Study On The Mechanism Of H₂S Reducing The Level Of Hepatobiliary Sterol Through SIRT1

Atherosclerosis?AS?is one of the most fatal and disabling diseases at present,and hypercholesterolemia is recognized as a major risk factor for atherosclerosis.The liver is the main organ for the synthesis of cholesterol and plays an important role in the regulation of blood cholesterol level.Hydrogen sulfide?H₂S?is a newly discovered gas signal molecule which can upregulate the expression of silent information regulation 2 homolog 1?SIRT1?,then inhibit intrahepatic cholesterol synthesis and lower blood cholesterol concentration.It is expected to be used to treat atherosclerosis,but the mechanism remains unclear.Purpose:This study aims to explore the mechanism of H₂S promoting SIRT1 to regulate cholesterol metabolism,finally to provide a basis for the development of relevant anti-atherosclerosis drugs.Methods:HepG2 cells was used in the experiment.NaHS is exogenous H₂S donor and LY294002 is the inhibitor of PI3K/AKT.Firstly,Cell counting kits?CCK-8?and Western blot were used to determine the safety concentration and the optimum concentration and time to promote the protein expression of SIRT1 of H₂S intervention cells.Subsequently,to investigate the effects of H₂S and LY294002 on the expression of SIRT1 including its downstream cholesterol regulating genes,western blot was used to detecte the protein expression of P-AKT?SIRT1,RT-qPCR was used to detect the gene expression of SIRT1?SREBP-1c?SREBP-2?CYP7A1?HMGCR and the intracellular cholesterol level of each group was detected by relevant kit.Result:Compared with the control group?0?mol/L?,all the three different concentrations?50?mol/L,100?mol/L and 200?mol/L?of NaHS had no effect on cell activity and could significantly increase SIRT1 protein expression.When the concentration was 100?mol/L,the SIRT1 expression reached to maximum.Compared with the control group?0h?,with the gradual increase of NaHS intervention time,the expression of SIRT1 protein increased in a time-dependent manner and reached the highest level at 24h.Compared with the control group,H₂S could significantly increase the protein expression of P-AKT?SIRT1 and gene expression of SIRT1?CYP7A1,inhibit the gene expression of SREBP-1c?SREBP-2 and HMGCR,reduce the intracellular cholesterol concentration.After pretreatment with LY294002,the expression of P-AKT?SIRT1 and CYP7A1 were down regulated and the expression of SREBP-1c?SREBP-2 and HMGCR increased,also intracellular cholesterol concentration increased.Conclusion:H₂S can increase SIRT1 through the PI3K/AKT pathway,inhibit downstream SREBP-1c and then up regulate CYP7A1;simultaneously inhibit SREBP-2 and further down regulate HMGCR,ultimately reduce the level of hepatobiliary sterol.