20-羟基蜕皮激素对高糖诱导HepG2细胞氧化损伤的作用及机制研究

Role and Mechanism of 20-Hydroxyecdysone in Oxidative Damage of HepG2 Cells Induced by High Glucose

目的:探究20-羟基蜕皮激素(20-Hydroxyecdysone,20-HE)对高糖诱导HepG2细胞氧化损伤的保护作用及相关分子机制。方法:利用高糖(50 mmol/L葡萄糖)建立HepG2细胞氧化损伤模型,分别采用CCK-8法、caspase-3活性检测实验、荧光探针法和比色法检测细胞的活力、凋亡、活性氧(Reactive Oxygen Specie,ROS)、超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)和丙二醛(Malondialdehyde,MDA)的水平。基于生物信息学分析的方法对参与20-HE调控作用的相关信号通路进行预测,采用Western blot检测Akt蛋白的磷酸化水平,评价PI3K/Akt信号通路的激活水平,利用PI3K/Akt信号通路的抑制剂(LY294002)验证其是否参与20-HE发挥的调控作用。结果:20-HE的浓度低于20μmol/L对HepG2细胞没有显著毒性作用;20-HE可以显著提高损伤细胞的活力(P<0.05),显著抑制损伤细胞的凋亡(P<0.05),显著下调损伤细胞的ROS水平(P<0.05),显著提高SOD和CAT的水平(P<0.05),显著下调MDA水平(P<0.05);PI3K/Akt信号通路是20-HE发挥调控作用的潜在下游机制;20-HE可以显著上调损伤细胞中PI3K/Akt信号通路的水平(P<0.05);LY294002可以逆转20-HE对损伤细胞发挥的保护作用。结论:20-HE通过激活PI3K/Akt信号通路发挥对高糖诱导HepG2细胞氧化损伤的保护作用。

Objective:To explore the protective effects of 20-Hydroxyecdysone(20-HE)on high glucose induced HepG2 cells and its related molecular mechanism.Methods:In this study,high glucose(50 mmol/L glucose)was used to establish the oxidative damage model in HepG2 cells.The CCK-8 assay,caspase-3 assay,fluorescent probe method,and colorimetric method were used to assess the levels of cell viability,apoptosis,oxygen species(ROS),superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA),respectively.The signaling pathways involved in the regulation of 20-HE were predicted using bioinformatics analysis.The phosphorylation level of Akt protein was detected by Western blot to evaluate the activation level of the PI3K/Akt signaling pathway.The involvement of the PI3K/Akt signaling pathway in the regulatory effects of 20-HE was verified using the inhibitor LY294002.Results:Treatment with 20-HE had no significant toxic effect on HepG2 cells at concentrations lower than 20μmol/L.In the injured cells,20-HE could significantly improve the viability(P<0.05),inhibit the apoptosis(P<0.05),down-regulate the level of ROS,improve the levels of SOD and CAT(P<0.05),and down-regulate the level of MDA(P<0.05).PI3K/Akt signaling pathway was the potential downstream mechanism of regulatory effects exerted by 20-HE.20-HE could significantly up-regulate the level of PI3K/Akt signaling pathway in the injured cells(P<0.05).LY294002 could reverse the protective effects exerted by 20-HE on the injured cells.Conclusion:20-HE exerted protective effects on high glucose induced oxidative damage in HepG2 cells by activating the PI3K/Akt signaling pathway.