SB431542对高糖诱导的RPE细胞自噬及上皮-间充质转化影响的实验研究  

Effect of SB431542 on autophagy and epithelial mesenchymal transition in retinal pigment epithelial cells induced by high glucose

作　　者：曹靖靖 韩菲菲 寇振宇 庄彤彤 东莉洁[1] 张红 滕贺 Cao Jingjing;Han Feifei;Kou Zhenyu;Zhuang Tongtong;Dong Lijie;Zhang Hong;Teng He(Tianjin Medical University Eye Hospital,Eye Institute and School of Optometry,Tianjin Branch of National Clinical Research Center for Ocular Disease,Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院、天津医科大学眼视光学院、天津医科大学眼科研究所、国家眼耳鼻喉疾病临床医学研究中心天津市分中心、天津市视网膜功能与疾病重点实验室,天津300384

出　　处：《中华眼科杂志》2025年第3期202-210,共9页Chinese Journal of Ophthalmology

基　　金：天津市教委科研计划项目(2024ZD033)。

摘　　要：目的探究转化生长因子(TGF)-β受体抑制剂SB431542在高糖状态下对视网膜色素上皮(RPE)细胞自噬及视网膜上皮-间充质转化(EMT)形成的影响。方法实验研究。RPE细胞分为正常对照组(N组), 甘露醇组(M组), 高糖组(HG组), 高糖联合二甲基亚砜(DMSO)处理组(HG+DMSO组)和高糖联合SB431542处理组(HG+SB431542组)。噻唑蓝(MTT)实验检测细胞增殖;细胞划痕实验检测细胞迁移情况;鬼笔环肽染色检测纤维状肌动蛋白;实时荧光定量PCR(qPCR)和细胞免疫荧光(IF)检测EMT相关蛋白波形蛋白和上皮型钙黏附蛋白的表达;细胞自噬染色检测试剂盒检测自噬体的表达, Western印迹、qPCR和IF检测重组人自噬效应蛋白(Beclin1)的表达。采用单因素方差分析和 LSD-t检验进行统计分析。结果 MTT实验显示, HG+DMSO组和HG+SB431542组的细胞光密度值分别为2.02±0.10和1.35±0.04(t=15.39, P<0.001);细胞划痕实验显示, HG+DMSO组和HG+SB431542组细胞的迁移率分别为58.33%±2.07% 和28.17%±1.94%(t=26.07, P<0.001);鬼笔环肽染色显示, HG+DMSO组细胞呈梭形样改变, 而HG+SB431542组细胞可恢复正常的多边形结构;qPCR和IF结果显示, HG+DMSO组和HG+SB431542组的波形蛋白mRNA水平分别为1.03±0.04和0.93±0.05, 荧光强度分别为(61 828±760)和(46 680±671)AU(任意单位)(tqPCR=3.85, P=0.003;tIF=36.62, P<0.001);而上皮型钙黏附蛋白mRNA水平分别为0.86±0.03和1.00±0.04, 荧光强度分别为(38 637±880)和(54 988±1 264)AU(tqPCR=8.89, P<0.001;tIF=26.01, P<0.001);细胞自噬染色显示HG+DMSO组和HG+SB431542组自噬体的荧光强度分别为(22.75±1.39)和(33.21±1.95)AU(t=10.70, P<0.001);Western印迹、qPCR和IF结果显示, HG+DMSO组和HG+SB431542组Beclin1的蛋白水平分别为0.38±0.04和0.75±0.08, mRNA水平分别为0.77±0.08和 1.05±0.05, 荧光强度分别为荧光强度分别为(42 639±1 713)和(49 027±1 024)AU(tWB=9.51, P<0.001;tqPCR=6.90, P<0.001;tIF=7.84, P<0.001)。结论 SB431542可抑制高Objective:Exploring the effect of transforming growth factorβreceptor inhibitor SB431542 on autophagy and the formation of retinal epithelial mesenchymal transition(EMT)in retinal pigment epithelial cells under high glucose conditions.Methods:This study was an experimental research.RPE cells were divided into normal group(N group),mannitol group(M group),high glucose group(HG group),high glucose combined with DMSO group(HG+DMSO group)and high glucose combined with SB431542 group(HG+SB431542 group).MTT assay was used to detect cell proliferation.Cell scratch assay was used to detect cell migration.Phalloidin staining was used to detect Fibrotic actin,real-time quantitative PCR(qPCR)and immunofluorescence were used to detect the expression of EMT related proteins Vimentin and E-cadherin.The cell autophagy staining kit detects the expression of autophagosomes.Western blotting(WB),qPCR,and immunofluorescence were used to detect the expression of Beclin1.One-way ANOVA and LSD-t test were used for statistical analysis.Results:The MTT assay showed that the cell optical density values of HG+DMSO group and HG+SB431542 group were 2.02±0.10 and 1.35±0.04,respectively(t=15.39,P<0.001);The results of cell scratch assay showed that the migration rates of cells in HG+DMSO group and HG+SB431542 group were 58.33%±2.07%and 28.17%±1.94%,respectively(t=26.07,P<0.001);Ghost pen cyclic peptide staining revealed spindle shaped changes in HG+DMSO group cells,while HG+SB431542 group cells were able to restore their normal polygonal structure;The qPCR and cell immunofluorescence results showed that the mRNA levels of vimentin in the HG+DMSO group and HG+SB431542 group were 1.03±0.04 and 0.93±0.05,respectively,with fluorescence intensities of(61828±760)and 46680±671 AU(tqPCR=3.85,P=0.003;tIF=36.62,P<0.001);The mRNA levels of epithelial calcium adhesion protein were 0.86±0.03 and 1.00±0.04,respectively,with fluorescence intensities of(38637±880)and(54988±1264)AU(tqPCR=8.89,P<0.001;tIF=26.01,P<0.001);Autophagy staining showed th

关 键 词：苯甲酰胺类 二恶茂类 视网膜色素上皮 上皮细胞 自噬 上皮-间质转化 

分 类 号：R73[医药卫生—肿瘤]