不可分型流感嗜血杆菌诱导NCI-H292细胞产生MUC5AC的分子机制

目的:探讨不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae,NTHi)诱导气道上皮细胞表达黏蛋白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NTHi感染后,采用ELISA检测MUC5AC和基质金属蛋白酶9(MMP-9)的水平;明胶酶谱实验分析MMP-9的酶活性;同时分别采用表皮生长因子受体(EGFR)、磷脂酰肌醇3-激酶(PI3K)、NADPH氧化酶、活性氧簇(ROS)、和MMP-9特异性抑制剂AG1478、LY294002、DPI、NAC和GM6001预处理NCI-H292细胞,检测MUC5AC以及MMP-9的水平。结果:NTHi能以时间依赖性方式诱导NCI-H292细胞产生MMP-9,并上调其酶活性,同时增加Rac1的活性并诱导ROS生成;采用AG1478和LY294002处理后,Rac1活性显著降低;采用DPI或Rac1抑制剂NSC23766处理后,ROS含量明显减少;当NCI-H292细胞用NAC或NSC23766预处理后,可显著下调MMP-9的表达与活性。此外,采用GM6001处理后,MUC5AC的分泌明显降低。结论:NTHi经EGFR/PI3K/Rac1/NADPH氧化酶/ROS/MMP-9通路诱导NCIH292细胞产生MUC5AC。

Molecular mechanism of nontypeable Haemophilus influenzae stimulated MUC5AC production in NCI-H292 cells

AIM: To investigate the molecular mechanism of nontypeable Haemophilus influenzae(NTHi)-induced MUC5AC expression in the human airway NCI-H292 cells.METHODS: Bronchial epithelial NCI-H292 cells were cultured in vitro. The production of MUC5AC and matrix metalloproteinase 9(MMP-9) after stimulation with NTHi was analyzed by enzyme-linked immunosorbent assay(ELISA). The enzymatic activity of MMP-9 was detected by gelatin zymography. In addition, the cells were pretreated with different inhibitors such as AG1478, LY294002, DPI, NAC and GM6001 before stimulation, which specifically inhibit epidermal growth factor receptor(EGFR), phosphatiadylinositol 3-kinase(PI3K), NADPH oxidase, reactive oxygen species(ROS) and MMP-9, respectively, and then the production of MUC5AC or MMP-9 was determined.RESULTS: NTHi-stimulated NCI-H292 cells exhibited a time-dependent increase in MMP-9 secretion and activity. NTHi also increased the activity of Rac1 and the production of ROS. Pretreatment of AG1478 and LY294002 decreased the Rac1 activity, and preincubation of DPI or Rac1 inhibitor significantly abrogated ROS production. In addition, secretion of MMP-9 and the enzymatic activity was decreased by treatment of NAC and NSC23766. Furthermore, inhibition of the MMP-9 activity by GM6001 significantly inhibited MUC5AC production.CONCLUSION: EGFR/PI3K/Rac1/NADPH oxidase/ROS/MMP-9 regulates MUC5AC production in NTHi-challenged NCI-H292 cells.