Dong Shu-Qian, Xu Hui-Zhuo, Xia Xiao-Bo, Wang Sha, Zhang Li-Xin, Liu Shuang-Zhen
Department of Ophthalmology, Xiangya Hospital of Central South University, Changsha 410006, Hunan Province, China.
Int J Ophthalmol. 2012;5(2):138-42. doi: 10.3980/j.issn.2222-3959.2012.02.04. Epub 2012 Apr 18.
To evaluate the influence of hydrogen peroxide (H(2)O(2)) on mouse photoreceptor-derived 661W cell survival and to determine the effect of PD98059, an inhibitor for MEK1 (the direct upstream activator of ERK1/2), and S3I201, a STAT3- specific inhibitor on 661W cell survival after H(2)O(2) exposure.
The mouse photoreceptor-derived 661W cells were cultured. 661W cells were treated for 12 hours with different concentrations (0, 0.25, 0.50, 0.75, 1mmol/L) of H(2)O(2) and cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ) (MTT) assay. 661W cells were treated with different concentrations H(2)O(2) (0, 5, 10, 50, 500, 1000 µmol/L) for 15 minutes or 1mmol/L H(2)O(2) for different time points (0,5,10,15,30 minutes), and p-Tyr705-STAT3, STAT3, Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2 were surveyed by immunoblot analysis. After treatment with 50µmol/L PD98059, or S3I201 for 1 hour, the inhibition efficiency of cell signal pathways was analyzed by immunoblot analysis and the effects of inhibitors on cell viability were determined by MTT.
After treating with different concentrations of H(2)O(2) for 12 hours, the cell viability of 661W cells decreased in concentration-dependent manner (P<0.05). Moreover, H(2)O(2) induced phosphorylation of ERK1/2 and STAT3 in 661W cells (P<0.05). After pretreatment with 50µmol/L PD98059 or S3I201 for 1 hour, H(2)O(2)-induced phosphorylation of ERK1/2 or STAT3 was suppressed separately (P<0.05). Using PD98059 or S3I201 to inhibit ERK1/2 or STAT3 signal pathway, the cell viability of 661W cells decreased significantly (P<0.05).
We demonstrated that the exposure of 661W cells to H(2)O(2) increased the activation of ERK1/2 and STAT3 signal pathways. Activation of these pathways is required for 661W cell survival following oxidant injury.
评估过氧化氢（H₂O₂）对小鼠光感受器来源的661W细胞存活的影响，并确定MEK1（ERK1/2的直接上游激活剂）抑制剂PD98059和STAT3特异性抑制剂S3I201对H₂O₂暴露后661W细胞存活的影响。
培养小鼠光感受器来源的661W细胞。用不同浓度（0、0.25、0.50、0.75、1mmol/L）的H₂O₂处理661W细胞12小时，并用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）法测定细胞活力。用不同浓度的H₂O₂（0、5、10、50、500、1000µmol/L）处理661W细胞15分钟或用1mmol/L H₂O₂处理不同时间点（0、5、10、15、30分钟），通过免疫印迹分析检测p-Tyr705-STAT3、STAT3、磷酸化-p44/42 MAPK（Thr202/Tyr204）、ERK1/2。用50µmol/L PD98059或S3I201处理1小时后，通过免疫印迹分析分析细胞信号通路的抑制效率，并用MTT法测定抑制剂对细胞活力的影响。
用不同浓度的H₂O₂处理12小时后，661W细胞的细胞活力呈浓度依赖性下降（P<0.05）。此外，H₂O₂诱导661W细胞中ERK1/2和STAT3的磷酸化（P<0.05）。用50µmol/L PD98059或S3I201预处理1小时后，H₂O₂诱导的ERK1/2或STAT3磷酸化分别受到抑制（P<0.05）。使用PD98059或S3I201抑制ERK1/2或STAT3信号通路，661W细胞的细胞活力显著下降（P<0.05）。
我们证明661W细胞暴露于H₂O₂会增加ERK1/2和STAT3信号通路的激活。这些通路的激活是氧化损伤后661W细胞存活所必需的。
