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目的构建PPARγ和PPARγ应答元件(PPRE)荧光素酶系统,并确定大黄单体成分大黄素是否能够通过激活PPARγ促进HepG2肝细胞葡萄糖摄取。方法(1)构建PPARγ和PPRE荧光素酶系统并对20余种中药成分进行筛选;(2)将能够激活PPARγ和PPRE系统的大黄素与HepG2肝细胞进行培养,分别用RT-PCR/Southern杂交测定PPARγmRNA的表达;(3)用Western印迹法测定大黄素处理后的HepG2细胞的PPARγ和葡萄糖转运蛋白2(Glut2)的表达水平;(4)测定大黄素作用后的HepG2细胞对2-脱氧-[~3H]-D-葡萄糖摄取率。结果(1)在筛查的中药成分中,大黄素作用24 h后,呈剂量(0.04～180μmol/L)依赖性地增强COS-7细胞PPRE荧光素酶活性,其中90μmol/L浓度时达到最高值,为对照组的4倍(P<0.01)。而10μmol/L浓度的吡格列酮作用强度为对照组的6倍(P<0.01);(2)大黄素在90μmol/ L浓度时刺激HepG2细胞PPARγmRNA表达水平增加2.7倍(P<0.01);(3)大黄素的作用呈剂量和时间依赖性地刺激HepG2细胞PPARγ和Glut2蛋白的表达水平。其中,PPARγ蛋白水平在90μmol/L和作用16 h刺激作用最强,约为对照组的3.1～3.8倍(P<0.01);Glut2蛋白水平在90μmol/L和作用16 h刺激作用最强,约为对照组的2.5～4.3倍(P<0.01);(4)HepG2细胞的葡萄糖摄取率在90μmol/L浓度的大黄素作用24 h后,约为对照组的5倍(P<0.01)。结论研究结果显示大黄素刺激HepG2肝细胞PPARγ和Glut2蛋白表达,并促进葡萄糖的摄取。
Objective To construct the PPARγ and PPARγ response element (PPRE) luciferase system and determine whether the rhubarb monomer component emodin can promote HepG2 hepatocyte glucose uptake by activating PPARγ. Methods (1) PPARγ and PPRE luciferase systems were constructed and more than 20 kinds of Chinese medicinal ingredients were screened; (2) Emodin and HepG2 liver cells that could activate PPARγ and PPRE system were cultured, respectively, by RT-PCR/Southern hybridization. The expression of PPARγ mRNA was measured. (3) The expression of PPARγ and glucose transporter 2 (Glut2) in HepG2 cells treated with emodin was determined by Western blotting. (4) The HepG2 cells treated with emodin were tested for 2-deoxy- [~3H]-D-glucose uptake rate. Results (1) The luciferase activity of COS-7 cells was enhanced in a dose-dependent manner (0.04-180 μmol/L) after emodin treatment for 24 h, among which the highest concentration reached 90 μmol/L. The value was 4 times that of the control group (P<0.01). The pioglitazone concentration of 10μmol/L was 6-fold higher than that of the control group (P<0.01); (2) The emodin-stimulated HepG2 cells had a 2.7-fold increase in PPARγmRNA expression at a concentration of 90μmol/L (P<0.01); (3) The effect of emodin stimulated the expression of PPARγ and Glut2 proteins in HepG2 cells in a dose- and time-dependent manner. Among them, the PPARγ protein level was the highest at 90 μmol/L and 16 h after stimulation, which was about 3.1-3.8-fold higher than that of the control group (P<0.01). The level of Glut2 protein was the strongest at 90 μmol/L and 16 h after stimulation. It was 2.5-4.3 times that of the control group (P<0.01); (4) The glucose uptake rate of HepG2 cells was about 5 times that of the control group after 24 hours of the emodin concentration of 90 μmol/L (P<0.01). Conclusions The results showed that emodin stimulated the expression of PPARγ and Glut2 proteins in HepG2 hepatocytes and promoted glucose uptake.