TGF-β1-Smad2/3信号转导通路在百草枯中毒致肺纤维化中的作用  

The role of TGF-β1-Smad2/3 signaling pathway in paraquat-induced pulmonary fibrosis

作　　者：王亚鹏[1] 杨惠芳[1] 蔡倩[1] 朱玲勤[1] 刘贺荣[1] 黄敏[1] 

机构地区：[1]宁夏医科大学公共卫生学院,宁夏银川750004

出　　处：《工业卫生与职业病》2016年第3期167-171,共5页Industrial Health and Occupational Diseases

基　　金：国家自然科学基金项目(NO:81360435)

摘　　要：目的观察百草枯(PQ)染毒对人肺成纤维细胞的影响,探讨PQ中毒致肺纤维化中转化生长因子β1-Smad2/3蛋白(TGF-β1)-Smad2/3信号转导通路的作用。方法体外培养人胚肺成纤维细胞(MRC-5),以终浓度为0、12.5、25、50、100、200、400μmol/L的PQ处理细胞24h,四甲基偶氮唑蓝(MTT)检测细胞存活率。使用终浓度50μmol/L PQ处理细胞6、8、12、24、48、72h,检测PQ在不同时间点对细胞的损伤程度。终浓度0、25、50、100μmol/L PQ处理细胞24h,酶联免疫吸附(ELISA)检测细胞TGF-β1蛋白表达水平,聚合酶链反应(RT-PCR)检测细胞TGF-β1 mRNA表达水平。为了观察PQ染毒对TGF-β1/Smad信号通路的影响,将MRC5细胞分成6组:正常对照组、25μmol/L PQ染毒组、50μmol/L PQ染毒组、100μmol/L PQ染毒组、TGF-β1阳性对照组、TGF-β1刺激组(100μmol/L PQ+50μmol/L TGF-β1),免疫印迹法(Western blot)检测磷酸化Smad2/3蛋白(p-Smad2/3)表达水平。结果与对照组比较,MRC-5细胞存活率随着PQ剂量和时间的增加明显降低(P<0.05),呈剂量和时间依赖性;ELISA显示,PQ作用MRC-5细胞24h后,TGF-β1蛋白表达升高至37.4、48.8、39.3μg/ml,但诱导作用呈非剂量依赖方式;RT-PCR显示,随着PQ浓度增加TGF-β1mRNA表达水平明显升高,分别是对照组的3.5、6.9和9.7倍,诱导作用呈剂量依赖方式(P<0.05);Westem blot显示,浓度为25、50、100μmol/L PQ作用细胞24h后,p-Smad2、p-Smad3蛋白表达水平明显升高(P<0.05),给予细胞100μmol/L PQ+50μmol/L TGF-β1共同刺激下,p-Smad2、p-Smad3蛋白量达到最多(P<0.05)。结论TGF-β1、pSmad2、p-Smad3的异常表达参与了PQ致MRC5细胞损伤过程,TGF-β1/Smad信号通路激活在PQ致肺纤维化中发挥重要作用。Objective To investigate the effects of Paraquat（PQ）on human embryonic lung fibroblasts（MRC5）and explore the role of TGF-β1-Smad2/3signaling pathway in paraquat-induced pulmonary fibrosis.Methods MRC5 cells were cultured with different concentration of PQ（0,12.5,25,50,100,200,400μmol/L）for 24 hand different time（6,8,12,24,48,72h）following 50μmol/L PQ treatment.The viability of cells was measured by MTT.The protein and mRNA levels of TGF-β1were analyzed by ELISA,Real time RTPCR,respectively.To examine whether TGF-β1/Smad signaling pathway was involved in paraquat-induced cytotoxicity,cells was divided into 6groups：（1）control;（2）25μmol/L PQ group;（3）50μmol/L PQ group;（4）100μmol/L PQ group;（5）TGF-β1positive control group;（6）stimulate group（100μmol/L PQ＋50μmol/L TGF-β1）.The protein levels of p-Smad2 and p-Smad3 were assayed by western blot.Results MTT showed that cell viability decreased with increasing PQ concentration and time（P〈0.05）.The protein expression of TGF-β1treated with PQ（25、50、100μmol/L）significantly increased to 37.4,48.8,and39.3μg/ml in a dose-independent manner.Real time RT-PCR showed TGF-β1 mRNA in PQ group also significantly up-regulated in a dose-dependent manner.Exposure to PQ（25、50、100μmol/L）induced increase of protein levels of p-Smad2 and p-Smad3.Noteworthy,the expression of p-Smad2 and p-Smad3 were dramatically increased following PQ plus TGF-β1 stimulation（P〈0.05）.Conclusions Abnormal expressions of TGF-β1,phosphorylated Smad2/3 were involved in PQinduced cytotoxicity.It suggests that TGF-β1/Smad signaling pathway plays an important role in PQ-induced pulmonary fibrosis.

关 键 词：百草枯 MRC-5细胞 肺纤维化 TGF-β1/Smad信号转导通路 

分 类 号：R114[医药卫生—卫生毒理学]