线粒体靶向肽SS31及RIP抑制剂Necrostatin-1(Nec-1)对H2O2诱导的氧化应激损伤ARPE-19细胞的保护作用  

Protective effect of mitochondrial targeted peptide SS31 and the RIP1 inhibitor necrostatin-1(Nec-1)on ARPE-19 cells after oxidative stress injury induced by H2O2

作　　者：许雲 陈泽君 权卓娅 张瑞雪 何蓓蕾 何媛[1] XU Yun;CHEN Zejun;QUAN Zhuoya;ZHANG Ruixue;HE Beilei;HE Yuan(Department of Ophthalmology,the Second Affiliated Hospital of Xi’an Medical University,Xi’an 710038,Shaanxi Province,China;Xi’an Medical University,Xi’an 710068,Shaanxi Province,China)

机构地区：[1]西安医学院第二附属医院眼科,陕西省西安市710038 [2]西安医学院,陕西省西安市710021

出　　处：《眼科新进展》2020年第7期607-611,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号81100665,81770929);陕西省教育厅2018年服务地方科学研究计划资助(编号18JC026);陕西省科技厅项目资助(编号2019SF-162)。

摘　　要：目的探讨线粒体靶向肽SS31及RIP抑制剂Necrostatin-1(Nec-1)对H2O2诱导的氧化应激损伤ARPE-19细胞的保护作用。方法选择400μmol·L-1 H2O2构建氧化应激损伤模型;根据MTT结果筛选出1μmol·L-1 SS31、40μmol·L-1 Nec-1作为实验最佳浓度。将培养的细胞分为对照组、SS31+Nec-1组、H2O2组、SS31+H2O2组、SS31+Nec-1+H2O2组、Nec-1+H2O2组。利用MTT测定细胞存活率,双氯荧光素染色测定细胞活性氧自由基(ROS)水平,JC-1染色测定线粒体膜电位,AnnexinV/PI双染检测细胞PI阳性率,Western blot法检测RIP3蛋白表达水平。结果与对照组相比,H2O2组细胞存活率明显降低(P<0.05)。SS31+H2O2组、Nec-1+H2O2组细胞存活率高于H2O2组,差异均有统计学意义(均为P<0.05)。荧光显微镜下双氯荧光素染色结果显示,与H2O2组相比,SS31+H2O2组、Nec-1+H2O2组绿色荧光明显减弱,细胞内ROS含量明显减少。流式细胞仪结果显示与对照组相比,H2O2组线粒体膜电位降低的细胞比例升高,差异有统计学意义(P<0.05);与H2O2组相比,SS31+H2O2组、Nec-1+H2O2组线粒体膜电位降低的细胞比例降低,差异均有统计学意义(均为P<0.05)。AnnexinV/PI双染色结果显示与对照组相比,H2O2组PI阳性率增加;与H2O2组相比,SS31+H2O2组、Nec-1+H2O2组PI阳性率均明显降低,差异均有统计学意义(均为P<0.05)。Western blot检测结果显示,与对照组相比,H2O2组RIP3蛋白表达水平明显升高,差异有统计学意义(P<0.05);与H2O2组相比,SS31+H2O2组、Nec-1+H2O2组RIP3蛋白表达上调作用显著减弱,差异均有统计学意义(均为P<0.05)。结论SS31及Nec-1对H2O2诱导的ARPE-19细胞氧化应激损伤具有明显的保护作用。Objective To investigate the protective effect of mitochondrial targeted peptide SS31 and the RIP1 inhibitor necrostatin-1(Nec-1)on ARPE-19cells against oxidative stress injury induced by H2O2.Methods The cell model of oxidative damage was established by adding 400μmol·L-1 hydrogen peroxide(H2O2),According to the MTT results,1μmol·L-1 SS31 and 40μmol·L-1 Nec-1 were the optimal concentration for sequential study.The cells were divided into control group,H2O2 group,SS31+Nec-1 group,SS31+H2O2 group,SS31+Nec-1+H2O2 group,and Nec-1+H2O2 group.MTT assay was used to detect the survival rate of the cells,while the level of reactive oxygen species(ROS)was measured by dichlorodihydrofluorescin diacetate(DCFH-DA)staining.Mitochondrial membrane potential was measured using the JC-1 assay,and the PI positive rate was determined by flow cytometry after Annexin V/Propidium-dide(PI)staining.In addition,the protein levels of RIP3(an indicator of necroptosis)were determined by Western blot.Results The rate of cell viability was lower in the H2O2 group than in the control group(P<0.05).The viability of the cells improved following treatment with SS31 and Nec-1 compared with H2O2 group,and the differences were statistically significant(all P<0.05).DCFH-DA staining showed that green fluorescence cells were much more in the H2O2 group compared with SS31+H2O2 group and Nec-1+H2O2 group,suggesting that treatment with SS31 and Nec-1 significantly decreased the quantity of ROS.Flow cytometry analysis confirmed that the proportion of cells with decreased mitochondrial membrane potential in the H2O2 group was higher than those in the control group,SS31+H2O2 group and Nec-1+H2O2 group,and the difference was statistically significant(all P<0.05).AnnexinV/PI double staining results showed that the PI-positive rate of ARPE-19 cells in H2O2 group was significantly higher than that in the control group,SS31+H2O2 group and Nec-1+H2O2 group(all P<0.05).Western blot analysis showed that compared with the control group,the relative expression l

关 键 词：线粒体靶向肽SS31 氧化应激 ARPE-19细胞 程序性坏死 

分 类 号：R774[医药卫生—眼科]