乌司他丁抑制肿瘤坏死因子-α诱导血管内皮细胞高通透性的机制研究

Inhibitory role of Ulinastatin in TNF-α induced hyperpermeability of endothelial cells via Rho/ROCK pathway

目的探讨乌司他丁(UTI)对肿瘤坏死因子-α(TNF-α)诱导的血管内皮细胞通透性增高的影响及其主要分子机制。方法离体培养人脐静脉内皮细胞系EA.hy926,分别以UTI和TNF-α作用于EA.hy926,小室法测单层细胞通透性,免疫荧光法测磷酸化肌球蛋白磷酸酶靶向亚基1(p-MYPT1)的表达;分别采用免疫细胞化学法、Western Blot法测与通透性相关的关键分子(RhoA、ROCK2、MYPT1、p-MYPT1及VE-cadherin)表达的变化情况。结果 TNF-α作用下EA.hy926单层细胞通透性增加,细胞内p-MYPT1的表达量较正常对照组明显增加,而UTI可改善EA.hy926细胞的上述变化情况;免疫细胞化学结果显示,与正常对照组比较,TNF-α作用下RhoA、ROCK2的表达明显增加,而UTI可抑制RhoA、ROCK2的高表达;Western Blot结果显示,与正常对照组比较,TNF-α作用下p-MYPT1、RhoA和ROCK2的表达明显增加,VE-cadherin的表达明显降低(均P<0.05),而UTI可抑制p-MYPT1、RhoA和ROCK2蛋白的高表达,增加VE-cadherin的表达。结论 UTI可能通过Rho/ROCK信号通路抑制TNF-α引起的EA.hy926细胞通透性增加。

Objective To investigate the role of Ulinastatin（UTI） on the hyper-permeability of human umbilical vein endothelial cell by TNF-α and possible underlying mechanism. Methods Human umbilical vein endothelial cell（EA.hy926） were cultured in vitro and exposed to UTI and tumor necrosis factor alpha（TNF-α） respectively. The permeability of EA.hy926 cells were detected by a transwell chamber system. Immunofluorescence was used to assay the expression of p-MYPT1. The expression of the key molecules related to endothelial permeability（RhoA, ROCK2, MYPT1, p-MYPTl and VE-cadherin） were detected by immunocytochemistry assays and western-blot. Results Compared with the control group, the permeability of cell exposed to TNF-awas increased significantly, the expression of p-MYPT1 was higher, but UTI could attenuate the phenomena. The immunocytochemistry assays showed that the expression of RhoA and ROCK2 was significantly upregulated in cells treated with TNF-α; however, UTI could inhibit the high expression of these two proteins. Western blots showed that the expression of p-MYPT1, RhoA and ROCK2 were significantly higher（P〈0.05） and VE-cadherin was significantly lower（P〈0.05） after incubation with TNF-α, but UTI treatment could moderate the above changes. Conclusion The serum from septic patients induced hyperpermeability of EA.hy926 cells can be attenuated by ulinastatin in a dose-dependent manner.