黄芪甲苷调节PI3K/Akt/FoxO1通路抑制糖尿病大鼠肝糖异生

Effect of Astragaloside Ⅳ in Regulating PI3K/Akt/FoxO1 Pathway and Inhibiting Hepatic Gluconeogenesis in Diabetic Rats

目的:探讨黄芪甲苷对2型糖尿病(T2DM)大鼠肝损伤保护潜在机制及肝组织磷酯酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/叉头框转录因子1(FoxO1),磷酸烯醇式丙酮酸羧基酶(PEPCK)和葡萄糖6-磷酸酶(G6Pase)蛋白表达的影响。方法:6周高糖高脂饮食后,链脲佐菌素(STZ)一次性腹腔注射(0.035 g·kg^-1)建立T2DM大鼠模型,随机分为正常组、模型组、黄芪甲苷低、中、高剂量组及二甲双胍组。黄芪甲苷低、中、高剂量组灌胃黄芪甲苷0.02,0.04,0.08 g·kg^-1·d^-1,二甲双胍组灌胃二甲双胍0.2 g·kg^-1·d^-1;给药8周后,于末次灌胃24 h禁食不禁水后处死,测血清肝生化指标,肝脏指数等;采用苏木素-伊红(HE)染色观察肝脏组织病理形态学变化;马松(Masson)染色观察纤维化程度;过碘酸希夫(PAS)染色反应观察细胞内糖原等变化;免疫组化及蛋白免疫印迹法(Western blot)检测各组肝中PI3K/Akt/FoxO1信号蛋白及PEPCK,G6Pase蛋白表达水平。结果:与正常组比较,模型组肝脏指数显著升高(P<0.01),肝功能指标丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),总胆固醇(TC),甘油三酯(TG)的含量显著升高(P<0.01),高密度脂蛋白胆固醇(HDL-C)显著降低(P<0.01),大鼠体质量显著减轻(P<0.01),PI3K/Akt/Fox O1信号减弱(P<0.01),PEPCK,G6Pase蛋白表达水平显著增强(P<0.01);与模型组比较,黄芪甲苷中、高剂量组及二甲双胍组肝功能指标ALT,AST,TC,TG的含量明显降低(P<0.05,P<0.01),大鼠体质量明显增加(P<0.05,P<0.01),肝组织Fox O1,PEPCK及G6Pase蛋白水平明显降低(P<0.05,P<0.01),Fox O1的磷酸化水平明显增强(P<0.05,P<0.01)。结论:黄芪甲苷可能通过调节PI3K/Akt/Fox O1信号通路抑制高脂高糖加小剂量STZ诱导T2DM肝糖异生,从而起到延缓T2DM大鼠肝脏损伤作用。

Objective:To investigate the potential mechanism of astragalosideⅣin protecting liver injury and phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt)/forkheadbox transcription factor 1(FoxO1),phosphoenolpyruvate carboxykinase(PEPCK)and glucose 6-phosphatase(G6 Pase)protein expressions in type 2 diabetic(T2 DM)rats.Method:After 6 weeks of high-sugar and high-fat diet,a model of type 2 diabetes was established through intraperitoneal injection of streptozotocin(STZ,0.035 g·kg^-1).The rats were randomly divided into normal group,model group,low,medium and high-dose astragalosideⅣgroups and metformin group,0.02,0.04,0.08 g·kg^-1·d^-1 astragaloside crude drug and 0.2 g·kg^-1·d^-1 metformin were administered in the low,middle and high-dose astragalosideⅣand metformin groups.After 8 weeks of continuous administration,and24 hours later after the last gavage,the rats were executed.Serum and liver tissues were collected to detect serum liver biochemical indexes,liver index HDL-C.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue.Masson staining was used to observe the degree of liver fibrosis.The changes of glycogen,glycoprotein,or mucopolysaccharide in tissue cells were observed by periodic acid Schiff(PAS)reaction staining.Immunohistochemistry and Western blot analysis were used to detect the expression levels of PI3 K/Akt/FoxO1 signaling protein and PEPCK and G6 Pase in liver tissues of each group.Result:Compared with normal group,the liver index of the model group increased significantly(P<0.01),the levels of liver function indicators alanine aminotransferase(ALT),aspartate aminotransferase(AST),TC and TG were significantly increased(P<0.01),while HDL-C and body weight were significantly reduced(P<0.01).The results of immunohistochemistry and Western blot showed that the signal of PI3 K/Akt/FoxO1 was weakened(P<0.01),and PEPCK and G6 Pase were increased(P<0.01)in model group.Compared with model group,the contents of ALT,AST,TC and TG in middle and high-dose astragalosideⅣgroups were significantly decreased(P<0.05,P<0.01),while the body weight was significantly increased(P<0.05,P<0.01),the middle and high dose of astragalosideⅣsignificantly inhibited the levels of FoxO1,PEPCK and G6 Pase in liver tissue(P<0.05,P<0.01),and enhanced the phosphorylation of FoxO1(P<0.05,P<0.01).Conclusion:AstragalosideⅣmay inhibit T2 DM hepatic gluconeogenesis by regulating PI3 K/Akt/FoxO1 signaling pathway,and inhibiting highfat,high-sugar and low-dose STZ,thereby protecting liver damage in T2 DM rats.