去乙酰化酶3在中波紫外线诱导视网膜色素上皮细胞凋亡中的保护作用及机制研究  

Protective effect of SIRT3 on UVB induced apoptosis of retinal pigment epithelial cells and its mechanism

作　　者：刘思维 张勇 齐佳 袁均 LIU Si-wei;ZHANG Yong;QI Jia;YUAN Jun(Department of Ophthalmology,Shiyan Taihe Hospital,Affiliated Hospital of Hubei Medical College,Shiyan,Hubei 442000,China)

机构地区：[1]湖北医药学院附属医院太和医院眼科,湖北十堰442000

出　　处：《环境与健康杂志》2024年第11期948-953,F0003,共7页Journal of Environment and Health

基　　金：湖北省科技厅科技计划项目(202201AY070001-219)。

摘　　要：目的探讨去乙酰化酶3(sirtuins 3,SIRT3)在中波紫外线(ultraviolet B,UVB)诱导的人视网膜色素上皮细胞凋亡中的影响及作用机制。方法将人视网膜色素上皮-19(adult retinal pigment epithelial cell line-19,ARPE-19)细胞分别在0(对照)、10、20、30、40、50、60 mJ/cm2强度UVB下照射12 h、24 h,采用4-甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法测定细胞活性,采用蛋白免疫印迹法和实时荧光定量PCR(quantitative PCR,qPCR)法分别测定SIRT3蛋白和mRNA的表达水平。将ARPE-19细胞在无血清OPTI-MEM中与空载体+siRNA对照序列(阴性+UVB组)、SIRT3特异性pBABEPuro载体(SIRT3+UVB组)或SIRT3特异性小干扰RNA序列(20 nmol/L siSIRT3+UVB组)孵育,同时加入Lipofectamine RNAiMAX转染试剂,培养24 h后,将阴性+UVB组、siSIRT3+UVB组、SIRT3+UVB组细胞用50 mJ/cm2UVB照射12 h后,继续培养24 h;未经转染处理,但同样经UVB照射的细胞作为UVB组,而未经转染和UVB照射处理的细胞作为空白组。测定细胞中SIRT3蛋白和mRNA的表达水平,采用流式细胞术检测细胞活性氧(reactive oxygen species,ROS)水平和细胞凋亡;采用CaTM-2/AM探针和Rhod-2/AM探针检测细胞内和线粒体内钙离子(calcium ion,Ca^(2+))浓度。采用RNA测序和基因集富集分析(gene set enrichment analysis,GSEA)确定受SIRT3影响的途径。结果与对照组相比,不同照射强度UVB下ARPE-19细胞处理12 h和24 h后,细胞存活率均下降,差异有统计学意义(P<0.05);且随着UVB照射强度的升高,ARPE-19细胞的存活率呈下降趋势。与对照组相比,不同照射强度UVB下ARPE-19细胞处理12 h后,细胞SIRT3mRNA和蛋白表达水平均下降,除10 mJ/cm2UVB组SIRT3蛋白外,差异均有统计学意义(P<0.05);且随着UVB照射强度的升高,ARPE-19细胞SIRT3 mRNA和蛋白表达水平均呈下降趋势。与空白组相比,UVB组ARPE-19细胞凋亡率和ROS生成量均增加,差异有统计学意义(P<0.05);然而阴性+UVB组细胞凋亡率和ROS生成量与UVB组Objective To understand the effect and mechanism of deacetylase 3(SIRT3)on UVB induced apoptosis of human retinal pigment epithelial cells.Methods Human retinal pigment epithelium-19(ARPE-19)cells cultured in vitro were divided into blank group,UVB group,negative+UVB group,siSIRT3+UVB group and SIRT3+UVB group.Except the blank group,the cells in UVB exposed groups were treated with UVB through ultraviolet lamp,and the expression level of SIRT3 in the cells of each group was determined.The cell viability was measured by methyl thiazolyl tetrazolium(MTT).The level of reactive oxygen species(ROS)and apoptosis were detected by flow cytometry.CaTM-2/AM probe and Rhod-2/AM probe were used to detect intracellular and mitochondrial calcium(Ca^(2+))concentrations.SIRT3 mRNA and protein expression were detected by western blot and real-time quantitative fluorescent PCR(qPCR),respectively.RNA sequencing and gene set enrichment analysis(GSEA)were used to identify the pathways affected by SIRT3.Results After UVB irradiation,the survival rate of ARPE-19 cells was significantly decreased in a dose-dependent manner,and the mRNA and protein expression levels of SIRT3 were significantly down-regulated(P<0.05).Compared with blank group,the apoptosis rate and ROS production in UVB group were significantly increased,and the concentration of Ca^(2+)in cytoplasm and mitochondria was significantly increased(P<0.05).There was no significant change in negative+UVB group(P<0.05).Compared with the negative+UVB group,the apoptosis rate,endogenous ROS production,cytoplasmic and mitochondrial Ca^(2+)concentration in siSIRT3+UVB group were further increased.SIRT3+UVB group effectively reversed the above reaction(P<0.05).GESA showed that genes related to retinal morphological maintenance and JAK-STAT cascade pathway in AMD were enriched in ARPE-19 cells over expressing SIRT3.Conclusion UVB can induce the down-regulation of SIRT3 expression in ARPE-19 cells.SIRT3 over expression can protect ARPE-19 cells from UVB-induced damage through antioxidan

关 键 词：视网膜色素上皮 去乙酰化酶3 年龄相关性黄斑变性 钙超载 氧化应激 

分 类 号：R994.6[医药卫生—毒理学]