苦菊提取物对HepG2细胞的抗氧化作用及其机制研究

Study on anti-oxidative effect of extracts from Cichorium endivia on HepG2 cells and its mechanism

目的:考察苦菊提取物(CEE)对H2O2致HepG2细胞氧化应激损伤的保护作用,并探讨苦菊提取物在HepG2细胞中抗氧化作用机制。方法:通过MTT法和DCFH-DA荧光染色,检测H2O2处理HepG2细胞的活性和胞内ROS水平;在抗氧化反应元件(ARE)报告基因转染稳定的HepG2细胞内检测ARE-Luciferase活性;并通过荧光定量RT-PCR测定HepG2细胞内含有ARE序列相关基因的mRNA表达水平。结果:H2O2处理HepG2细胞后,细胞存活率下降,胞内ROS水平上升,而加入不同浓度的CEE则可明显改善上述结果。不同浓度的CEE处理转染ARE报告基因的HepG2细胞,可使细胞内的ARE活性呈浓度依赖性增强。此外,CEE可使HepG2细胞中含ARE序列的调控基因GCLC,GCLM和HMOX-1的mRNA表达水平上调,并呈浓度依赖性。结论:CEE通过降低ROS水平,抑制H2O2诱导的HepG2细胞损伤,而CEE对HepG2细胞的抗氧化保护作用机制可能与其激活HepG2细胞内Nrf2-ARE通路相关的抗氧化防御系统密切相关。

Objective： To investigate the protective effect of extracts from Cichorium endivia（ CEE） in H2O2-induced HepG2cell oxidative stress injury,and explore the antioxidant mechanism of CEE in HepG2 cells. Method： The viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidantresponse element（ ARE）-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells. Result： The cell viability reduced,while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE,the intracellular ARE activity could increase in a concentration-dependent manner. In addition,the mRNA expressions of regulatory genes GCLC,GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE. Conclusion：CEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE＇s antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.