PI3K/AKT/SIRT1信号通路介导H<sub>2</sub>S调节肝胆固醇代谢-东南大学学报医学版

PI3K/AKT/SIRT1信号通路介导H₂S调节肝胆固醇代谢

单位：1. 东南大学医学院, 江苏南京 210009;
2. 东南大学附属中大医院心血管内科, 江苏南京 210009

分类号：R-33;R589.2

出版年·卷·期（页码）：2019·38·第二期（230-237）

摘要：

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、200 μmol·L^-1）NaHS处理HepG2细胞24 h，CCK8检测各组细胞活性，免疫印迹法检测各组SIRT1蛋白表达；100 μmol·L^-1 NaHS分别处理细胞0、6、12、24 h，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、200 μmol·L^-1 NaHS对HepG2细胞活性无影响（P>0.05）；与对照组相比，50、100、200 μmol·L^-1 NaHS都能促进SIRT1蛋白表达并在100 μmol·L^-1时达到最大（P<0.05）；SIRT1蛋白表达呈时间依赖性升高，在24 h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.

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