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目的探讨膜连接蛋白Ezrin在中性粒细胞弹性蛋白酶(NE)诱导气道黏液高分泌的作用及相关调节机制。方法以中性粒细胞弹性蛋白酶刺激培养的人气道上皮HBE16细胞,ELISA法、real-time PCR法测定黏蛋白(MUC)5AC的蛋白及mRNA水平。免疫荧光法检测Ezrin蛋白含量。结果 NE刺激30 min后MUC5AC mRNA表达增强,细胞中及培养上清中黏蛋白MUC5AC的含量均显著高于对照组(P<0.01);转染Ezrin永久磷酸化载体pEGFPN1-Ezrin-T567D的细胞经NE刺激后,胞质内磷酸化Ezrin表达明显增强,且胞膜分布增多。同时,细胞培养上清液中的MUC5AC蛋白含量亦显著高于单纯NE组(P<0.01)。遏制Ezrin磷酸化pEGFP-N1-Ezrin-T567A载体转染组在NE刺激后,Ezrin蛋白与pEGFP-N1-Ezrin-T567D组相比有明显减少,且未出现向胞膜聚集的趋势,同时分泌至培养上清的MUC5AC蛋白也有明显减少(P<0.01),同时伴随胞质内MUC5AC蛋白有所增加(P<0.05)。结论 Ezrin蛋白参与了NE诱导的MUC5AC蛋白分泌,是气道上皮细胞MUC5AC分泌的重要调控分子。
Objective To investigate the role of membrane-bound Ezrin in the induction of airway mucus hypersecretion by neutrophil elastase (NE) and its related regulatory mechanism. Methods The cultured human airway epithelial HBE16 cells were stimulated with neutrophil elastase and the protein and mRNA levels of mucins (MUC) 5AC were determined by ELISA and real-time PCR. Immunofluorescence assay Ezrin protein content. Results The expression of MUC5AC mRNA increased after 30 min of NE stimulation, and the content of mucin MUC5AC in cells and culture supernatants was significantly higher than that in control group (P <0.01). The cells transfected with Ezrin permanent phosphorylation vector pEGFPN1-Ezrin-T567D After NE stimulation, phosphorylation of Ezrin in cytoplasm significantly increased, and the distribution of membrane increased. Meanwhile, the content of MUC5AC in the cell culture supernatant was also significantly higher than that in the NE group (P <0.01). Inhibition of Ezrin phosphorylation in EEG pEGFP-N1-Ezrin-T567A vector transfected group under NE stimulation, Ezrin protein and pEGFP-N1-Ezrin-T567D group significantly reduced, and did not appear to the membrane aggregation trend, while secreting The level of MUC5AC protein in the culture supernatant also decreased significantly (P <0.01), accompanied by increased MUC5AC protein in the cytoplasm (P <0.05). Conclusion Ezrin protein is involved in NE-induced MUC5AC protein secretion and is an important regulator of MUC5AC secretion in airway epithelial cells.