Calcium Overload Is A Critical Step in Programmed Necrosis of ARPE-19 Cells Induced by High-Concentration H2O2
Calcium Overload Is A Critical Step in Programmed Necrosis of ARPE-19 Cells Induced by High-Concentration H2O2
复制标题
钙超载是高浓度 H2O2 诱导的 ARPE-19 细胞程序性坏死的关键步骤。
DOI：
10.1016/s0895-3988(10)60078-5
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发表时间：
2010-10-01
影响因子：
3.5
通讯作者：
Zheng, Yong-Chen
中科院分区：
文献类型：
--
作者：
Li, Guang-Yu;Fan, Bin;Zheng, Yong-Chen
目的氧化应激在视网膜色素上皮（RPE）老化死亡和老年性黄斑变性的发生发展中起重要作用。尽管早期的报道表明包括H2 O2在内的活性氧（reactive oxygen species，ROS）在较低浓度下可引发细胞凋亡，在较高浓度下可引发细胞坏死，但RPE死亡的确切分子机制仍不清楚。本研究的目的是探讨外源性活性氧（特别是高浓度活性氧）诱导RPE死亡的分子途径。方法用不同浓度的H2 O2处理培养的ARPE-19细胞，MTT法检测细胞活力。通过显微镜使用APOPercentage测定和PI染色对细胞死亡进行形态学研究。此外，氧化应激对ARPE-19细胞的影响通过HO-1和PARP-1蛋白质印迹法以及抗氧化剂EGCG的保护来评估。使用Fura-2钙指示剂测定钙内流，并在钴处理以阻断钙效应后评价细胞内钙超载在ARPE-19细胞死亡中的作用。结果H2 O2浓度依赖性地降低ARPE-19细胞的存活率，呈典型的S形曲线。高浓度H2 O2引起的细胞死亡被证实为程序性坏死。形态学上，死亡的ARPE-19细胞极度肿胀，失去了其质膜的完整性，APO百分比测定和PI染色阳性。24-500 μ mol/L H2 O2处理ARPE-19细胞1h，HO-1和PARP-1表达明显上调。此外，使用EGCG的抗氧化剂处理有效地保护细胞免受H2 O2诱导的损伤，使细胞活力从14.17%+/- 2.31%增加到85.77%+/-4.58%。H2 O2处理后，细胞内钙水平高度升高，最大浓度为1200 nM。值得注意的是，钙通道抑制剂钴能够钝化这种钙内流并阻断坏死途径，将ARPE-19细胞从H2 O2诱导的死亡中拯救出来。结论高浓度H2 O2诱导ARPE-19细胞死亡是通过一个受调节的坏死途径，其中钙超载是细胞死亡程序中的关键步骤。
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 mu 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-induced death. Conclusions At high concentrations, H2O2 induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.