The Role Of Exogenous Hydrogen Sulfide In Hepatocytes Proliferation And Apoptosis Via PI3k/Akt Pathway

Objective: Discuss the influence of Hydrogen sulfide(H2S) and LY294002 on rat hepatic cells proliferation and apoptosis; research the role of PI3K/Akt signaling pathway that in hydrogen sulfide influence the hepatic cells proliferation and apoptosis. Explore whether H2 S through PI3K/Akt signaling pathway to postpone the onset of liver fibrosis.Methods: Female SD rat were subcutaneously injected with carbon tetrachloride to induce hepatic fibrosis; two-step flow method isolated primary hepatocytes in rats; hepatic cell proliferation detected by MTT assay: The cells were divided in control group and experimental group, the experimental grouping according to the dosing concentration gradient. The Na HS concentration control at 25 μmol/L、50μmol/L、75μmol/L、100μmol/L、200μmol/L, the concentration of LY294002 control at 25 μmol/L、50μmol/L、75μmol/L、100μmol/L、200μmol/L. Drugs intervention for 48 hours then affected by MTT 20μl response 4 hours in the dark and affected by DMSO 150μl to terminatied the reaction. Use microplate reader to detection opticaldelnsity(A) at 570 nm wavelength; Using Hoechst 33342 to detect the influence of H2 S and LY294002 on hepatic cell apoptosis: The cells were divided in 3 groups randomly, which were normal control group(N), Na HS 50μmol/L group(S), LY294002 50μmol/L +Na HS50μmol/L group(LYS), cells cultivate for 48 h, colouration by, then apoptosis rate observed and counted by Leica DMIRB, The expressions of PI3 k and Phospho-bad were tested by western blot; Using PCR method to detect collagen typeⅠand collagen type Ⅲ m RNA expression in hepatic cell: The cells were also divided in groups randomly: which were group N, S, LYS, LY, total RNA was isolated from hepatic cells, PCR method to detect collagen typeⅠand collagen type Ⅲ m RNA expression in hepatic cells.Results: 50μmol/L Sodium Hydrosulfide promoted cell proliferation(P<0.05, compared with control group), 50μmol/L LY294002 inhibited cell proliferation(P<0.05, compared with control group). Compared with the control group and Sodium Hydrosulfide group, LY294002 with hydrogen sulfide group had high rate of cell apoptosis(P<0.05). The expression of PI3 k and Phospho-bad protein in Sodium Hydrosulfide group were higher than the control group and LY294002 with hydrogen sulfide group(P<0.05).However, the PI3 k and Phospho-bad protein expression of LY294002 with hydrogen sulfide group was less than the control group and Sodium Hydrosulfide group(P<0.05). The expression of COL-I、COL-III m RNA of sodium hydrosulfide group were more than other groups(P<0.05). However, compared with control group, the COL-I m RNA expression of Na HS group showed no statistically significant. Collagen I and collagen III m RNA expression in LY group was less than other groups(P<0.01). The COL-I m RNA expression of LY with sodium hydrosulfide group were higher than LY group, lower than the control group and sodium hydrosulfide group(P<0.01).Conclusions:H2S may promoted primary hepatocytes proliferation and inhibited its apoptosis through PI3k/Akt pathway. H2 S promoted the liver cells to synthesize the collagen I and collagen III. When PI3k/Akt pathway was blocked, the synthesis of COL-I and COL-III were sharp decrease. The relevant mechanism may involve that H2 S regulated hepatocytes proliferation through PI3k/Akt pathway.