PI3K/Akt信号转导通路在硫化氢影响肝星状细胞Ⅰ和Ⅲ型胶原表达中的作用

摘要

目的 观察PI3K/Akt信号转导通路在硫化氢影响肝星状细胞Ⅰ、Ⅲ型胶原表达中的作用.方法 体外培养大鼠肝星状细胞-T6,通过外源性硫化氢预处理体外培养的肝星状细胞,同时给予PI3K/Akt信号转导通路的特异性阻断剂LY294002,RT-PCR测定肝星状细胞的Ⅰ型、Ⅲ型胶原蛋白mRNA的表达,Western blot法检测PI3K/Akt信号转导通路上游蛋白PI3K、p-Akt的表达情况.用SPSS17.0统计软件进行单因素方差分析,多个样本均数间的多重比较采用LSD-t检验分析数据.结果 正常对照组(N组)、二甲基亚砜溶质组(D组)、硫化氢组(S组)、LY294002抑制剂组(LY组)、LY拮抗硫化氢组(LY+S组)Ⅰ型胶原蛋白mRNA的灰度值分别为0.950±0.201、0.970±0.205、1.270±0.213、0.680±0.161、0.420±0.154,Ⅲ型胶原蛋白mRNA的灰度值分别为0.740±0.164、0.730±0.177、1.040±0.215、0.510±0.133、0.290±0.101,与正常对照组相比,单独给予硫化氢组肝星状细胞的Ⅰ型胶原蛋白mRNA的表达增强(F=14.635,P＜0.05),Ⅲ型胶原蛋白mRNA的表达增强(F=14.620,P＜ 0.05),与硫化氢组相比,同时给予硫化氢和LY294002组肝星状细胞的Ⅰ型胶原蛋白mRNA的表达减弱(F=14.635,P＜ 0.05),Ⅲ型胶原蛋白mRNA的表达减弱(F=14.620,P＜0.05).N组、D组、S组、LY组、LY+S组PI3K蛋白的灰度值分别为0.150±0.019、0.170±0.019、0.240±0.027、0.110±0.024、0,060±0.020,p-Akt蛋白的灰度值分别为1.020±0.097、0.940±0.153、1.330±0.236、0.610±0.055、0.330±0.043,与正常对照组相比,单独给予硫化氢组肝星状细胞的PI3K蛋白的表达增强(F=26.672,P＜0.05),p-Akt蛋白的表达增强(F=23.522,P＜0.05),与硫化氢组相比,同时给予硫化氢和LY294002组肝星状细胞的PI3K蛋白的表达减弱(F=26.672,P＜0.05),p-Akt蛋白的表达增强(F=23.522,P＜0.05).结论 硫化氢可能通过激活肝星状细胞PI3K/Akt信号转导通路,增强肝星状细胞Ⅰ型、Ⅲ型胶原蛋白mRNA的表达.%Objective To investigate the role of the PI3K/Akt signaling pathway in hydrogen sulfide-induced alterations in expression of collagen Ⅰ and collagen Ⅲ in hepatic stellate cells.Methods In vitro cultured rat hepatic stellate cells (HSC-T6) were treated with hydrogen sulfide,or left untreated for use as controls,and divided into groups for treatment with different inhibitors for the various factors involved in the PI3K/Akt signaling pathway.Reverse transcription-PCR was used to measure Collagen Ⅰ and collagen Ⅲ mRNA expression.Western blotting was used to detect the protein expression of PI3K and p-Akt,which are upstream proteins of the PI3K/Akt pathway.Results Compared with the untreated control cells,the hydrogen sulfide treated cells showed elevated expression of collagen Ⅰ mRNA (F =14.635,P ＜ 0.05),collagen Ⅲ mRNA (F =14.620,P ＜ 0.05),PI3K protein (F =26.672,P ＜ 0.05),and p-Akt (F =23.522,P ＜ 0.05).Compared to the cells treated with hydrogen sulfide alone,the cells treated with the various inhibitors showed lower expression of collagen Ⅰ mRNA (F =14.635,P ＜ 0.05),collagen Ⅲ mRNA (F=14.620,P ＜ 0.05),PI3K protein (F =26.672,P ＜ 0.05),and p-Akt protein (F =23.522,P ＜ 0.05).Conclusion Hydrogen sulfide can activate the PI3K/Akt pathway and elevate the expression of collagen Ⅰ and collagen Ⅲ in rat hepatic stellate cells.