DRP1调控线粒体稳态对人视网膜色素上皮细胞上皮-间充质转化的影响

Effect of dynamin-related protein 1 regulation of mitochondrial homeostasis on epithelial-to-mesenchymal transition in human retinal pigment epithelial cells

目的探讨线粒体动力相关蛋白(DRP1)对人视网膜色素上皮(ARPE-19)细胞上皮-间充质转化(EMT)进程的影响。方法构建H_(2)O_(2)干预ARPE-19细胞模型,将ARPE-19细胞分为3组,NG组:采用含体积分数10%胎牛血清的DMEM/F12培养基培养细胞6 h;H_(2)O_(2)组:先采用650μmol·L^(-1)H_(2)O_(2)干预细胞,此后培养方式及时间与NG组相同;H_(2)O_(2)+Mdivi-1组:先采用10μmol·L^(-1)Mdivi-1处理ARPE-19细胞2 h,再给予650μmol·L^(-1)H_(2)O_(2)干预,此后培养方式及时间与NG组相同。Western blot检测各组细胞p-DRP-1/DRP1、E-钙黏蛋白、N-钙黏蛋白、α-平滑肌肌动蛋白(α-SMA)、波型蛋白(Vimintin)及紧密连接蛋白(ZO-1)表达水平;线粒体红色荧光探针检测各组细胞线粒体形态;线粒体超氧化物红色荧光探针检测各组细胞线粒体活性氧(ROS)水平;JC-1染色试剂盒检测各组细胞线粒体膜电位;免疫荧光检测各组细胞中ZO-1表达水平。结果H_(2)O_(2)组细胞p-DRP-1/DRP1蛋白表达比值高于NG组,H_(2)O_(2)+Mdivi-1组细胞p-DRP-1/DRP1蛋白表达比值低于H_(2)O_(2)组,差异均有统计学意义(均为P<0.05)。H_(2)O_(2)+Mdivi-1组细胞较H_(2)O_(2)组线粒体碎片化程度得到改善。H_(2)O_(2)组细胞线粒体ROS水平(4.42±0.29)与NG组(1.00±0.17)及H_(2)O_(2)+Mdivi-1组(2.15±0.18)比较,差异均有统计学意义(均为P<0.05)。H_(2)O_(2)组细胞红/绿荧光强度比值(0.16±0.12)与NG组(1.00±0.09)及H_(2)O_(2)+Mdivi-1组(0.42±0.05)比较,差异均有统计学意义(均为P<0.05)。H_(2)O_(2)组细胞上皮样标志物表达下降,间质样标志物表达上升,H_(2)O_(2)+Mdivi-1组细胞上皮样标志物表达上升,间质样标志物表达下降。各组细胞α-SMA、N-钙黏蛋白、E-钙黏蛋白、Vimintin及ZO-1相对表达量比较,H_(2)O_(2)组与NG组及H_(2)O_(2)+Mdivi-1组比较,差异均有统计学意义(均为P<0.05)。ZO-1免疫荧光染色实验显示,H_(2)O_(2)+Mdivi-1组的细胞连接紧密程度优于H_(2)O_(2)组。结论DRP1可调控线粒体动态平衡,靶向DRP1可改善线粒体功能并抑制EMT进展,从而减轻H_(2)O_(2)诱导的RPE细胞功能障碍。

Objective To investigate the effect of dynamin-related protein 1(DRP1)on epithelial-to-mesenchymal transition(EMT)in human retinal pigment epithelial(ARPE-19)cells.Methods In this study,H_(2)O_(2)-induced ARPE-19 cell models were established.ARPE-19 cells were divided into three groups.NG group:ARPE-19 cells were cultured in DMEM/F12 medium containing 10%fetal bovine serum for 6 hours;H_(2)O_(2)group:ARPE-19 cells were first treated with 650μmol·L^(-1)H_(2)O_(2),and the subsequent culture method and time were the same as those of the NG group;H_(2)O_(2)+Mdivi-1 group:ARPE-19 cells were first treated with 10μmol·L^(-1)Mdivi-1 for 2 hours,followed by intervention with 650μmol·L^(-1)H_(2)O_(2),and afterwards,the culture method and time were the same as those of the NG group.For each group of cells,the expression levels of p-DRP-1/DRP1,E-cadherin,N-cadherin,α-smooth muscle actin(α-SMA),vimentin,and tight junction protein(ZO-1)were detected using Western blot;the mitochondrial morphology was evaluated using the mitochondrial red fluorescence probe;the level of mitochondrial reactive oxygen species(ROS)was measured using the mitochondrial superoxide red fluorescence probe;the mitochondrial membrane potential was detected using the JC-1 staining kit;and the expression level of ZO-1 was detected using the immunofluorescence.Results The ratio of p-DRP-1/DRP1 protein expression in the H_(2)O_(2)group was higher than that in the NG group,while the ratio of p-DRP-1/DRP1 protein expression in the H_(2)O_(2)+Mdivi-1 group was lower than that in the H_(2)O_(2)group,and the differences were statistically significant(both P<0.05).The mitochondrial fragmentation in the H_(2)O_(2)+Mdivi-1 group improved compared to the H_(2)O_(2)group.The level of mitochondrial ROS in the H_(2)O_(2)group(4.42±0.29)was significantly higher than that in the NG group(1.00±0.17)and the H_(2)O_(2)+Mdivi-1 group(2.15±0.18)(both P<0.05).The red/green fluorescence intensity ratio in the H_(2)O_(2)group(0.16±0.12)was significantly lower than that in the NG group(1.00±0.09)and the H_(2)O_(2)+Mdivi-1 group(0.42±0.05)(both P<0.05).The expression of epithelial markers decreased and the expression of mesenchymal markers increased in the H_(2)O_(2)group,while the expression of epithelial markers increased and the expression of mesenchymal markers decreased in the H_(2)O_(2)+Mdivi-1 group.There were significant differences in the relative expression levels ofα-SMA,N-cadherin,E-cadherin,vimentin,and ZO-1 proteins between the H_(2)O_(2)group and the NG group,H_(2)O_(2)+Mdivi-1 group(all P<0.05).The ZO-1 immunofluorescence staining assay showed that the cell connection in the H_(2)O_(2)+Mdivi-1 group was tighter than that in the H_(2)O_(2)group.Conclusion DRP1 can regulate mitochondrial homeostasis,and targeting DRP1 can improve mitochondrial function and inhibit EMT progression,thereby alleviating H_(2)O_(2)-induced RPE cell dysfunction.