肝糖异方改善肝细胞过氧化损伤致糖代谢异常的体外研究

Gantangyi Recipe improving abnormal glucose metabolism of hepatocyte peroxidative damage in vitro

目的探讨肝糖异方改善氧化应激、通过调控糖原合成信号通路糖原合成激酶-3β(GSK-3β)/FoxO1转录因子(FoxO1)调节糖代谢的作用机制。方法肝细胞以500μmol/L过氧化氢(H_(2)O_(2))孵育30 min后分为模型组、肝糖异方组及二甲双胍组,肝糖异方组及二甲双胍组分别以肝糖异方(200μg/L)及二甲双胍(5 mmol/L)与肝细胞共孵育48 h,同时设正常组以对照。采用CCK-8法检测细胞活力;酶联免疫吸附试验(ELISA)法检测细胞上清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)活性及丙二醛(MDA)水平;2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针法检测活性氧(ROS)水平;过碘酸雪夫染色(PAS)法检测肝细胞内糖原含量;葡萄糖氧化酶法检测细胞上清中葡萄糖浓度;Western blot法检测细胞p-GSK-3β/GSK-3β、p-FoxO1/FoxO1蛋白表达。结果与正常组比较,H_(2)O_(2)可诱导模型组肝细胞过氧化损伤,且细胞上清中ALT、AST活性及MDA含量明显升高(P<0.05),ROS生成速率明显升高(P<0.05),细胞上清中葡萄糖含量明显升高(P<0.05),糖原染色结果显示糖原阳染颗粒显著减少、细胞着色较浅,p-GSK-3β/GSK-3β及p-FoxO1/FoxO1蛋白表达明显降低(P<0.05)。与模型组比较,肝糖异方及二甲双胍干预后,ALT、AST、MDA水平均有不同程度的降低,ROS生成速率均明显降低(P<0.05),且肝糖异方均优于二甲双胍(P<0.05);肝糖异方组细胞着色较模型组加深,细胞上清中葡萄糖含量显著下降(P<0.05),p-GSK-3β/GSK-3β及p-FoxO1/FoxO1蛋白表达升高(P<0.05)。结论肝糖异方可减轻H_(2)O_(2)诱导的肝细胞氧化应激损伤致糖代谢异常,其作用机制与激活GSK-3β/FoxO1糖原合成信号通路有关。

Objective To investigate the mechanism of Gantangyi Recipe in ameliorating oxidative stress and regulating(GSK-3β/FoxO1)glycogen synthesis signaling pathway to regulate glucose metabolism.Methods The hepatocytes were incubated with 500μmol/L H_(2)O_(2) for 30 min and then divided into the model group,Gantangyi Recipe group,and metformin group.Gantangyi Recipe group and metformin group were respectively incubated with Gantangyi Recipe(200μg/L)or metformin(5 mmol/L)for 48 h.The normal group was set up.The cell viability was detected by the CCK-8 method;supernatant glutathione aminotransferase(ALT),glutathione aminotransferase(AST)activity,and malondialdehyde(MDA)content were detected by enzyme-linked immunosorbent assay(ELISA);reactive oxygen species(ROS)levels were detected by DCFH-DA fluorescent probe method;glycogen content was stained by PAS staining method;cell supernatant glucose concentration in hepatocytes was detected by glucose oxidation enzyme method;and cell p-GSK-3β/GSK-3βand p-FoxO1/FoxO1 protein expressions were analyzed by Western blot.Results Compared with the condition of normal group,H_(2)O_(2) could induce cell peroxidative damage model,the ALT,AST activity,and MDA content were significantly higher in the model group(P<0.05),ROS generation rate was significantly higher(P<0.05),there was a significant increase in supernatant glucose content(P<0.05),the glycogen staining results showed significant reduction and lighter coloration of cell positive staining granules,and p-GSK-3β/GSK-3βand p-FoxO1/FoxO1 protein expressions were significantly reduced(P<0.05).Compared with the condition of model group,the levels of ALT,AST and MDA were reduced to different degrees after the intervention of Gantangyi Recipe and metformin,the rate of ROS production was significantly reduced(P<0.05),and the effect of Gantangyi Recipe was superior to that of metformin(P<0.05).In the Gantangyi Recipe group,cells stained darker in glycogen staining than those in the model group,the supernatant glucose concentration decreased significantly(P<0.05),and the p-GSK-3β/GSK-3βand p-FoxO1/FoxO1 protein expressions increased(P<0.05).Conclusion Gantangyi Recipe can alleviate the oxidative stress injury and abnormal glucose metabolism in hepatocytes induced by H_(2)O_(2),and the mechanism is related to the activation of the GSK-3β/FoxO1 glycogen synthesis signaling pathway.