地塞米松在中性粒细胞弹力酶致气道黏液高分泌中的作用机制

Mechanism of Dexamethasone on Airway Mucus Hypersecretion Induced by Neutrophil Elastase

目的探讨糖皮质激素在中性粒细胞弹力酶(NE)引起气道黏液高分泌中的作用机制。方法用NE刺激A549细胞,给予地塞米松处理后,以分裂原激活的蛋白激酶磷酸酶-1(MKP-1)特异性抑制剂Ro-31-8220、糖皮质激素受体(GR)拮抗剂RU-486为干预因素。采用RT-PCR法检测黏蛋白5AC(MUC5AC)mRNA和MKP-1 mRNA的表达,ELISA法检测细胞中MUC5AC蛋白含量,Western blotting法检测磷酸化细胞外调节激酶(ERK)水平和MKP-1蛋白的表达。结果 NE刺激后引起MUC5AC基因转录和蛋白表达水平明显高于未给予NE刺激的对照组,同时伴随P-ERK水平的增高;给予地塞米松处理后,上述指标的水平明显降低,同时MKP-1的蛋白及mRNA水平增高;而给予Ro-31-8220或RU-486干预后,ERK及MUC5AC蛋白、MUC5AC mRNA水平则增高,而MKP-1的蛋白及其mRNA水平降低。结论NE可通过ERK信号通路引起气道黏液的高分泌,而地塞米松可阻断这一过程,这是通过上调MKP-1的水平使ERK去磷酸化失活而实现的,且MKP-1的上调呈GR依赖性。

Objective To investigate the mechanism of glucocorticoids on the airway mucus hypersecretion induced by neutrophil elastase（NE）.Methods A549 cells were stimulated by NE,and dealt with dexamethasone.The intervention factors were Ro-31-8220 and RU-486.Mucoprotein 5AC（MUC 5AC） mRNA and mitogen-activated protein kinase phosphatase-1（MKP-1） mRNA expression were determined by RT-PCR.MUC 5AC protein contained in cells was detected by ELISA.Phosphorylated extracellular signal-regulated kinase（ERK） level and MKP-1 protein expression were measured by Western blotting.Results In the group stimulated by NE,gene transcription level and protein expression of MUC 5AC were significantly higher than those in the control group,and P-ERK level was higher than that in the control group.Glucocorticoids significantly reduced P-ERK level,MUC 5AC protein expression and gene transcription level,while increased MKP-1 protein and mRNA expression.Ro-31-8220 or RU-486 significantly increased the levels of ERK,MUC 5AC protein and MUC 5AC mRNA expression,whereas significantly decreased MKP-1 protein and mRNA expression.Conclusion NE induces airway mucus hypersecrtion via EPK signaling pathway,and dexamethasone may block this process.Dexamethasone could inactivate EPK dephosphorylation by increasing MKP-1 level.The up-regulation of EPK may be glucocorticoids receptor dependent.