灵芝多糖及其菌群代谢产物对HepG2细胞胰岛素抵抗的改善作用及机制

Improvement and Mechanism of Ganoderma lucidum Polysaccharides and Its Flora Metabolites on Insulin Resistance in HepG2 Cells

目的:探讨灵芝多糖及其菌群代谢产物对HepG2细胞胰岛素抵抗状态的影响及其作用机制。方法:以胰岛素(10^(-3)μmol/L)和地塞米松(10μmol/L)联合建立HepG2细胞胰岛素抵抗模型(insulin resistant HepG2,IRHepG2);使用CCK-8法评价灵芝多糖(Ganoderma lucidum polysaccharides,GLP)和灵芝多糖肠道菌群代谢物(Ganoderma lucidum polysaccharide flora metabolite,GLP-F)的细胞毒性;使用葡萄糖试剂盒、糖原试剂盒法评价GLP和GLP-F对IR-HepG2细胞葡萄糖消耗及糖原合成的影响;使用Western blot法检测了GLP与GLP-F对IR-HepG2细胞胰岛素信号级联中的关键蛋白IRS-1、AKT、GSK-3β、GLUT2、以及PEPCK的磷酸化或表达量的影响。结果:GLP和GLP-F均能显著增加IR-HepG2细胞的葡萄糖摄取量及糖原合成量(P<0.05),且GLP-F对IR-HepG2细胞葡萄糖消耗促进作用显著高于GLP(P<0.05);Western blot实验显示,GLP和GLP-F均能促进IR-HepG2细胞IRS-1、P-AKT、P-GSK-3β、GLUT2蛋白的表达,抑制PEPCK蛋白的表达,且GLP-F对PEPCK的抑制效用显著高于GLP(P<0.05)。结论:GLP与其在肠道菌群的介导下代谢产生的GLP-F具有同样的缓解肝脏胰岛素抵抗的生物学作用,并且在促进IR-HepG2细胞葡萄糖消耗以及抑制其糖异生限速酶活性方面,GLP-F比GLP具有更加显著的效果。

Objective:To investigate the effects of Ganoderma lucidum polysaccharides and its flora metabolites on the insulin resistance status of HepG2 cells and its mechanisms.Methods:Insulin resistant HepG2(IR-HepG2)model was established with the combination of insulin(10^(−3)μmol/L)and dexamethasone(10μmol/L).The cytotoxicity of Ganoderma lucidum polysaccharides(GLP)and Ganoderma lucidum polysaccharide flora metabolite(GLP-F)was evaluated using the CCK-8 method.The effects of GLP and GLP-F on glucose consumption and glycogen synthesis in IR-HepG2 cells were evaluated using the glucose kit and glycogen kit methods.Western blot assay was used to detect the effects of GLP and GLP-F on the phosphorylation or expression of IRS-1,AKT,GSK-3β,GLUT2,and PEPCK,key proteins in the insulin signaling cascade in IR-HepG2 cells.Results:Both GLP and GLP-F significantly increased glucose uptake and glycogen synthesis in IR-HepG2 cells(P<0.05).GLP-F promoted glucose consumption in IR-HepG2 cells significantly more than GLP(P<0.05).Western blot experiments showed that both GLP and GLP-F promoted IR-HepG2 cells IRS-1,P-AKT,P-GSK-3β,GLUT2 protein expression and inhibited PEPCK protein expression,and the inhibitory utility of GLP-F on PEPCK was significantly higher than that of GLP(P<0.05).Conclusions:GLP and its metabolism by intestinal flora-mediated production of GLP-F have the same biological effect of alleviating hepatic insulin resistance,and GLP-F has a more significant effect than GLP in promoting glucose consumption in IR-HepG2 cells and inhibiting their gluconeogenic rate-limiting enzyme activity.