从自噬角度研究雷公藤甲素引起HepG2细胞肝毒性的机制  

Triptolide-induced HepG2 Cells Hepatotoxicity Through Autophagy

作　　者：刘春晖[1] 周玲玲[1] 马可迅[1] 王婧[1] 冯哲[1] 周学平[1] LIU Chun-hui ZHOU Ling-ling MA Ke-xun WANG Jing FENG Zhe ZHOU Xue-ping(Nanjing University of Chinese Medicine, Nanjing 210023, China)

机构地区：[1]南京中医药大学,南京210023

出　　处：《中国实验方剂学杂志》2017年第10期99-103,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81573869;81673937)

摘　　要：目的:探讨雷公藤甲素(TP)对HepG2细胞的过氧化损伤及对自噬的调控作用。方法:将质量浓度为0.003 2,0.016,0.08,0.4,2 g·L^(-1)的TP作用于体外培养的HepG2细胞48 h,另设空白组做对比,采用噻唑蓝(MTT)法检测细胞生长活性,采用酶联免疫吸附测定(ELISA)法检测超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px)的活性,采用蛋白质免疫印迹(Western blot)法检测自噬相关蛋白微管相关蛋白轻链3(LC3Ⅱ)与Beclin1蛋白的表达情况,用免疫荧光法检测自噬相关蛋白LC3的表达情况。结果:与空白组比较,随着TP质量浓度的增加,HepG2活性下降(P<0.05,P<0.01);检测细胞上清中SOD,GSH-Px的活性,均较空白组降低(P<0.05,P<0.01);Western blot表明TP可上调LC3Ⅱ与Beclin1蛋白水平的表达(P<0.05,P<0.01),免疫荧光法同样表明TP能够引起自噬相关蛋白LC3表达的增多(P<0.05,P<0.01),均具有一定的浓度依赖性。结论:一定质量浓度的TP可造成肝细胞损伤,其损伤机制可能与过氧化损伤以及过氧化损伤导致的自噬过度激活有关。Objective： To investigate the toxicity of triptolide on HepG2 cells and its modulating effects on autophagy. Method： HepG2 cells were cultured in vitro,and triptolide with the concentrations of 0. 003 2,0.016,0. 08,0. 4,2 g·L-1were added into the cells for 48 h. Compared with the control group,the cells viability was detected by MTT assay,ELISA methods was used to determine superoxide dismutase（SOD）,glutathion peroxidase（GSH-Px） activity,the expressions of the autophagy-related protein LC3 Ⅱ and Beclin1 were detected by Western blot,and the expression of the autophagy-related protein LC3 was detected by immunofluorescence.Result： Compared with the control group,with the increase in the dose of triptolide,the HepG2 activity was decreased（P〈0. 05,P〈0.01）; the levels of SOD and GSH-Px expressions were lower than the control group（P〈0. 05,P〈0.01）; the expressions of autophagy-related protein LC3Ⅱ and Beclin1 were detected by Western blot（P〈0. 05, P〈0.01）, and the expression of autophagy-related protein LC3 was also detected by immunofluorescence in a dose-dependent manner（P〈0. 05,P〈0.01）. Conclusion： Certain concentrations of triptolide could cause hepatotoxicity, which was related to peroxidation and overactivating autophagy caused peroxidation.

关 键 词：雷公藤甲素 HEPG2细胞 自噬 肝毒性 过氧化损伤 

分 类 号：R285.5[医药卫生—中药学]