The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)
目的：探讨蛋白酶激活受体2（protease-activated receptor 2，PAR2）在人支气管上皮细胞向间充质表型转化中的作用及其可能机制。方法：16HBE细胞予以不同浓度类胰蛋白酶（PAR2激动剂）处理，采用划痕、transwell小室、蛋白质免疫印迹等方法观察类胰蛋白酶对细胞上皮间充质转化（epithelial-mesenchymal transition，EMT）过程的影响；通过DCFH-DA探针测定细胞活性氧（reactive oxygen species，ROS）的水平；外源性加入ROS清除剂N-乙酰半胱氨酸（N-acety-1-cysteine，NAC）后，划痕、transwell小室观察类胰蛋白酶诱导的细胞修复与迁移功能。结果：类胰蛋白酶呈剂量依赖性地促进16HBE修复与迁移；下调E-cadherin、上调N-cadherin及α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）表达（P<0.05）。类胰蛋白酶增加16HBE胞内ROS的水平，而PAR2抑制剂FSLLRY-NH2可逆转上述改变（P<0.05）。NAC可有效抑制类胰蛋白酶诱导的细胞修复与迁移（P<0.05）。结论：激活PAR2可经由ROS信号、调控人支气管上皮细胞EMT过程，是细胞表型转化与重塑调控的潜在靶标。
Objective: To investigate the role of protease-activated receptor 2 (PAR2) involved in epithelial-mesenchymal transition (EMT) of human bronchial epithelial (HBE) cells. Method: 16HBE cells were stimulated with various concentrations of tryptase (a nature agonist of PAR2). Cell migration and repair were assessed by transwell and scratch assay. Western blot was used to examine the expressions of EMT associated biomarkers including E-cadherin, N-cadherin and α-smooth muscle actin (α-SMA). DCFH-DA probe was employed to measure the generation of reactive oxygen species (ROS). The effects of N-acety-1-cysteine (NAC) on 16HBE induced by tryptase were also examined by transwell and scratch assay. Results: Tryptase dramatically promoted cell migration and repair with loss of E-cadherin and increase of N-cadherin and α-SMA in a dose-dependent manner (P<0.05). Tryptase enhanced generation of ROS in 16HBE cells, and this effect can be inhibited by PAR2 antagonist FSLLRY-NH2 (FS) (P<0.05). ROS scavenger N-acety-1-cysteine (NAC) significantly inhibited the EMT changes induced by tryptase (P<0.05). Conclusion: Activation of PAR2 triggered HBE EMT process via ROS signal, which highlights a potential target for the regulation of HBE phenotype shift involved in airway remodeling.