葛根素通过PI3K/Akt/GSK-3β信号通路调节HepG2细胞胰岛素抵抗

Effect of Puerarin in Reducing Insulin Resistance in HepG2 Cells via PI3K/Akt/GSK-3β Signal Transduction Pathway

目的:观察葛根素对胰岛素抵抗Hep G2细胞内磷脂酰肌醇3激酶(PI3K),蛋白激酶B(Akt),糖原合成酶激酶-3β(GSK-3β)信号分子的影响。方法:通过0.5 mmol·L^(-1)棕榈酸联合9×10^(-4)U·L^(-1)胰岛素孵育24 h诱导Hep G2胰岛素抵抗细胞模型,噻唑蓝(MTT)比色法测定细胞存活率以确定葛根素给药浓度。实验设正常组、模型组和葛根素各剂量组(40,80,160,320μg·L^(-1))。葡萄糖检测试剂盒检测细胞培养液上清葡萄糖的含量,酶联免疫吸附测定(ELISA)检测细胞培养液上清中肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6)的含量,肝糖原测定试剂盒测定Hep G2细胞内肝糖原含量,蛋白免疫印迹法(Western blot)检测细胞内PI3K,Akt,磷酸化(p)-Akt,GSK-3β,p-GSK-3β的蛋白表达水平。结果:与正常组比较,模型组Hep G2细胞葡萄糖消耗率明显下调(P<0.05),细胞内糖原含量减少(P<0.05),细胞培养液上清中TNF-α和IL-6的含量增加(P<0.05),PI3K和p-Akt蛋白表达下降(P<0.05),GSK-3β蛋白表达增加(P<0.05);与模型组比较,葛根素可呈剂量依赖性增加细胞葡萄糖消耗率(P<0.05),增加细胞内肝糖原含量(P<0.05),降低细胞培养液上清中TNF-α和IL-6的含量(P<0.05),增加PI3K蛋白表达,上调Akt蛋白磷酸活化水平(P<0.05),下调GSK-3β蛋白表达并增强其磷酸失活水平(P<0.05)。结论:葛根素可通过增强PI3K/Akt/GSK-3β信号转导过程,增加肝细胞内糖原含量从而改善Hep G2细胞的胰岛素抵抗状态。

Objective:To observe the effect of puerarin on phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt)and glycogen synthase kinase-3β(GSK-3β)in insulin resistant HepG2 cells.Method:HepG2cells were treated with palmitic acid 0.5 mmol·L-1and insulin 9×10^-4U·L^-1to induce insulin resistant condition for 24 h.Cell viability was detected by methyl thiazolyl tetrazolium(MTT)assay to determine the concentration of puerarin.This experiment included normal control group,model control group and puerarin groups of different doses(40,80,160,320μg·L^-1).Glucose detection kit was used to detect the content of glucose in cell culture supernatant.Tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6)levels in supernatant of cell culture medium were detected by enzyme-linked immunosorbent assay(ELISA).Hepatic glycogen assay kit was used for detecting the hepatic glycogen content in HepG2 cells.Western blot was applied to detect protein expression levels of PI3K,Akt,p-Akt,GSK-3βand p-GSK-3β.Result:Compared with those in the normal control group,the glucose consumption rate was significantly down-regulated in HepG2 cells in the model control group(P<0.05),the intracellular glycogen content was decreased(P<0.05),the content of TNF-αand IL-6 were increased in supernatant of cell culture medium(P<0.05),PI3K protein level was decreased,Akt phosphorylation level was down-regulated(P<0.05),and GSK-3βprotein expression was up-regulated(P<0.05).Compared with those in the model control group,in the puerarin groups,the glucose consumption rate was increased in a dose-dependent manner(P<0.05),the intracellular glycogen content was increased,the contents of TNF-αand IL-6 were reduced in supernatant of cell culture medium(P<0.05),PI3K protein expression was enhanced,Akt phosphorylation level was up-regulated(P<0.05),GSK-3βprotein expression was down-regulated,but its phosphorylation inactivation was increased(P<0.05).Conclusion:Puerarin alleviates the insulin resistance of HepG2 cells by strengthening the PI3K/Akt/GSK-3βsignal transduction process and increasing the glycogen content in hepatocytes.