类胰蛋白酶通过激活 PAR-2促进肠黏膜上皮 IEC-6细胞损伤

摘要

[ ABSTRACT] AIM:To investigate the role of protease activated receptor-2 ( PAR-2 ) in the process of tryptase mediated IEC-6 cell injury.METHODS:The rat intestinal epithelial cell line IEC-6 was treated with tryptase at different concentrations (1 μg/L, 10 μg/L, 100μg/L and 1 000μg/L) in the presence or absence of PAR-2 antagonist FSLLRY-NH2 for 12 h respectively.The cell survival rate was detected by MTT assay.The protein levels of PAR-2 and cleaved-caspase 3 were determined by Western blotting.The LDH activity was also measured.RESULTS:Compared with control group, the cell survival rates were significantly decreased in 100 μg/L and 1 000 μg/L tryptase treated groups, the LDH activities were significantly increased in 10 μg/L to 1 000 μg/L tryptase treated groups, and the protein levels of PAR-2 and cleaved caspase 3 were significantly increased in 100μg/L and 1 000μg/L tryptase treated groups (P<0.05).Com-pared with 1 000 μg/L tryptase treated group, the LDH activity and cleaved caspase 3 protein level were dramatically de-creased while the survival rate was significantly increased in the presence of PAR-2 antagonist FSLLRY-NH2 (P<0.05). CONCLUSION:Tryptase induces IEC-6 cell injury in a dose-dependent manner by activating PAR-2.%目的：观察类胰蛋白酶（ tryptase）对小肠黏膜上皮细胞IEC-6的作用及机制。方法：体外培养IEC-6细胞，随机分为对照（control）组、tryptase处理组、蛋白酶激活受体2（PAR-2）抑制剂（FSLLRY-NH2，FS）处理组以及tryptase＋FS处理组。采用MTT法检测细胞生存率；测定分泌的乳酸脱氢酶（ LDH）活性；Western blotting测定PAR-2和cleaved-caspase 3的表达。结果：与对照组比，100μg／L和1000μg／L类胰蛋白酶导致细胞生存率明显降低（P＜0.05）。与对照组相比，10μg／L到1000μg／L类胰蛋白酶导致LDH的活性明显增加（P＜0.05）。与对照组相比，100μg／L和1000μg／L类胰蛋白酶导致细胞的PAR-2及cleaved-caspase 3表达明显升高（ P＜0.05）。与1000μg／L tryptase处理组比较， FS能够明显增加细胞生存率、降低LDH活性及cleaved-caspase 3表达（ P＜0.05）。结论：类胰蛋白酶通过激活PAR-2促进IEC-6细胞损伤，且这种损伤呈浓度依赖性。