Li Yu, Luo Xin-Xin, Yan Feng-Dong, Wei Zhang-Bin, Tu Jun
Jiangxi Province Key Laboratory of Traditional Chinese Medicine Etiopathogenisis, Research Center for Differentiation and Development of Traditional Chinese Medicine Basic Theory, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China.
Pharmacy Department, Pingxiang Maternal and Child Health-Care Hospital, Pingxiang 337000, China.
Zhongguo Zhong Yao Za Zhi. 2017 May;42(10):1939-1944. doi: 10.19540/j.cnki.cjcmm.2017.0081.
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.
观察葛根在肝细胞胰岛素抵抗（IR）模型中的降血糖作用，并探讨其初步分子机制。按照本研究组优化方案，用1×10⁻⁹ mol•L⁻¹胰岛素加3.75×10⁻⁶ mol•L⁻¹地塞米松处理48小时，稳定建立IR-HepG2细胞模型。用不同浓度（5%、10%和15%）含葛根血清处理IR-HepG2细胞后，采用CCK-8法检测细胞活力；采用葡萄糖氧化酶法在不同时间点（12、15、18、21、24、30、36小时）分别检测IR-HepG2细胞中的葡萄糖消耗量；采用蒽酮法检测细胞内糖原含量；采用蛋白质免疫印迹法检测瘦素受体（Ob-R）、胰岛素受体底物-2（IRS2）、葡萄糖转运蛋白1（GLUT1）和葡萄糖转运蛋白2（GLUT2）的蛋白表达水平。结果显示，含葛根血清（5%、10%和15%）对IR-HepG2细胞活力无显著影响；5%和10%含葛根血清在18、21和24小时显著增加IR-HepG2细胞的葡萄糖消耗量（P<0.01）；15%含葛根血清在15小时提高IR-HepG2细胞的葡萄糖消耗量（P<0.05），并在18、21、24和30小时以剂量依赖方式显著提高葡萄糖消耗量（P<0.01）。将降血糖作用的最佳时间定义为24小时，进一步研究表明，含葛根血清处理24小时后可增加细胞内糖原含量（P<0.01），并上调IRS2 Ob-R、GLUT1和GLUT2蛋白表达水平。我们的结果表明，含葛根血清可通过重要的PI3K/PDK信号通路部分上调IR-HepG2细胞中Ob-R、IRS2、GLUT1和GLUT2的表达水平，加速葡萄糖转运入肝细胞并增加肝糖原合成，从而增强IR-HepG2细胞的降血糖作用，实现降血糖效果。
