尖叶假龙胆乙酸乙酯部位对胰岛素抵抗HepG2细胞IRS-1、Akt的影响

Effects of Ethyl Acetate Extracts of Gentianella acuta on IRS-1 and Akt in Insulin Resistance HepG2 Cells

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+50μg·mL^(-1)组、IR+500μg·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500μg·mL^(-1)时,存活率达到95%。浓度高于500μg·mL^(-1)时,存活率下降;与IR组比较,IR+50μg·mL^(-1)组与IR+500μg·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+50μg·mL^(-1)组与IR+500μg·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+50μg·mL^(-1)组与IR+500μg·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^-6mol·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^-1group, 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^-1group and the IR ＋ 500 μg·mL^-1group 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^-1and IR ＋ 500 μg·mL^-1compared 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^-1and IR ＋ 500 μg·mL^-1was 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.