PI3K/AKT/SIRT1信号通路介导H2S调节肝胆固醇代谢

PI3K/AKT/SIRT1 signal path can mediate the effect of H2S on cholesterol regulation in liver

目的:探讨硫化氢(H2S)上调SIRT1表达调控肝内胆固醇代谢过程,进而降低胆固醇水平的机制。方法:使用HepG2细胞建模,NaHS作为H2S供体,LY294002是PI3K/AKT通路抑制剂。利用不同浓度(0、50、100、200μmol·L^-1)NaHS处理HepG2细胞24h,CCK8检测各组细胞活性,免疫印迹法检测各组SIRT1蛋白表达;100μmol·L^-1NaHS分别处理细胞0、6、12、24h,检测SIRT1蛋白表达;将细胞分成对照组、NaHS干预组及LY294002+NaHS干预组3组,免疫印迹法检测P-AKT、AKT和SIRT1,RT-qPCR检测SIRT1、SREBP-1c、SREBP-2、CYP7A1、HMGCR;将细胞分为空白对照组、油酸干预组(阳性对照)、油酸+NaHS组、油酸+NaHS+LY294002组,试剂盒检测细胞内胆固醇含量。结果:与对照组相比,50、100、200μmol·L^-1NaHS对HepG2细胞活性无影响(P>0.05);与对照组相比,50、100、200μmol·L^-1NaHS都能促进SIRT1蛋白表达并在100μmol·L^-1时达到最大(P<0.05);SIRT1蛋白表达呈时间依赖性升高,在24h达到最高(P<0.05);与对照组相比,NaHS可以促进细胞内P-AKT/AKT和SIRT1蛋白表达,下调SREBP-1c、SREBP-2和HMGCR基因表达,上调CYP7A1基因表达,降低胞内胆固醇含量(P<0.05),联合应用LY294002具有相反的效应(P<0.05)。结论:PI3K/AKT/SIRT1通路参与了H2S对肝内胆固醇的调节作用。

Objective: To investigate the mechanism of H2S up-regulate SIRT1 to regulate the metabolism of cholesterol in the liver, thereby reducing the cholesterol level. Methods: HepG2 cells was used in the experiment. NaHS was exogenous H2S donor and LY294002 was the inhibitor of PI3K/AKT. HepG2 cells were cultivated with 4 different concentrations (0, 50, 100 and 200 μmol·L^-1 ) of NaHS for 24 hours. Then the viability of HepG2 cells was detected by CCK-8 kits and the protein expressions of SIRT1were detected by western blot. Then 100 μmol·L^-1 NaHS was selected to deal with HepG2 cells for 0 h, 6 h, 12 h, and 24 h respectively, western blot was used to detect SIRT1 protein expression. HepG2 cells were divided into blank control group, NaHS drug intervention group and LY294002+NaHS intervention group, then P-AKT, AKT and SIRT1 protein expressions were tested with western blot, gene expressions of SIRT1, SREBP-1c,SREBP-2,CYP7A1,HMGCR were detected with RT-qPCR. The cells were divided into blank control group, oleic acid intervention group (positive control),oleic acid+NaHS group and oleic acid+NaHS+LY294002 group, then intracellular cholesterol concentration were tested by using cholesterol testing kits. Results: Compared with the control group, 50,100,200 μmol·L^-1 NaHS all had no effects on the viability of HepG2( P >0.05).The protein expressions of SIRT1 were increasing in a concentration and time dependence way and reached the maximum when the concentration was 100 μmol·L^-1 and the time was 24 h( P <0.05). Compared with control group,H2S could increase P-AKT/AKT and SIRT1 protein expressions and CYP7A1 gene expression ( P <0.05), inhibite SREBP-1c,SREBP-2 and HMGCR gene expressions ( P <0.05), meanwhile, intracellular cholesterol levels reduced( P <0.05),using LY294002 processing cells in advance had the opposite effect. Conclusion: The PI3K/AKT/SIRT1 pathway is involved in the function of H2S in regulating cholesterol metabolism in the liver.