Protective effect of SIRT3 on UVB induced apoptosis of retinal pigment epithelial cells and its mechanism
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(Ca2+)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 Ca2+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 Ca2+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 antioxidant effect and maintenance of intracellular Ca2+homeostasis.
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