax表达上调,Bcl-2/Bax蛋白比值降低,差异有统计学意义(P<0.05)。与高糖组相比,NEP1-40组细胞抑制NgR表达后,氧化应激反应减弱,Bcl-2/Bax提高,细胞状态改善、细胞活力提高、凋亡率降低,差异有统计学意义(P<0.05)。结论:高浓度葡萄糖能通过NgR介导氧化应激反应诱导RGC-5细胞凋亡。

AIM：To investigate the effect of NgR mediated oxidative stress on the apoptosis of retinal ganglion cells（RGCs） induced by glucose.METHODS：RGC-5 cell were divided into 3 groups：control group（DMEM high glucose medium＋10%fetal calf serum）,high glucose group（DMEM high glucose medium＋ 10%fetal calf serum ＋30mmol/L glucose）,NEP1-40 group（DMEM high glucose medium ＋10%fetal calf serum＋30mmol/L glucose＋1μmol/L NEP1-40）.Detections were performed after 3d culture：the state of cell growth was observed by microscopy.Cell viability was detected by CCK-8 kit.The apoptosis rate of RGC cells was detected by flow cytometry（FCM）.The intensity of ROS of the cells were detected by fluorescence microscopy.Intracellular MDA levels and SOD activity were measured by related kits.Western blot was used to detect the expressions of Bcl-2 and Bax proteins.· RESULTS：Compared with control group,high glucose group had a poor state and cell viability decreased,cell apoptosis rate significantly increased,ROS and MDA levels were significantly enhanced,SOD activity decreased,and the expression of anti- apoptotic protein Bcl-2 was decreased and the expression of pro apoptotic protein Bax was up-regulated.Compared with glucose group,after NgR expression was inhibited by NEP1-40,the oxidative stress reaction was reduced,Bcl-2/Bax was increased,the cell status was improved,the cell viability was increased,and the apoptosis rate was decreased in the NEP1-40group（P〈0.05）.CONCLUSION：High concentration of glucose can induce apoptosis of RGC- 5 cells by NgR mediated oxidative stress reaction.