衍生物Fla-CN的抗糖尿病作用及其作用机制研究

Study on the anti-diabetic effect and mechanism of the derivative Fla-CN

目的:体内外研究黄酮衍生物Fla-CN的抗糖尿病作用及其机制。方法:体外实验以人肝HepG2细胞为研究对象,不同浓度Fla-CN孵育后,利用荧光标记2-脱氧葡萄糖(2-NBDG),通过检测HepG2细胞内2-NBDG的荧光强度,观察Fla-CN对肝细胞葡萄糖摄取的影响;采用硫酸-蒽酮比色法检测HepG2细胞内糖原含量;通过检测一定时间内葡萄糖生成量,观察Fla-CN对肝细胞糖异生过程的影响。体内实验采用2型糖尿病模型db/db小鼠为研究对象,给予Fla-CN和二甲双胍干预4周后,测定db/db小鼠空腹血糖、糖耐量以及胰岛素耐量,计算糖耐量、胰岛素耐量实验血糖-曲线下面积(AUC)。通过Western印迹法检测化合物干预后小鼠肝组织中磷酸化AMP活化蛋白激酶(AMPK)、糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、葡萄糖-6-磷酸酶(G6Pase)及转录调控因子过氧化物酶体增殖物激活受体γ协同刺激因子1α(PGC-1α)的表达。进一步通过Western印迹检测AMPK抑制剂Compound C或AMPK激动剂AICAR与Fla-CN联用后,HepG2细胞内PEPCK、G6Pase、PGC-1α的蛋白表达。结果:Fla-CN能够促进HepG2细胞对葡萄糖的摄取(F=33.50,P<0.01)和糖原合成(F=93.63,P<0.01),抑制糖异生(F=44.19,P<0.01)。同时,Fla-CN显著降低2型糖尿病db/db小鼠空腹血糖以及时间-血糖曲线下面积(F=98.40,P<0.05)。Western印迹显示Fla-CN能够激活db/db小鼠肝组织中AMPK,抑制PEPCK、G6Pase以及PGC-1α的表达,并呈现一定剂量依赖性。Compound C能在一定程度上阻断Fla-CN对AMPK的激活和对PEPCK、G6Pase以及PGC-1α的抑制作用。结论:黄酮衍生物Fla-CN通过激活AMPK,有效调节肝HepG2细胞糖代谢,抑制肝糖异生,降低2型糖尿病db/db小鼠空腹血糖,改善其受损的糖耐量,提高胰岛素敏感性。

Objective:To study the anti-diabetic effect and mechanism of the flavonoid derivative Fla-CN in vitro and in vivo.Methods:In vitro experiment,HepG2 cells were incubated with different concentrations of Fla-CN,and the fluorescence intensity of 2-NBDG in HepG2 cells was detected to observe the effect of Fla-CN on glucose uptake in hepatocytes.The glycogen content in HepG2 cells was measured by sulfuric acid anthronecolorimetry.The glucose production was measured to investigate the effect of Fla-CN on gluconeogenesis of hepatocytes.In vivo experiments,db/db mice model of type 2 diabetes were used as the subjects of study.After intervention with Fla-CN and metformin for 4 weeks,fasting blood glucose,glucose tolerance and insulin tolerance of db/db mice were investigated,and AUC of glucose tolerance and insulin tolerance were calculated.Western blotting was used to detect the expression of AMP dependent protein kinase(AMPK)phosphorylation,phosphoenolpyruvate carboxykinase(PEPCK),glucose-6-phosphatase(G6Pase)and peroxisome proliferator activated receptorγcoactivator-1α(PGC-1α)in the liver tissue of mice.The protein expression of PEPCK,G6Pase and PGC-1αin HepG2 cells was further detected by Western blotting after an incubation of AMPK inhibitor Compound C or AMPK agonist AICAR combined with Fla-CN.Results:Fla-CN could promote glucose uptake(F=33.50,P<0.01),glycogen synthesis(F=93.63,P<0.01)and inhibit gluconeogenesis(F=44.19,P<0.01)in HepG2 cells.Meanwhile,Fla-CN significantly reduced fasting blood glucose and its AUC in db/db mice(F=98.40,P<0.05).Western blotting showed that Fla-CN could activate AMPK,inhibit the expression of PEPCK,G6Pase and PGC-1αin the liver of db/db mice in a dose-dependent manner.Compound C could impair the activation of AMPK and the inhibition of PEPCK,G6Pase and PGC-1αby Fla-CN.Conclusion:Fla-CN could effectively regulate the glucose metabolism of HepG2 cells,reduce the fasting blood glucose of db/db mice,and improve the impaired glucose tolerance and insulin sensitivity.Further studies have shown that Fla-CN promotes glucose uptake and inhibits glyconeogenesis by activating AMPK in liver.