活性氧在实验性视网膜脱离后坏死性凋亡中的表达及与RIP-1的关系

ROS expression and its relationship with RIP-1 in photoreceptor necroptosis after experimental retinal detachment

目的:观察实验性视网膜脱离后活性氧(ROS)和受体相互作用蛋白1(RIP-1)表达以及z-VAD和Necrostatin-1干预下对其表达的影响。方法:健康雄性SD大鼠首先分为正常对照组和单纯视网膜脱离组观察视网膜脱离不同时间点ROS和RIP-1的表达情况,借此选择最佳时间点为药物干预做准备。再将大鼠分为无视网膜脱离组(attached)、单纯视网膜脱离组(untreated)、z-VAD-FMK组、z-VAD-FMK+Necrostatin-1组,观察z-VAD和Necrostatin-1干预下对ROS表达的影响。通过视网膜下注入透明质酸钠构建视网膜脱离模型。利用蛋白质免疫印迹法(western blot)测出内部RIP-1蛋白表达情况,活性氧检测试剂盒检测各组内视网膜ROS表达情况。结果:与attached组相比,实验性视网膜脱离后RIP-1、ROS表达增加,并且均于视网膜脱离后第3天达峰值,d3组与其他组相比,差异有统计学意义(P<0.05)。药物干预实验结果显示z-VAD-FMK诱导的坏死性凋亡组ROS表达最高,而Necrostatin-1干预后,与untreated组及z-VAD-FMK组相比,ROS表达降低(P<0.05)。结论:ROS作为RIP-1的下游参与了光感受器细胞的坏死性凋亡,Necrostatin-1可以通过降低ROS的水平,对视网膜脱离后的视网膜起到一定程度的保护作用。

Objective: To observe reactive oxygen species (ROS) expression and receptor interacting protein 1 (RIP-1) in phhotoreceptor necroptosis after experimental retinal detachment as well as the effect of z-VAD-FMK and necrostatin-1 on ROS expression. Methods :Healthy male Sprague-Dawley rats were initially divided into control group (attached) and retinal detachment group to observe the expression of ROS and RIP-1 at different time points by which to determine optimal time points for drug intervention.Then the rats were subgrouped into attached,untreated,z-VAD-FMK and z-VAD-FMK+necrostatin-1 to observe ROS expression following z-VAD-FMK and necrostatin-1 intervention.Retinal detachment model was established by injection of sodium hyaluronate beneath retina.Western blot was performed to detect the expression of RIP-1 protein,and reactive oxygen species detection kit was used to measure ROS level. Results :Expression of RIP-1 and ROS was significantly increased,and peaked on the third day after retinal detachment as compared with attached group.The difference was also significant between d3 group and other groups ( P< 0.05).The results by tentative drug intervention showed that ROS was highly expressed in z-VAD-FMK group,whereas significantly decreased after necrostatin-1 treatment compared with the untreated group and z-VAD -FMK group ( P< 0.05). Conclusion :After experimental retinal detachment,ROS possibly participates in photoreceptor necroptosis and acts as downstream of RIP-1.Necrostatin-1 could protect the retina to a certain extent by reducing ROS level.