OGR1籍ERK信号通路参与酸性微环境致气道上皮细胞黏蛋白5AC表达

Study of OGR1/ERK Mediated Mechanism for the Acid-induced Mucin 5AC Expression and Secretion in Human Airway Epithelial Cells

目的探讨气道酸性微环境诱导气道上皮细胞黏蛋白5AC表达的上游信号通路调节机制。方法体外培养人气道上皮细胞(16HBE),以氯化氢创造细胞酸性环境(pH 6.4),以细胞外信号调节激酶(ERK)特异性抑制剂PD88059及卵巢癌G蛋白耦联受体1(OGR1)siRNA进行干预,将细胞随机分为8组:对照组、酸刺激组、酸刺激+转染OGR1 siRNA组、pH 7.4+OGR1siRNA组、酸刺激+阴性siRNA组、pH 7.4+阴性siRNA组、酸刺激+PD98059组、pH 7.4+PD98059组。采用RT-PCR、ELISA法分别观察细胞黏蛋白(MUC)5AC转录水平及培养上清液中MUC5AC蛋白水平的改变;Western blot法检测OGR1蛋白及p-ERK蛋白的相对含量。结果酸刺激组内细胞MUC5AC转录及蛋白水平显著高于对照组(P值均<0.05),其OGR1及p-ERK蛋白含量较对照组亦明显增加,酸刺激+转染OGR1 siRNA组MUC5AC mRNA转录及蛋白水平显著低于酸刺激组(P值均<0.05),其p-ERK含量亦明显降低。而pH 7.4+OGR1 siRNA组MUC5AC蛋白含量及mRNA水平较pH7.4对照组无明显差别,其p-ERK含量也变化不大。酸刺激+PD98059组MUC5AC mRNA转录及蛋白水平显著低于酸刺激组(P值均<0.05),而pH 7.4+PD98059组较对照组MUC5AC mRNA转录及蛋白水平无明显差异。结论 OGR1可能通过激活ERK信号通路参与气道酸性微环境所致的人气道黏膜上皮细胞MUC5AC表达。

Objective To explore the related signal transduction mechanism in acid-induced expression of MUC5AC mRNA by airway epithelial cells. Methods 16HBE cells were stimulated by hydrogen chloride, and treated with OGR1 siRNA and an inhibitors to ERK （PD98059）. Cells were divided into 8 groups ： the control group （pH 7.4）, the acidic stimulation group （pH 6.4）, the acidic stimulation＋ OGR1 siRNA group, the pH 7.4＋ OGRI siRNA group, the acidic stimulation＋control siRNA group, the pH 7.4＋control siRNA group, the acidic stimulation＋PD98059 group, and the pH 7.4＋PD98059 group. The transcription level of mucin （MUC）5AC was evaluated with reverse transcription-polymerase chain reaction （RT-PCR）. The MUC5AC secreted in supernatant was measured by enzyme-linked immunosorbent assay （ELISA）. The expression of OGR1 pro- tein and the protein levels of phosphorylation extracellular signal-regulated kinase （p-ERK） were measured by Western blot. Results The expres- sion of MUC5AC mRNA and MUC5AC protein levels were remarkably higher in the acidic stimulation group compared with those in the control group（P〈 0.05）. The expression of OGRI and p-ERK protein also significantly increased. The expression of MUC5ACmRNA and MUC5AC pro- tein levels in the acidic stimulation＋OGR1 siRNA group were also significantly lower than those in the control group and its p-ERK decreased re- markably. There is no significant difference in expression of MUC5AC mRNA and mRNA level between the control group and p-ERK also showed no significant change after transfection of OGR1 siRNA. The secretion level of MUC5AC and the expression of MUC5AC mRNA were much lower in the acidic stimulation＋PD98059 group than in the acidic stimulation group （P〈0.05）, while there were no significant difference between the control group and the pH 7.4＋PD98059 group in the secretion level of MUC5AC and expression of MUC5AC mRNA （P 〉 0.05 ）. Conclusion OGR1/ERK transduction cascade may be upper signal regulators in acid-induced MUC5AC expression in 16HBE cells.