葛根上调肝胰岛素抵抗HepG2细胞OB-R,IRS2,GLUT1和GLUT2蛋白调节糖代谢的研究

Puerariae Lobatae Radix elevated expression levels of OB-R,IRS2,GLUT1 and GLUT2 to regulate glucose metabolism in insulin-resistance HepG2 cells

研究中药葛根对人肝癌Hep G2细胞胰岛素抵抗(IR)模型糖代谢的改善作用并初步探讨葛根降糖的分子作用机制。该实验采用课题组优化方法,1×10^(-9)mol·L^(-1)胰岛素加3.75×10^(-6)mol·L^(-1)地塞米松联合给药Hep G2细胞48 h建立稳定的IR模型;CCK-8法检测葛根含药血清对细胞活性的影响;葡萄糖氧化酶法检测多个时间点(12,15,18,21,24,30,36 h)IRHep G2细胞葡萄糖消耗量;蒽酮法检测细胞内糖原含量;Western blot法检测胰岛素受体底物2(IRS2)、瘦素受体(Ob-R)、葡萄糖转运蛋白1(GLUT1)和GLUT2蛋白表达水平。研究结果显示葛根含药血清(5%,10%,15%)对IR-Hep G2细胞的活力没有明显的影响;5%和10%葛根含药血清在给药18,21,24 h均显著上调IR-HepG2细胞葡萄糖消耗量(P<0.01),15%葛根含药血清除给药15 h上调葡萄糖消耗量较弱(P<0.05),18,21,24,30 h都显著上调葡萄糖消耗量(P<0.01),呈现一定程度剂量依赖性。葡萄糖消耗时效实验确认24 h是最佳时间点,进一步研究发现葛根含药血清作用24 h增加胞内糖原含量(P<0.01);上调IRS2,Ob-R,GLUT1和GLUT2蛋白表达量。葛根含药血清降糖作用可能部分通过上调Ob-R和IRS2蛋白表达来调节以PI3K/PDK为中心的胰岛素信号通路;上调IR-HepG2细胞GLUT1和GLUT2表达量,加速葡萄糖转运进入肝细胞中并增加糖原合成来增强IR-HepG2细胞的胰岛素敏感性来实现的。

To observe the anti-hyperglycemic effect of Puerariae Lobatae Radix in hepatocyte insulin resistance（IR） models, and investigate its preliminary molecular mechanism. IR-HepG2 cell model was stably established with 1×10^-9 mol·L^-1 insulin plus 3.75×10^-6 mol·L^-1 dexamethasone treatment for 48 h according to optimized protocol in our research group. After IR-HepG2 cells were treated with different concentrations（5%,10% and 15%） of Puerariae Lobatae Radix-containing serum, cell viability was detected by CCK-8 assay; the glucose consumptions in IR-HepG2 cells were separately detected at different time points （12, 15, 18, 21, 24, 30, 36 h） by using glucose oxidase method; intracellular glycogen content was detected by anthrone method; and the protein expression levels of leptin receptor （Ob-R）, insulin receptor substrate-2 （IRS2）, glucose transporter 1（GLUT1） and GLUT2 were detected by Western blot assay. The results showed that Puerariae Lobatae Radix-containing serum （5%, 10% and 15%） had no significant effect on IR-HepG2 cell viability; 5% and 10% Puerariae Lobatae Radix-containing serum significantly increased glucose consumption of IR-HepG2 cells （P〈0.01） at 18, 21 and 24 h; 15% Puerariae Lobatae Radix-containing serum elevated the glucose consumption of IR-HepG2 cells at 15 h （P〈0.05）, and significantly elevated the glucose consumption at 18, 21, 24 and 30 h （P〈0.01） in a dose-dependent manner. The optimized time of anti-hyperglycemic effect was defined as 24 h, and further study showed that Puerariae Lobatae Radix-containing serum could increase intracellular glycogen content after 24 h treatment （P〈0.01）, and up-regulate IRS2, Ob-R, GLUT1 and GLUT2 protein expression levels. Our results indicated that Puerariae Lobatae Radix-containing serum could achieve the anti-hyperglycemic effect through important PI3K/PDK signaling pathway partially by up-regulating the expression levels of Ob-R and IRS2, GLUT1 and GLUT2 in IR-HepG2 cells, accelerating the glucose transport into hepatocytes and increasing hepatic glycogen synthesis to enhance the anti-hyperglycemic effect of IR-HepG2 cells.