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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 effects of triptolide (TP) on peroxidation injury and the regulation of autophagy in HepG2 cells. METHODS: HepG2 cells were treated with TP at concentrations of 0.003, 0.016, 0.08, 0.4 and 2 g · L -1 for 48 h, and blank control group was also established. MTT assay The cell growth activity was measured. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by enzyme linked immunosorbent assay (ELISA) The expression of autophagy-related protein LC3Ⅱ and Beclin1 protein was detected by immunohistochemistry. The expression of autophagy-related protein LC3 was detected by immunofluorescence. Results: Compared with the blank group, the activity of HepG2 decreased (P <0.05, P <0.01) with the increase of TP concentration. The activity of SOD and GSH-Px in the supernatant of the blank group was lower than that of the blank group , P <0.01). Western blot showed that TP up-regulated the expression of LC3Ⅱ and Beclin1 protein (P <0.05, P <0.01). Immunofluorescence assay also showed that TP increased the expression of LC3 (P < P <0.01), all with a certain concentration-dependent. Conclusion: A certain concentration of TP can cause hepatocellular injury, which may be related to the over-activation of autophagy caused by peroxidation and peroxidation.