黄芪多糖抑制糖原合成酶激酶3β活性与核转位及其调控糖稳态的作用研究

摘要

Objective To observe the effect of astragalus polysaccharides ( APS) on glucose homeostasis regulation and focus on glycogen synthase kinase 3 beta (GSK3 beta) activity and subcellular localization (nuclear translocation).Methods HepG2 human hepatoma cells were cultured in vitro and treated with high glucose of different concentrations (30, 40 mM) to induce hepatocyte endoplasmic reticulum stress model, then acquire optimum operating concentration.The HepG2 cells were treated with APS of different concentrations (50, 100, 200, 400 μg/mL) to select the most effective concentration.The HepG2 cells were divided into seven groups with different treatment: negative control group (C), positive control group (Tm), 30 mM high glucose-induced group (G30), 45 mM high glucose-induced group (G45), negative control+APS group (CA), positive control+APS group ( TA) and high glucose-induced+APS group ( GA).Effect of APS at different concentrations on proliferation activity of HepG2 cells were detected by MTT assay, transcription and shear levels of XBPlmRNA in HepG2 cells by quantitative real-time PCR, and phosphorylation levels of GSK3βin cytoplasm and nucleus by immunoblotting techniques.Results The optimum operating glucose concentration was 30 mM.The most effective APS concentration was 200μg/mL.The transcription and shear levels of XBPlmRNA in HepG2 cells of GA group were lower than those of G30 group ( P<0.05), respectively, but there were no significant differences between TA and Tm group.The phosphorylation levels of GSK3βin cytoplasm and nucleus of GA group were higher than those of G group(P<0.05), respectively, but there were no significant differences between TA and Tm group. Conclusion APS could improve hepatic steatosis, and its mechanism might be that APS inhibits the activity and nuclear localization of GSK3β, then alleviate endoplasmic reticulum stress.%目的：观察黄芪多糖（ astragalus polysaccharide，APS）对糖稳态的调节作用，研究糖原合成酶激酶3β（ glycogen synthase kinase 3β，GSK3β）活性及其亚细胞定位（核转位）情况。方法体外培养HepG2人肝癌细胞，用不同浓度高糖（30、40 mM）诱导肝细胞内质网应激模型，选择最佳工作糖浓度，用不同浓度的APS（50、100、200、400μg／mL）处理肝细胞以选择最大有效浓度。按照不同处理方法将细胞分为：阴性对照组（ C）、阳性对照组（ Tm）、30 mM高糖诱导组（ G30）、45 mM高糖诱导组（ G45）、阴性对照＋APS处理组（CA）、阳性对照＋APS处理组（TA）与高糖应激＋APS处理组（GA），共7组。采用MTT法检测不同浓度APS对HepG2细胞增殖的影响，实时定量PCR反应检测HepG2细胞内XBPl mRNA的剪切水平与转录水平，免疫印迹技术检测组胞浆和胞核内GSK3β磷酸化水平。结果选择30 mM高糖作为工作糖浓度，APS最大有效浓度200μg／mL，对细胞进行干预处理。 GA组细胞内质网应激细胞XBP1的转录和剪切水平均显著低于G30组（P＜0.05），但TA组与Tm组XBP1的转录和剪切水平差异无统计学意义。与G30组比较， GA组胞浆和胞核内GSK3β磷酸化水平均显著增加（P＜0.05），但TA组与Tm组组胞浆和胞核内GSK3β无显著性变化。结论 APS能显著改善肝脏脂肪变性，其机理与APS减少肝脏GSK3β的活性特别是核定位并减轻肝脏内质网应激有关。