Objective Oxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration.Although early reports indicate that reactive oxygen species (ROS) including H2O2 can trigger apoptosis at lower concentrations and necrosis at higher concentrations,the exact molecular mechanism of RPE death is still unclear.The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS,especially at higher concentrations.Methods Cultured ARPE-19 cells were treated with H2O2 at different concentrations and cell viability was measured with the MTT assay.Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining.Furthermore,the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG.Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.Results H2O2 reduced the viability of ARPE-19 cells in a concentration-dependent manner,which was presented as a typical s-shaped curve.Cell death caused by high concentrations of H2O2 was confirmed to be programmed necrosis.Morphologically,dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane,positively detected with APOPercentage assay and PI staining.24-hour treatment with 500 ?mol/L H2O2 induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells.Moreover,antioxidant treatment using EGCG effectively protected cells from H2O2-induced injury,increasing cell viability from 14.17%±2.31% to 85.77%±4.58%.After H2O2 treatment,intracellular calcium levels were highly elevated with a maximum concentration of 1200nM.Significantly,the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway,rescuing the ARPE-19 cell from H2O2-in
目的研究核酶PARP-1在光诱导的视网膜神经节细胞(RGCs)凋亡中的作用。方法应用1000 Lux光诱导视网膜神经节细胞-5(RGC-5)光损伤模型;利用MTT、APOPercentageTM实验及原位TUNEL确定光诱导RGC-5细胞死亡的模式;通过半定量RT-PCR和Western blot方法评估光对RGC-5细胞中核酶PARP-1表达的影响;探讨PARP-1抑制剂尼克酰胺和NU1025对光损伤中RGC-5细胞的保护作用。结果1000 Lux光以时间依赖的方式降低体外培养的RGC-5细胞活性,光暴露4d明显诱导RGC-5细胞凋亡。RGC-5细胞光损伤导致核酶PARP-1转录和表达的上调(F=224.59,P<0.01,n=12,ANOVA,Dunnett t test),PARP-1抑制剂尼克酰胺和NU1025显著提高细胞活性(F=312.87,P<0.01,n=12,ANOVA,Dunnett t test)。结论1000 Lux光可以诱导体外培养的RGC-5细胞凋亡,核酶PARP-1在该细胞凋亡分子机制中起重要作用。
