香烟烟雾提取物对A549细胞MUC5AC分泌影响的机制研究

Effects of Cigarette Smoke Extract on Signal Transduction Pathway of MUC5AC Mucin Expression in A549 Cells

目的探讨香烟烟雾提取物(CSE)所致人气道上皮细胞黏液高分泌的分子机制及血红素加氧酶-1(HO-1)对该分子机制的阻断作用。方法通过CSE刺激人肺A549细胞,构建气道黏液高分泌的细胞模型,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HO-1、双功能氧化酶1(Duox1)、黏蛋白5AC(MUC5AC)、表皮生长因子受体(EGFR)及磷酸化EGFR(p-EGFR)的表达。将A549细胞分为对照组、CSE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MUC5AC的mRNA水平;Western blot法检测p-EGFR、EGFR、Duox1及HO-1的蛋白水平;并用酶联免疫吸附测定法(ELISA)检测各组上清液及细胞裂解液中的MUC5AC蛋白含量。结果 CSE刺激组MUC5AC的mRNA积分吸光度值、培养上清液及细胞裂解液中MUC5AC蛋白水平分别为(0.660±0.044)、(62.80±6.85)μg/mg及(157.00±3.26)μg/mg,EGFR mRNA和蛋白的积分吸光度值分别为(0.596±0.061)和(0.620±0.051),均较对照组明显升高(P<0.05),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增高(P<0.05)。给予Hemin预处理后,与CSE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和蛋白水平降低(P<0.05)。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和蛋白水平明显增高(P<0.05)。结论 HO-1可通过下调Duox1的表达,阻断香烟烟雾诱导的EGFR活化上游的配体依赖信号途径,减少EGFR活化,从而抑制黏蛋白MUC5AC的合成及分泌,此结果为研究香烟烟雾诱导的气道黏液高分泌的信号机制提供了新的线索。

Objective To investigate the effects of heme oxygenase-1 （HO-1） on the signal transduction pathway of cigarette smoke-induced mucin （MUC） expression in airway mucous hypersecretion. Methods The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by cigarette smoke extract （CSE）, and treated with hemin or ZnPPIX, an inductor or inhibitor of HO-1. The expression of HO-1, dual oxidase 1 （Duoxl）, MUCSAC, epidermal growth factor receptor （EGFR） and p-EGFR were detected. The cell viability treated with hemin or ZnPPIX was assessed by methyl thiazolyl tetrazolium method （MTT）. The cells were divided into 4 groups： a negative control group, a CSE treatment group, an hemin pre-treatment group and a ZnPPIX pre-treatment group. The changes of EGFR mRNA, Duoxl mRNA, HO-1 mRNA and MUC5AC mRNA were examined by reverse transcriptase-polymerase chain reaction （RT-PCR）. The protein expression changes of Duox1, HO-1, p-EGFR and EGFR were measured by Western blot, while the protein of MUCSAC in supernate and endoehylema were detected by enzyme-linked immunosorbent assay （ELISA）. Results The expression levels of MUCSAC mRNA and its protein in supernate liquid and endochylema in the CSE group were （0.660±0.044）, （62.80±6.85） μg/mg and （157.00±3.26） μg/mg, and those of EGFR were 0.596±0.061 and 0.620±0.051, both of them increased significantly （P〈0.05） compared with the control group [（0.412±0.043）, （42.57±7.23） μg/mg, （105.28±7.73） μg/mg and 0.426±0.064, 0.340±0.052]. The protein of p-EGFR, the mRNA and protein of Duoxl, HO-linereased significantly compared with the control group （P〈0.05）. After the cells were pre-treated with hemin, the mRNA and protein of HO-1 increased significantly, while the protein of p-EGFR, the mRNA and protein of EGFR, Duoxl and MUCSAC decreased significantly compared with the CSE group（P〈0.05）. After the cells were pre-treated with ZnPPIX, the increase of HO-1 mRNA and protein were not significant compared with the control group （P〉0.05）, while the protein of p-EGFR, the mRNA and protein of EGFR, Duoxl and MUCSAC increased significantly （P〈0.05）. Conclusion HO-1 may decrease the expression levels of MUCSAC through down-regulating the expression of Duox 1, blocking ligand-dependent EGF-R activation, which maybe is a new clue for the mechanism of airway mucous hypersecretion induced by cigarette smoke.