脂联素通过腺苷酸激活蛋白激酶途径抑制大鼠肝星状细胞氧化应激并调控转化生长因子β1和Ⅰ型胶原表达

Adiponectin inhibits oxidative stress and modulates TGF-β1 and COL-1 expression via the AMPK pathway in HSC-T6 cells

目的 观察外源性脂联素对体外培养的大鼠肝星状细胞HSC-T6氧化应激水平及转化生长因子（TGF）β 1和Ⅰ型胶原（COL-1）表达的影响,并探讨脂联素的抗氧化应激作用是否与腺苷酸激活蛋白激酶（AMPK）信号途径相关. 方法 采用实时荧光定量PCR法检测TGFβ1和COL-1mRNA的表达情况,酶联免疫吸附法检测上清液中TGFβ1和COL-1的表达水平,Western blot方法检测AMPK和p-AMPK的蛋白表达情况.对数据行两独立样本均数t检验、重复测量方差分析. 结果 与H2O2刺激组相比,脂联素＋H2O2组超氧化物歧化酶活力增加（17.78±1.87与11.30±1.98,P＜0.05）、丙二醛含量下降（1.55±0.25与1.91±0.23,P＜0.05）,TGFβ1、COL-1的基因及蛋白相对表达水平均下降（P＜0.05）.1.0 μ g/ml脂联素处理HSC-T6细胞60、120 min后,AMPK激酶磷酸化水平较空白对照组（0 min）增加（P＜0.05）,当脂联素作用时间为120 min时达最大值.AMPK抑制剂可逆转脂联素的抗氧化应激作用和抗纤维化作用. 结论 抑制氧化应激为脂联素抗肝纤维化机制之一,脂联素可通过激活AMPK信号通路改善HSC-T6氧化应激水平,从而调控TGFβ1和COL-1的表达.

Objective To investigate the anti-oxidative stress and anti-fibrotic mechanisms of adiponectin by examining effects on oxidative stress levels and expression of fibrosis-related signaling factors,including transforming growth factor-beta 1 （TGFβ1）,collagen Ⅰ （COL-1）,and the adenosine monophosphate-activated protein kinase （AMPK） pathway by using an in vitro HSC-T6 cultured cell system.Methods Activated HSC-T6 cells were pre-treated with 1.0 μg/mL adiponectin for 0,30,60 and 120 min,or left untreated to serve as controls,and both groups were then exposed to 5 μmol/L H2O2; a portion of the adiponectin-treated oxidative stress-induced cells were treated with an AMPK inhibitor （Compound C）.The effects on mRNA levels of TGFβ1.and COL-1 were analyzed by real-time PCR,in the levels of secreted TGF-β1 and COL-1 were detected by enzyme-linked immunosorbent assay of supematants,and in the phosphoAMPK and AMPK protein expressions were detected by Western blotting.Results Compared to the H2O2 group without adiponectin pre-treatment,the H2O2 group with adiponectin pre-treatment showed significantly increased activity of superoxide dismutase （SOD）,decreased content of malondialdehyde （MDA）,and decreased gene and protein expressions of TGF-β1 and COL-1 （P ＜ 0.05）.Moreover,inhibition of the AMPK pathway inhibited these adiponectin-mediated effects.The H2O2 group with adiponectin pre-treatment also showed increased levels of phospho-AMPK protein expression,with the maximum effect detected after 120 min of the adiponectin pre-treatment （P ＜ 0.01）.Conclusion Inhibition of oxidative stress is one of the mechanisms of the anti-fibrotic effects of adiponectin.Adiponectin can attenuate oxidative stress levels,resulting in down-regulation of TGFβ1 and COL-1 expression through activation of the AMPK pathway.