双氢青蒿素对HepG2细胞氧化损伤和能量代谢的影响及其与索拉非尼的协同作用

Effect of Dihydroartemisinin on Oxidative Damage and Energy Metabolism of HepG2 Cells and Its Synergistic Effect with Sorafenib

目的:探讨双氢青蒿素(DHA)对HepG2细胞的增殖抑制作用,通过细胞氧化损伤及能量代谢交互通路的影响阐明其作用机制;并通过与索拉非尼(Sora)联用,探讨其联合用药的可能性。方法:选用HepG2细胞和SW480细胞,细胞增殖与活性检测(CCK-8)法分别得到DHA与Sora的半数抑制浓度(IC50);Chou-Talalay法分析DHA与Sora联用的联用指数(CI)。选用HepG2细胞,分为正常组,DHA单用组(10μmol·L^(-1)),Sora单用组(5μmol·L^(-1))和DHA与Sora联用组(DHA 10μmol·L^(-1),Sora 5μmol·L^(-1)),药物孵育8~12 h后,糖酵解速率试剂盒检测细胞糖酵解功能,线粒体压力试剂盒检测线粒体氧化磷酸化功能;DCFH-DA活性氧(ROS)探针检测细胞内ROS水平变化;脂质过氧化物丙二醛(MDA)检测试剂盒检测细胞内MDA水平变化;蛋白免疫印迹法(Western blot)检测细胞内血红素氧合酶1(HO-1)和谷氨酸半胱氨酸连接酶催化亚基(GCLC)蛋白的水平变化。结果:与正常组比较,DHA单用组能够显著抑制HepG2细胞的线粒体氧化磷酸化ATP合成能力和糖酵解速率(P<0.01),升高细胞内ROS和MDA水平(P<0.05),降低HO-1,GCLC水平(P<0.05);DHA与Sora联用在HepG2和SW480细胞中均具有较好的协同抑制细胞增殖作用,其CI值<0.9;与DHA单用组比较,DHA与Sora联用组对HepG2细胞的线粒体氧化磷酸化ATP合成和糖酵解的抑制程度均显著增强(P<0.01);细胞内的ROS和MDA水平均显著升高(P<0.01);细胞内抗氧化相关蛋白HO-1,GCLC水平均显著降低(P<0.01)。结论:DHA可能通过降低HepG2细胞内的HO-1,GCLC水平,升高ROS使MDA水平增加,并造成细胞线粒体氧化损伤,抑制细胞糖酵解能力以及氧化磷酸化能力,从而抑制HepG2细胞增殖;DHA与Sora联用具有协同抑制HepG2细胞增殖的作用,其机制可能与协同造成细胞氧化损伤从而影响线粒体电子传递链,抑制细胞能量代谢有关。

Objective:To explore the inhibitory effect of dihydroartemisinin (DHA) on the proliferation of HepG2 cells,elucidate the mechanism from the perspectives of oxidative damage and energy metabolism,and discuss the possibility of combined use of DHA with sorafenib (Sora).Method:Cell counting kit-8 (CCK-8)assay was used to obtain the 50%inhibitory concentration(IC50)of DHA and Sora on HepG2 and SW480 cells and Chou-Talalay method was used to obtain the combination index(CI)of DHA and Sora.HepG2 cells were classified into the control group,DHA group(10μmol·L^(-1)),Sora group(5μmol·L^(-1)),and DHA+Sora group(DHA 10μmol·L^(-1),Sora 5μmol·L^(-1))and then incubated with corresponding drugs for 8-12 h.Seahorse XF glycolytic rate assay kit and cell mito stress test kit were employed to respectively detect the glycolysis function of cells and oxidative phosphorylation function of mitochondria.DCFH-DA and lipid peroxidation MDA assay kit were separately used to analyze the intracellular levels of reactive oxygen species(ROS)and malondialdehyde(MDA).Western blot was applied to determine the intracellular levels of heme oxygenase-1(HO-1)and glutamate-cysteine ligase catalytic subunit(GCLC).Result:Compared with the control group,DHA alone inhibited the ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis(P<0.01),increased the levels of intracellular ROS and MDA(P<0.05),and decreased the levels of HO-1 and GCLC(P<0.05)in HepG2 cells.DHA and Sora had synergistic inhibitory effect on proliferation of HepG2 and SW480 cells,with CI<0.90.The DHA+Sora group showed stronger suppression of ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis(P<0.01),higher levels of intracellular ROS and MDA(P<0.01),and lower levels of intracellular antioxidation-related proteins HO-1 and GCLC in HepG2 cells(P<0.01)than the DHA group.Conclusion:DHA may increase the level of MDA by reducing HO-1 and GCLC and increasing ROS in HepG2 cells,which results in mitochondria oxidative damage,restricts cell glycolysis and mitochondrial oxidative phosphorylation,and thus finally inhibits the proliferation of HepG2 cells.DHA and Sora have synergistic inhibitory effect on the proliferation of HepG2 and SW480 cells,and the mechanism may be related to the synergistic oxidative damage that affects the mitochondrial electron transport chain and suppresses cell energy metabolism.