白藜芦醇通过TFEB调节细胞自噬的机制研究

THE AUTOPHAGY INDUCED EFFECT OF RESVERATROL THROUGH TRANSCRIPTIONAL FACTOR TFEB

目的研究白藜芦醇对HeLa细胞自噬的影响,探讨白藜芦醇通过转录因子TFEB调节自噬的可能机制。方法用0、12.5、25、50、100μmol/L白藜芦醇处理HeLa细胞后,CCK-8法检测细胞活性;Western blot检测凋亡相关蛋白Caspase-3、自噬相关蛋白LC3-Ⅱ和TFEB总蛋白水平以及TFEB在核提取液中的水平;荧光显微镜观察GFP-LC3融合蛋白在细胞内分布;荧光定量PCR检测TFEB及其靶基因mRNA水平。结果白藜芦醇处理24 h后HeLa细胞活性降低,激活型Caspase-3蛋白水平增加。白藜芦醇处理增加细胞LC3-Ⅱ蛋白水平,在12h达到高峰,且呈剂量依赖性变化,同时GFP-LC3荧光在细胞内呈点状聚集。白藜芦醇处理增加总细胞和核提取液中TFEB蛋白水平,并增加TFEB自身mRNA及其靶基因mRNA水平。结论白藜芦醇可能通过TFEB在转录水平调节细胞自噬。

Objective To exmaine the effect of resveratrol on the autophagy of HeLa cell and the possible involvement of transcriptional factor TFEB in resveratrol induced-autophagy.Methods After treatment with 12.5,25,50 and 100μmol/L resveratrol,CCK-8 method was used to examine the survival of HeLa cells.Western blot was conducted to examine the protein levels of apoptois-related protein caspase-3,antophagy-related protein LC3-Ⅱand TFEB levels in whole cell lysate and in nuclear extract.Fluorescent microscopy was used to exmaine the intracellular distribution of GFP-LC3 fusion protein,and real-time quantitative PCR was used to measure the mRNA levels of TFEB and transcriptional target genes of TFEB.Results 24-hour treatment with resveratrol resulted in dicreased HeLa cell survival and increased protein levels of activated caspase-3.Resveratrol treatment increased protein levels of LC3-Ⅱ,which reached the peak at 12 hours,and the effect was dose-dependent.Meanwhile,resveratrol treatment caused a dot-distribution of GFP-LC3 fluorescence.Furthermore,resveratrol treatment increased TFEB protein levels in both whole cell lysate and nuclear extract and increased mRNA levels of TFEB itself as well as TFEB target genes.Conclusion Resveratrol may regulate autophagy at transcriptioanl levels through transcriptional factor TFEB.