[中性粒细胞弹性蛋白酶诱导黏液高分泌的上游信号通路]
[Upstream signaling pathway of neutrophil elastase-induced mucous hypersecretion].
作者信息
Li Qi, Zhou Xiang-dong
机构信息
Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
出版信息
Zhonghua Yi Xue Za Zhi. 2007 Jan 30;87(5):348-52.
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.
目的
研究中性粒细胞弹性蛋白酶（NE）通过表皮生长因子受体（EGFR）诱导黏液高分泌的上游信号转导途径。
方法
培养BEAS-2B气道上皮细胞并分为6组：对照组，在无血清DMEM/Ham's F12混合液中培养；NE组，单独用NE孵育；转化生长因子（TGF）-α蛋白酶抑制剂（TAPI）-1+NE组，用NE和TAPI-1孵育；1,3-二甲基-2-硫脲（DMTU）+NE组，先用活性氧（ROS）清除剂DMTU预孵育，然后用NE孵育；TGF-α组，用TGF-α孵育；DMTU+TGF-α组，用DMTU和TGF-α孵育。通过逆转录聚合酶链反应（RT-PCR）分析黏蛋白（MUC）5AC mRNA水平，用酶联免疫吸附测定法检测MUC5AC蛋白和可溶性TGF-α，用蛋白质印迹法检测磷酸化EGFR（p-EGFR）蛋白表达。
结果
NE组的MUC5AC mRNA表达和蛋白表达分别为0.81±0.13和0.78±0.06，均显著高于对照组（分别为0.32±0.07和0.24±0.08，均P<0.01）。NE组的可溶性TGF-α蛋白表达和p-EGFR蛋白表达分别为0.83±0.14和0.88±0.07，均显著高于对照组（分别为0.25±0.13和0.28±0.11，均P<0.01）。TAPI-1组的MUC5AC mRNA、MUC5AC蛋白、可溶性TGF-α和p-EGFR的表达率分别为0.37±0.04、0.30±0.11、0.32±0.07和0.43±0.09，均显著低于NE组（均P<0.01）。DMTU+NE组的MUC5AC mRNA、MUC5AC黏蛋白和可溶性TGF-α水平分别为0.36±0.03、0.39±0.12和0.43±0.05，均显著低于NE组（均P<0.01），然而，DMTU+TGF-α组的MUC5AC mRNA表达和MUC5AC蛋白表达分别为0.58±0.13和0.55±0.11，与TGF-α组（分别为0.56±0.0并0度8和0.57±0.13，均P>0.05）均无显著差异。
结论
NE通过ROS/TACE/前体TGF-α/EGFR诱导气道上皮细胞中MUC5AC表达。
Zotero 插件