[1]汤中,唐云骢.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[J].眼科新进展,2024,44(6):443-448.[doi:10.13389/j.cnki.rao.2024.0086]
 TANG Zhong,TANG Yuncong.Effect of dynamin-related protein 1 regulation of mitochondrial homeostasis on epithelial-to-mesenchymal transition in human retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2024,44(6):443-448.[doi:10.13389/j.cnki.rao.2024.0086]
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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/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
44卷
期数:
2024年6期
页码:
443-448
栏目:
实验研究
出版日期:
2024-06-03

文章信息/Info

Title:
Effect of dynamin-related protein 1 regulation of mitochondrial homeostasis on epithelial-to-mesenchymal transition in human retinal pigment epithelial cells
作者:
汤中唐云骢
425000 湖南省永州市,永州职业技术学院附属医院眼科
Author(s):
TANG ZhongTANG Yuncong
Department of Ophthalmology,the Affiliated Hospital of Yongzhou Vocational Technical College,Yongzhou 425000,Hunan Province,China
关键词:
线粒体动力相关蛋白线粒体功能人视网膜色素上皮细胞上皮-间充质转化年龄相关性黄斑变性
Keywords:
mitochondrial dynamin-related protein 1 mitochondrial function human retinal pigment epithelial cells epithelial-to-mesenchymal transition age-related macular degeneration
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2024.0086
文献标志码:
A
摘要:
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H2O2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;H2O2组:先采用650 μmol·L-1 H2O2干预细胞,此后培养方式及时间与NG组相同;H2O2+Mdivi-1组:先采用10 μmol·L-1 Mdivi-1处理ARPE-19细胞2 h,再给予650 μmol·L-1 H2O2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 H2O2组细胞p-DRP-1/DRP1蛋白表达比值高于NG组,H2O2+Mdivi-1组细胞p-DRP-1/DRP1蛋白表达比值低于H2O2组,差异均有统计学意义(均为P<0.05)。H2O2+Mdivi-1组细胞较H2O2组线粒体碎片化程度得到改善。H2O2组细胞线粒体ROS水平(4.42±0.29)与NG组(1.00±0.17)及H2O2+Mdivi-1组(2.15±0.18)比较,差异均有统计学意义(均为P<0.05)。H2O2组细胞红/绿荧光强度比值(0.16±0.12)与NG组(1.00±0.09)及H2O2+Mdivi-1组(0.42±0.05)比较,差异均有统计学意义(均为P<0.05)。H2O2组细胞上皮样标志物表达下降,间质样标志物表达上升,H2O2+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,H2O2组与NG组及H2O2+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,H2O2+Mdivi-1组的细胞连接紧密程度优于H2O2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H2O2诱导的RPE细胞功能障碍。
Abstract:
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, H2O2-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; H2O2 group: ARPE-19 cells were first treated with 650 μmol·L-1 H2O2, and the subsequent culture method and time were the same as those of the NG group; H2O2+Mdivi-1 group: ARPE-19 cells were first treated with 10 μmol·L-1 Mdivi-1 for 2 hours, followed by intervention with 650 μmol·L-1 H2O2, 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 H2O2 group was higher than that in the NG group, while the ratio of p-DRP-1/DRP1 protein expression in the H2O2+Mdivi-1 group was lower than that in the H2O2 group, and the differences were statistically significant (both P<0.05). The mitochondrial fragmentation in the H2O2+Mdivi-1 group improved compared to the H2O2 group. The level of mitochondrial ROS in the H2O2 group (4.42±0.29) was significantly higher than that in the NG group (1.00±0.17) and the H2O2+Mdivi-1 group (2.15±0.18) (both P<0.05). The red/green fluorescence intensity ratio in the H2O2 group (0.16±0.12) was significantly lower than that in the NG group (1.00±0.09) and the H2O2+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 H2O2 group, while the expression of epithelial markers increased and the expression of mesenchymal markers decreased in the H2O2+Mdivi-1 group. There were significant differences in the relative expression levels of α-SMA, N-cadherin, E-cadherin, vimentin, and ZO-1 proteins between the H2O2 group and the NG group, H2O2+Mdivi-1 group (all P<0.05). The ZO-1 immunofluorescence staining assay showed that the cell connection in the H2O2+Mdivi-1 group was tighter than that in the H2O2 group.
Conclusion DRP1 can regulate mitochondrial homeostasis, and targeting DRP1 can improve mitochondrial function and inhibit EMT progression, thereby alleviating H2O2-induced RPE cell dysfunction.

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备注/Memo

备注/Memo:
永州市科技计划项目(编号:2023YZ076)
更新日期/Last Update: 2024-06-05