丝裂原活化蛋白激酶通路在百草枯诱导人肺Ⅱ型上皮样细胞A549凋亡中的作用

Role of MAPK Pathways in Paraquat Induced Apoptosis in Human Lung Type Ⅱ Alveolar Epithelial-Like A549 Cells

目的探讨百草枯(PQ)诱导人肺Ⅱ型上皮样细胞A549凋亡过程中丝裂原活化蛋白激酶(MAPK)的作用及其机制。方法通过PQ(200μmol/L)诱导A549细胞凋亡,选择c-JunN-末端激酶(JNK)MAPK特异性抑制剂(SP600125)与P38丝裂原活化蛋白激酶(P38MAPK)特异性抑制剂(SB203580)阻断相应MAPK通路。实验分为对照组、SP600125组、SB203580组、PQ组、SP600125+PQ组、SB203580+PQ组。各组细胞孵育48h后,MTT法分析细胞活性;Annexin V-FITC/PI双染法检测细胞凋亡率;采用检测试剂盒测定Caspase-3和Caspase-9活化程度;Western blotting检测MAPK与线粒体凋亡信号转导通路相关蛋白表达。结果与对照组比较,SP600125组与SB203580组各项检测指标比较差异无统计学意义(均P>0.05);与PQ组比较,SP600125+PQ组与SB203580+PQ组均显著提高了细胞活性、降低了细胞凋亡率、降低了Caspase-3和Caspase-9活化程度、降低促凋亡蛋白Bax表达和促进抗凋亡蛋白Bcl-2表达(均P<0.05);同时SP600125+PQ组降低p-JNK/JNK蛋白表达比例;SB203580+PQ组降低p-P38/P38蛋白表达比例(均P<0.05)。结论PQ通过MAPK途径诱导人肺Ⅱ型上皮样细胞A549凋亡。

Objective To investigate the role of mitogen-activated protein kinases( MAPK) and their mechanism in paraquat( PQ) induced apoptosis in human typeⅡalveolar epithelial-like A549 cells.Methods Apoptosis was induced in A549 cells by exposure to PQ (200 μmol/L).The c-Jun N-terminal kinase( JNK) specific inhibitor( SP600125) and P38 specific inhibitor( SB203580) blocked the corresponding MAPK pathway.Control group,SP600125 group,SB203580 group,PQ group,SP600125 + PQ group,and SB203580 + PQ groups were established.After a 48-h treatment cell survival was assessed using the MTT assay.The rate of apoptosis was determined using Annexin V-fluorescein isothiocyanate/propidium iodide( FITC/PI ) double staining.Caspase-3 and -9 activities were assayed. Western blotting was used to detect the expression of related proteins of MAPK and mitochondrial apoptosis signal transduction pathways. Results Compared with the control group,no significant difference of detection indicators were evident in the SP600125 and SB203580 groups( both P > 0.05).Compared with the PQ group,cell viability significantly increased,apoptotic rate significantly decreased,activation of Caspase-3 and -9 significantly decreased,expression of Bax significantly decreased,and the expression of Bcl-2 significantly increased in the SP600125 + PQ group and SB203580 + PQ group( all P < 0.05).The ratio of p-JNK/JNK significantly decreased in the P600125 + PQ group( P < 0.05).The ratio of p-P38 / P38 significantly decreased in the SB203580 + PQ group.Conclusion PQ induces apoptosis in human lung typeⅡalveolar epithelial-like A549 cells through the MAPK pathways.