中性粒细胞弹力蛋白酶引起黏蛋白5AC高表达的信号转导机制

目的研究中性粒细胞弹力蛋白酶（NE）通过表皮生长因子受体（EGFR）引起黏蛋白（MUC）5AC高表达的上游信号通路。方法培养人气道BEAS@B上皮细胞，给予NE刺激，以肿瘤坏死因子转换酶抑制剂-1（TAPI-1）及活性氧（ROS）清除剂DMTU为干预因素。RT-PCR检测干预前后细胞中MUCSACmRNA含量；酶联免疫吸附测定法（ELISA）检测培养上清中MUCSAC、可溶性转化生长因子（TGF）-α蛋白含量；Westem印迹分析磷酸化表皮生长因子受体（P-EGFR）蛋白水平。结果NE刺激后引起MUCSAC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（P〈0．01），同时伴随可溶性TGF-α及p-EGFR蛋白水平的增高，与对照组相比，差异有统计学意义（均P〈0．01）；加入浓度为20umol/L的肿瘤坏死因子转换酶抑制剂TAPI-1后可明显下调上述指标的表达水平（均P〈0．01），DMTU也同样降低了MUC5AC的基因转录、产物水平以及可溶性TGF-α蛋白相对含量，与单纯NE刺激组比较差异均有统计学意义（P〈0．01），但对单纯外源性TGF-α刺激组引起的MUCSAC基因转录和蛋白合成增加无明显作用（均P〉0．05）。结论NE能刺激细胞产生ROS，再活化肿瘤坏死因子转换酶（TACE）引起黏蛋白产生的增多，组成EGFR通路上游的主要信号分子，故ROS／TACE／TGF-α前体／EGFR转导途经是NE引起气道黏液高分泌的重要机制。

Upstream signaling pathway of neutrophil elastase-induced mucous hypersecretion

OBJECTIVE: To investigate the upstream signal transduction pathway of neutrophil elastase (NE)-induced mucous hypersecretion via epidermal growth factor receptor (EGFR). METHODS: The airway epithelial cells of the line BEAS-2B were cultured and divided into 6 groups: control group, cultured in serum-free DMEM/Ham's F12 mixture; NE group, incubated with NE alone, transforming growth factor (TGF)-alpha protease inhibitor (TAPI)-1 + NE group, incubated with NE and TAP I-1; 1, 3-dimethyl-2-thiourea (DMTU) + NE group, preincubated with DMTU, a reactive oxygen species (ROS) scavenger, and then incubated with NE; TGF-alpha group, incubated with TGF-alpha; and DMTU + TGF-alpha group, incubated with DMTU and TGF-alpha. The mucin (MUC) 5AC mRNA level was analyzed by RT-PCR, MUC5AC protein and soluble TGF-alpha were detected by enzyme-linked immunosorbent assay, and the phosphorylated EGFR (p-EGFR) protein production was assayed by Western blotting. RESULTS: The MUC5AC mRNA expression and protein expression of the NE group were 0.81 +/- 0.13 and 0.78 +/- 0.06 respectively, both significantly higher than those of the control group (0.32 +/- 0.07 and 0.24 +/- 0.08 both P < 0.01. The protein expression of soluble TGF-alpha and the protein expression of p-EGFR protein of the NE group were 0.83 +/- 0.14 and 0.88 +/- 0.07 respectively, both significantly higher than those of the control group (0.25 +/- 0.13 and 0.28 +/- 0.11 respectively, both P < 0.01). The expression rates of MUC5AC mRNA, MUC5AC protein, soluble TGF-alpha, and p-EGFR of the TAPI-1 group were 0.37 +/- 0.04, 0.30 +/- 0.11, 0.32 +/- 0.07, and 0.43 +/- 0.09 respectively, all significantly lower than those of the NE group (all P < 0.01). The level of MUC5AC mRNA, MUC5AC mucin protein, and soluble TGF-alpha of the DMTU + NE group were 0.36 +/- 0.03, 0.39 +/- 0.12, and 0.43 +/- 0.05 respectively, all significantly lower than those of the NE group (all P < 0.01), however, the MUC5AC mRNA expression and MUC5AC protein expression of the DMTU + TGF-alpha group were 0.58 +/- 0.13 and 0.55 +/- 0.11 respectively, both not significantly different from those of the TGF-alpha group (0.56 +/- 0.08 and 0.57 +/- 0.13 respectively, both P > 0.05). CONCLUSION: NE induces MUC5AC expression via ROS/TACE/pro-TGF-alpha/EGFR in the airway epithelial cells.