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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 investigate the regulation mechanism of upstream signal pathway of 5AC expression in airway epithelial cells induced by air acidic microenvironment. Methods Human airway epithelial cells (16HBE) were cultured in vitro. Hydrogen chloride was used to create a cellular acidic environment (pH 6.4). PD88059, an inhibitor of extracellular signal-regulated kinase (ERK), and OGR1 siRNA The cells were randomly divided into 8 groups: control group, acid stimulation group, acid stimulation + OGR1 siRNA transfection group, pH 7.4 + OGR1 siRNA group, acid stimulation + negative siRNA group, pH 7.4+ negative siRNA group, PD98059 group, pH 7.4 + PD98059 group. The transcription level of MUC 5AC and the level of MUC5AC protein in culture supernatants were detected by RT-PCR and ELISA. The relative contents of OGR1 protein and p-ERK protein were detected by Western blot. Results The mRNA and protein levels of MUC5AC in the acid-stimulated group were significantly higher than those in the control group (all P <0.05), and the protein levels of OGR1 and p-ERK were significantly increased as compared with those in the control group And protein levels were significantly lower than the acid-stimulated group (P <0.05), and its p-ERK content was also significantly reduced. The pH 7.4 + OGR1 siRNA group MUC5AC protein content and mRNA levels compared with the pH7.4 control group no significant difference, the p-ERK content also changed little. Acid stimulation + MUC5AC mRNA transcription and protein levels in PD98059 group were significantly lower than those in acid stimulation group (P <0.05), while there was no significant difference in MUC5AC mRNA and protein levels between pH 7.4 + PD98059 group and control group. Conclusion OGR1 may be involved in the expression of MUC5AC in human airway epithelial cells induced by airway acidic microenvironment by activating ERK signaling pathway.