孟鲁斯特对人支气管上皮细胞黏液蛋白分泌的影响

Effect of montelukast on the secretion of mucus protein in human bronchial epithelial cells

目的探讨孟鲁斯特对脂多糖(LPS)诱导的人支气管上皮细胞黏液蛋白分泌的影响。方法体外分离鉴定人原代支气管上皮细胞,LPS以1μg/ml剂量诱导细胞炎症反应,孟鲁斯特(50、20、10μmol/L)进行干预治疗,酶联免疫法(ELISA)检测分泌细胞上清黏蛋白5AC(MUC5AC)水平变化,并采用定量逆转录PCR(RT-PCR)、免疫印迹(Western-blot)检测MUC5AC m RNA和蛋白表达情况;2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针检测活性氧(ROS)变化;Western-blot检测孟鲁斯特干预治疗前后磷酸化细胞核转录因子κB(p-NF-κB)、磷酸化NF-κB抑制蛋白(p-IκBα)、磷酸化细胞外调节激酶1,2(ERK1/2)活化情况。结果孟鲁斯特以剂量依赖性降低LPS诱导的人支气管上皮细胞内MUC5AC mRNA水平和蛋白水平,抑制ROS产生,抑制NF-κB/p-IκBα、ERK活化。结论孟鲁斯特在体外能抑制MUC5AC的产生,其机制可能与抑制NF-κB、EKR的激活有关。

Objective To investigate the effect ofmontelukast on the secretion of mucus protein in lipopolysaccharide （LPS） induced human bronchial epithelial cells. Methods Primary human bronchial epithelial cells were isolated and identified in vitro. LPS （1 μg/mL） was used to induce cell inflammatory response. Montelukast （50 μmol/L, 20μmol/L, 10μmol/L） was used as intervention. The concertration of mucin-5 subtype AC （MUC5AC） in cell supematants was measured by ELISA. The expression levels of MUC5AC mRNA and protein were determined by RT-PCR and Western-blot. DCFH-DA fluorescent probe was used to detect reactive oxygen species （ROS）. To further elucidate the mechanism, NF-κB （2065）, IκBα, ERK1/2 phosphorylation before and after montelukast intervention were determined by Western-blot. Results Montelukast decreases the expression levels of MUC5AC mRNA and protein in a dose-dependent manner in LPS induced human bronchial epithelial cells. Meanwhile, monteluskast suppresses ROS generation and NF-κB （p65）, IκBα, ERK1/2 phosphorylation. Conclusions In response to LPS induced inflammation, montelukast decreases the expression level of MUC5AC in vitro, which may be related to NF-κB and ERK activation.