铜绿假单胞菌诱导气道上皮细胞产生MUC5AC的分子机制

Molecular mechanism of pseudomonas aeruginosa stimulating MUC5AC production in airway epithelial cells

目的探讨铜绿假单胞菌(Pseudomonas aeruginosa,Pa)诱导气道上皮细胞表达产生粘蛋白MUC5AC的影响,并探讨其可能的分子机制。方法体外培养NCI-H292细胞,用Pa感染后,采用ELISA检测MUC5AC的产生情况;并用商品化的MMP-9活性检测试剂盒检测其产生以及酶活性情况。同时,采用EGFR,PI3K,NADPH,ROS和MMP特异性抑制剂AG1478,LY294002,DPI,NAC和GM6001预处理NCI-H292细胞,观察MUC5AC以及MMP-9的产生情况。结果 Pa能以时间依赖性方式诱导NCI-H292细胞产生MMP-9,并增加其活性。EGFR活化后,经PI3K途径激活Rac1并诱导MMP-9的表达,最终诱导MUC5AC产生。结论 Pa经EGFR/PI3K/Rac1/NADPH/ROS/MMP-9通路诱导NCI-H292细胞产生MUC5AC。

Objective To investigate the molecular mechanism of Pseudomonas aeruginosa （Pa）-induced mucus MUC5AC expression in the human airway cells NCI-H292. Methods Bronchial epithelial NCI-H292 cells cells were cultured in vitro, MUC5AC production after stimulation with Pa was analyzed by enzyme-linked immunosorbent assay （ELISA）. Production and activity of matrix metalloproteinase 9（MMP-9）was analyzed by a commercial kit （MMP-9 Activity Biotrak Assay System kit）. For inhibition study, cells were pretreated with different inhibitors such as AG1478, LY294002,DPI,NAC and GM6001 before stimulation,which specifically inhibit EGFR,PI3K,NADPH,ROS,and MMP,and then production of MUC5AC or MMP-9 were annualized. Results Pa-stimulated NCI-H292 cells exhibited a time-dependent increase in total MMP-9 levels, and time-dependent increasing levels of active MMP-9. EGFR and PI3K mediated-Racl activation was required for an optimal secretion and activation of MMP-9,and EGFR is necessary for Pa to activate Racl. NADPH-generated ROS act downstream of Racl to promote MMP-9 secretion and activation, and then stimulated MUC5AC production. Conclusion EGFR/PI3K/Racl/NADPH/ROS/MMP-9 regulate MUC5AC production in Pa -challenged NCI-H292 cells.