目的：研究尖叶假龙胆乙酸乙酯部位对胰岛素抵抗HepG2细胞胰岛素关键信号IRS-1、Akt的基因、蛋白的影响。方法：以CCK-8法检测HepG2细胞活性；采用人肝癌HepG2细胞，并用高浓度胰岛素（10 -6 mol·L -1 ）培养HepG2细胞36 h，建立胰岛素抵抗细胞模型。根据CCK-8法结果分为正常组（Control组）、模型组（IR组）、尖叶假龙胆乙酸乙酯部位IR+50 μg·mL -1 组、IR+500 μg·mL -1 组及二甲双胍组，以葡萄糖试剂盒检测葡萄糖消耗量。尖叶假龙胆乙酸乙酯部位给药6 h后RT-PCR检测胰岛素抵抗HepG2细胞IRS-1、Akt基因表达；尖叶假龙胆乙酸乙酯部位给药6 h后，利用Western blot法检测IRS-1、Akt的蛋白表达。结果：尖叶假龙胆乙酸乙酯部位浓度为500 μg·mL -1 时，存活率达到95%。浓度高于500 μg·mL -1 时，存活率下降；与IR组比较，IR+50 μg·mL -1组与IR+500 μg·mL -1 组促进胰岛素抵抗HepG2细胞葡萄糖的消耗量，但其作用不及盐酸二甲双胍组。给药6 h后与IR组比较，IR+50 μg·mL -1 组与IR+500 μg·mL -1 组的RT-PCR检测胰岛素抵抗HepG2细胞IRS-1、Akt的基因表达显著升高，P<0.01。给药6 h后与IR组比较，IR+50 μg·mL -1 组与IR+500 μg·mL -1 组的Western blot法检测IRS-1、Akt的蛋白表达显著升高，P<0.01。结论：尖叶假龙胆乙酸乙酯部位上调HepG2细胞胰岛素信号通路关键靶点IRS-1、Akt基因表达，以及IRS-1、Akt蛋白表达从而可能是其改善胰岛素抵抗作用的机制。

This paper was aimed to study the effect of ethyl acetate extracts of Gentianella acuta on the gene and protein of insulin significant signal IRS-1 and Akt in insulin resistance (IR) HepG2 cells. The CCK-8 method was used to detect the HepG2 cell activity. HepG2 cells of human liver cancer were cultured with high concentration insulin (10 -6 mol·L -1 )for 36 hours to establish IR cell model. According to the results of CCK-8, the control group, model (IR) group, ethyl acetate extracts of Gentianella acuta IR + 50 μg·mL -1 , IR + 500 μg·mL -1 group, and the metformin group were divided. Glucose consumption was measured with a glucose assay kit. The expressions of IRS-1 and Akt gene in IR HepG2 cells were detected by RT-PCR after 6-hour using of ethyl acetate extracts of Gentianella acuta. Western blot was used to detect the expression of IRS-1 and Akt protein after 6-hour using of ethyl acetate extracts of Gentianella acuta. The results showed that when the concentration of ethyl acetate extracts of Gentianella acuta was 500 μg·mL -1 , the survival rate reached 95%. When the concentration was higher than 500 μg·mL -1 , the survival rate decreased. Compared with the IR group, the IR + 50 μg·mL -1 group and the IR + 500 μg·mL -1 group promoted glucose consumption of IR HepG2 cells,but its effect was less than that of the metformin hydrochloride group. The expression of IRS-1 and Akt in IR HepG2 cells was significantly increased by using RT-PCR in the group of IR + 50 μg·mL -1 and IR + 500 μg·mL -1 compared with the IR group after 6-hour using of ethyl acetate extracts of Gentianella acuta. The expression of IRS-1 and Akt protein in the group of IR + 50 μg·mL -1 and IR + 500 μg·mL -1 was significantly higher than that in the IR group after 6- hour medication detected by western blot. It was concluded that the ethyl acetate extracts of Gentianella acuta can increase the expression of IRS-1, Akt gene, the expression of IRS-1 and Akt protein in HepG2 cells, which may be the mechanism of IR improvement.

科学技术部国际科技合作项目（2010DFB33260）：中医药干预治胰岛素抵抗创新中药研究，负责人：徐暾海；北京中医药大学创新团队-中医药干预糖尿病及其并发症研究团队（2011-CXTD-19），负责人：刘铜华。