中文摘要：

目的探讨血红素加氧酶-1（HO-1）对炎性刺激时人气道上皮细胞黏液分泌的影响。方法通过人中性粒细胞弹性蛋白酶（HNE）刺激人肺腺癌细胞A549,构建炎性刺激下气道黏液高分泌模型,给予HO-1诱导剂氯化高铁血红素（Hemin）及HO-1抑制剂锌原卟啉（ZnPPⅨ）干预,观察黏蛋白5AC（MUC5AC）、表皮生长因子受体（EGFR）、磷酸化EGFR（p-EGFR）、HO-1及双功能氧化酶1（Duox1）的表达。将A549细胞分为对照组、HNE刺激组、Hemin干预组和ZnPPⅨ干预组。用四甲基偶氮唑盐法（MTT）测定Hemin及ZnPPⅨ对细胞活性的影响;逆转录-聚合酶链反应（RT-PCR）方法检测各组HO-1、Duox1、EGFR及MUC5ACmRNA的转录水平;Westernblot法检测p-EGFR、EGFR、Duox1和HO-1蛋白的表达;酶联免疫吸附测定法（ELISA）检测细胞裂解液中MUC5AC蛋白表达水平的变化;并用细胞免疫化学激光共聚焦显微镜观察不同条件下MUC5AC蛋白表达的变化。结果 HNE刺激组MUC5ACmRNA积分吸光度值和蛋白水平分别为（0.845±0.044）和（192.68±4.71）μg/mg,EGFRmRNA和蛋白的积分吸光度值分别为（0.868±0.039）和（0.623±0.052）,均较对照组[（0.462±0.053）、（104.95±6.82）μg/mg和（0.528±0.054）、（0.297±0.055）]明显升高（均P〈0.01）;p-EGFR蛋白水平亦明显升高,且HO-1及Duox1的mRNA和蛋白水平也较对照组显著增高（均P〈0.01）。给予Hemin预处理后,与HNE刺激组相比,HO-1mRNA及蛋白明显增多,p-EGFR蛋白水平、Duox1、EGFR及MUC5AC的mRNA和蛋白水平均降低（均P〈0.01）。给予ZnPPⅨ预处理后,与对照组相比,HO-1mRNA及蛋白水平无明显增高（P〉0.05）,而p-EGFR蛋白水平、Duox1、EGFR及MUC5AC的mRNA和蛋白水平均明显增高（均P〈0.01）。结论 HO-1可通过下调Duox1的表达,阻断EGFR活化上游的配体依赖信号途径,减少EGFR活化,抑制MUC5AC的表达,此机制为气道黏液高分泌疾病的防治提供了新的方向。

英文摘要：

Objective To investigate the effects of heme oxygenase-1（HO-1）on the signal transduction pathway of mucin in airway mucous hypersecretion.Methods The cell model of mucous hypersecretion was made by human lung A549 cells stimulated by human neutrophil elastae（HNE）,and treated with hemin or ZnPP Ⅸ,an inductor or inhibitor of HO-1.The expression of mucin（MUC）5AC,epidermal growth factor receptor（EGFR）,p-EGFR,HO-1 and dual oxidase 1（Duox1）was detected.The cell viability after hemin or ZnPP Ⅸ treatment was assessed by methyl thiazolyl tetrazolium method（MTT）.The cells were divided into 4 groups：a negative control group,an HNE treatment group,a hemin pre-treatment group and a ZnPP Ⅸ pre-treatment group.The mRNA changes of HO-1,Duox1,EGFR and MUC5AC were examined by using reverse transcriptase-polymerase chain reaction（RT-PCR）.The protein expression changes of EGFR,p-EGFR,Duox1 and HO-1 were measured by Western blot,and the protein expression changes of MUC5AC were detected by enzyme-linked immunosorbent assay（ELISA）.The protein changes of MUC5AC were observed by immunofluorescence and confocal laser technology.Results The expression levels of MUC5AC mRNA and its protein in the HNE group were（0.845±0.044）and（192.68±4.71）μg/mg,and those of EGFR were（0.868±0.039）and（0.623±0.052）,both of them increased significantly（all P0.01）as compared with those in the control group／[（0.462±0.053）,（104.95±6.82） μg/mg,and （0.528±0.054）,（0.297±0.055）／].And the mRNA and protein expression of Duox1 and HO-1,the protein expression of p-EGFR were increased significantly as compared with the control group（all P〈0.01）.After the cells were pre-treated with hemin,the mRNA and protein expression of HO-1 was increased significantly,while the mRNA and protein expression of Duox1,EGFR and MUC5AC,the protein of p-EGFR were decreased significantly as compared with the HNE group（all P〈0.01）.After the cells were pre-treated with ZnPP Ⅸ,the HO-1 mRNA and