CAJ | 학술논문

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.
목적 관찰외원성지련소대체외배양적대서간성상세포HSC-T6양화응격수평급전화생장인자(TGF)β 1화Ⅰ형효원(COL-1)표체적영향,병탐토지련소적항양화응격작용시부여선감산격활단백격매(AMPK)신호도경상관. 방법 채용실시형광정량PCR법검측TGFβ1화COL-1mRNA적표체정황,매련면역흡부법검측상청액중TGFβ1화COL-1적표체수평,Western blot방법검측AMPK화p-AMPK적단백표체정황.대수거행량독립양본균수t검험、중복측량방차분석. 결과 여H2O2자격조상비,지련소+H2O2조초양화물기화매활력증가(17.78±1.87여11.30±1.98,P＜0.05)、병이철함량하강(1.55±0.25여1.91±0.23,P＜0.05),TGFβ1、COL-1적기인급단백상대표체수평균하강(P＜0.05).1.0 μ g/ml지련소처리HSC-T6세포60、120 min후,AMPK격매린산화수평교공백대조조(0 min)증가(P＜0.05),당지련소작용시간위120 min시체최대치.AMPK억제제가역전지련소적항양화응격작용화항섬유화작용. 결론 억제양화응격위지련소항간섬유화궤제지일,지련소가통과격활AMPK신호통로개선HSC-T6양화응격수평,종이조공TGFβ1화COL-1적표체.
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.