氧化应激介导糖原合成酶激酶-3β促进肝细胞凋亡

Oxidative stress promotes hepatocyte apoptosis mediated by glycogen synthase kinase 3β

目的探讨糖原合成酶激酶-3β(GSK3β)在氧化应激诱导肝细胞凋亡中的作用。方法以人肝HL-7702细胞为研究对象,H2O2/抗霉素A诱导细胞产生氧化应激,建立肝细胞凋亡模型。SB216763为GSK3β特异性抑制剂,在H2O2/抗霉素A给药前2 h干预。采用钙黄绿素乙酰甲酯/碘化丙啶(PI)双染色观察细胞存活情况,采用annexinⅤ-FITC/PI联合流式细胞术检测细胞凋亡,同时对细胞培养上清进行乳酸脱氢酶(LDH)检测来评价细胞死亡程度;Western blot法检测p-GSK3β、GSK3β、caspase-3、cleaved caspase-3、c-Jun氨基末端激酶(JNK)和细胞色素C(Cyt C)蛋白的表达。结果 H2O2/抗霉素A诱导的氧化应激促进了GSK3β活性增加,抑制GSK3β活性缓解了氧化应激和由氧化应激引起的细胞凋亡。与氧化应激模型组相比,SB216763干预组中PI染色的细胞显著减少,流式细胞术检测细胞凋亡率降低,细胞培养上清中LDH显著降低,Western blot法结果显示干预组中cleaved caspase-3、JNK、Cyt C蛋白表达下降。结论 GSK3β是氧化应激诱导细胞凋亡通路中的一种重要信号分子,抑制其活性可减轻氧化应激而改善肝细胞凋亡。

Objective To analyze the role of glycogen synthase kinase 36 （GSK3~） in hepatocyte apoptosis induced by oxidative stress. Methods Human HL-7702 hepatoma cells were induced by H202/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H2O2/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide （ PI ） double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase （LDH） assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved cespase-3, c-Jun N-terminal kinase （JNK） and cytochrome C （CytC） proteins were examined using Western blotting. Results Oxidative stress triggered by H2O2/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased. Conclusion GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.