MAPK信号通路在百草枯诱导上皮-间充质改变中的作用  

Role of MAPK signaling pathway in epithelial-mesenchymal transition of type H alveolar epithelial cells induced by Paraquat

作　　者：朱晨笛 郭慕真 蔡倩 李颖颖 武坷鑫 黄敏 

机构地区：[1]宁夏医科大学公共卫生与管理学院、职业卫生与环境卫生学系,银川750004 [2]宁夏医科大学公共卫生与管理学院实验中心,银川750004

出　　处：《中华劳动卫生职业病杂志》2018年第8期561-567,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金项目（81360435）,宁夏高等学校一流学科建设（公共卫生与预防医学学科）资助项目（NXYIJXK2017808）

摘　　要：目的探讨MAPK家族中P38丝裂原活化蛋白激酶（p38MAPK）、细胞外调节蛋白激酶（ERK）、c—Jun氨基端激酶（JNK）信号通路在百草枯（Paraquat，PQ）诱导的肺泡上皮细胞上皮．间质转分化（epithelial-mesenchymal transition，EMT）中的作用。方法在体外培养的大鼠肺泡Ⅱ型上皮细胞（RLE-6TN）中加入不同浓度PQ（0、25、50、100μmol／L）后共培养6、12、24ho倒置相差显微镜观察细胞形态学改变，划痕实验评估细胞迁移能力，免疫印迹法（Westernblot）检测上皮及间质细胞表型蛋白表达（上皮细胞表型蛋白：E—cad、ZO-1，间质细胞表型蛋白：Vimentin、a—SMA）、MAPK信号通路关键蛋白的表达（P-p38MAPK、P-Erkl／2、P-JNK），实时荧光定量PCR（RT—PCR）检测成纤维细胞，肌成纤维细胞（MFB／FB）主要产物COL-Ⅰ、COL—Ⅲ、Fn和EMT来源的成纤维细胞标志物FSP-1的基因表达。结果100μmol／LPQ分别处理6、12、24h，细胞由卵圆形或多边形的上皮细胞变成长梭形的间质细胞，细胞间连接消失，且时间越长形态学改变越明显。与对照组比较，50、100、200、300μmol／LPQ处理24h的细胞存活率明显下降，200μmol／LPQ作用于细胞的时间越长（6、12、24、36、48h），细胞存活率越低；与对照组比较，50、100μmol／LPQ诱导上皮细胞标记蛋白E-cad、ZO-1蛋白表达下调，且下调的趋势随时间延长更加明显，差异有统计学意义（P〈0．05）；同时，随着PQ诱导剂量的升高、时间的延长，间质细胞标记蛋白α-SMA、vimentin蛋白表达逐渐升高，差异有统计学意义（P〈0．05）。划痕实验结果显示，与对照组比较，100μmol／LPQ处理24h后的细胞迁移能力明显增强，差异有统计学意义（P〈0．05）；与对照组比较，100μmol／LPQ诱导细胞24h后，P-p38MAPK、P-JNK及P-Erk1／2蛋白表达显著上调，差异有统计学意义（P〈0．05），分别给予上述�Objective To investigate the roles of p38 mitogen-activated protein kinases （p38 MAPK）, extracellular regulated protein kinases （ERK） and c-Jun N-tenninal kinases （JNK） of MAPK signaling pathway in Paraquat-induced epithelial to mesenchymal transition （EMT） of type Ⅱ alveolarepithelial cells. Methods RLE-6NT cells were incubated with different concentrations of PQ （0, 25, 50, 100μmol/L） for 6, 12 and 24 h. Cell morphology alteration was observed under phase-contrast microscopy. Cell viability was determined using an MIT assay. Cell migration ability was detected using scratch wound assay. Protein expression of P-p38 MAP, P-Erkl/2, P-JNK, E-cad, ZO-1, Vimentin and a-SMA were detected by western blot. The level of genes related to fibrosis （COL-Ⅰ, COL-Ⅲ, FN and FSP-1） were analyzed via quantitative real-time RT-PCR. Results Cell morphology started to undergo EMT changes with a phenotype characteristic of mesenchymal cells, including an elongated shape and a lack of tight cell-cell adhesions induced by 100μmol/L PQ treatment in a time-dependent manner. MTT showed that cell viability decreased with increasing PQ concentration （50,100,200,300 μmol/L PQ treatment for 24 h） and increasing treatment time （200 μmol/L PQ treatment for 6, 12, 24, 36, 48 h）. Compared to control group, the expressions of the epithelial phenotype marker E-cad and ZO-1 significantly decreased with PQ treatment （50, 100μmol/L） in a time-dependent manner（P〈0.05）. Additionally, the level of the mesenchymal marker （a-SMA, vimentin） dramatically increased with PQ treatment in the same concentration- and time-dependent manner （P〈0.05）. Cell migration ability was markedly increased after 24 h of 100 μmol/L PQ treatment compared to control （P〈0.05）. The phosphorylated forms of p38 MAPK, Erkl/2, and JNK were increased at 24 h after stimulation with PQ （P〈0.05）. This PQ induced （100 μmol/L） phosphorylation was markedly attenuated in the presence of the p38 MAPK, ERK and JNK

关 键 词：百草枯 肺泡上皮细胞 蛋白激酶类 

分 类 号：R595.4[医药卫生—内科学]