硫化氢在p38MAPK信号通路对大鼠肝星状细胞凋亡中的作用

目的探讨硫化氢(H2S)在p38MAPK信号通路对大鼠肝星状细胞(hepatic stellate cell,HSC)凋亡中的作用及磷酸化P38、Caspase-3蛋白表达的变化。方法实验设对照组(HSC加含10%胎牛血清的DMEM培养液)、二甲基亚砜(DMSO)组(对照组基础上加DMSO,使其终浓度为0.1%)、NaHS组(对照组基础上加NaHS,使其终浓度为50μmol/L)、SB组(DMSO组基础上加SB203580,使其终浓度为75μmol/L)、SB加NaHS(SB+NaHS)组;采用Hoechst荧光染色检测细胞凋亡;Western blotting法检测磷酸化p38MAPK表达及Caspase-3蛋白表达水平。结果与对照组比较,SB组和SB+NaHS组HSC-T6的凋亡率增加(P0.05),NaHS组p38MAPK磷酸化水平及Caspase-3表达均明显增高(P0.01);与NaHS组比较,SB组和SB+NaHS组细胞凋亡率增加明显(P0.01),p38MAPK磷酸化水平表达降低(P0.01);SB+NaHS组较SB组Caspase-3蛋白表达升高(P0.05)。结论 p38MAPK及Caspase-3在H2S刺激的HSC-T6中表达增强,H2S能促使SB203580诱导的HSC-T6细胞凋亡,其作用机制可能与活化p38MAPK的磷酸化途径,进而激活Caspase-3的表达有关。

Influence of hydrogen sulfide in p38MAPK signaling pathway on rat hepatic stellate cell apoptosis

Objective To investigate the influence of hydrogen sulfide in p38MAPK signaling pathway on rat hepatic stellate cells （HSC） apoptosis and the changes of phospho-P38,Caspase-3 protein.Methods Control group （HSC-T6 plus 10％ fetal bovine serum containing DMEM medium）,Dimethyl sulfoxide group （DMSO was added based on control group,final concentration for 0.1％）,Sodium Hydrosulfide group （Sodium Hydrosulfide was added based on control group,final concentration for 50 μmol/L）,SB group （SB203580 was added based on Dimethyl sulfoxide group,final concentration for 75 μmol/L）,SB with Sodium Hydrosulfide group were set up in this study.The apoptotic rate was detected by Hoechst 33342,the expression of phosphorylated p38MAPK（P-p38MAPK） and Caspase-3 protein level were determined by Western blotting.Results Compared with control group,the apoptotic rate of HSC-T6 cells in SB203580 group and SB with Sodium Hydrosulfide group was increased （P ＜ 0.05）,the levels of P-p38 and Caspase-3 positive expression in SB with Sodium Hydrosulfide group were significantly increased （P ＜ 0.01） ; Compared with SB with Sodium Hydrosulfide group,the apoptotic rate of HSC-T6 cells in SB group and SB with Sodium Hydrosulfide group was increased （P ＜ 0.01）,the expression of P-p38MAPK protein was decreased （P ＜ 0.01） ; the level of Caspase-3 positive expression in SB with Sodium Hydrosulfide group was more than SB group （P ＜ 0.05）.Conclusion Hydrogen sulfide could enhance the expression of p38MAPK and Caspase-3 protein and make SB203580 induced HSC-T6 cells apoptosis,the mechanism of action could be related to phosphorylation of p38MAPK and activation of Caspase-3.