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

doi:10.3877/cma.j.issn.2096-1537.2018.02.013

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。

关键词: 血管内皮细胞EA.hy926, 高通透性, 乌司他丁, 肿瘤坏死因子-α, Rho/ROCK信号通路

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-MYPT1 and VE-cadherin) were detected by immunocytochemistry assays and western-blot.

Results

Compared with the control group, the permeability of cell exposed to TNF-αwas 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.

Key words: Human umbilical vein endothelial cell line EA.hy926, Ulinastatin, High permeability, Tumor necrosis factor alpha, Rho/ROCK pathway

表1 不同UTI浓度对TNF-α作用下EA.hy926细胞高通透性的影响（n=3）

组别	相对通透性（吸光值A值）
1 U/ml UTI＋TNF-α组	1.854±0.064
10 U/ml UTI＋TNF-α组	1.500±0.081^b
100 U/ml UTI＋TNF-α组	1.234±0.013^b
1000 U/ml UTI＋TNF-α组	1.654±0.155
TNF-α组	2.097±0.308^a
正常对照组	1.346±0.038

表1 不同UTI浓度对TNF-α作用下EA.hy926细胞高通透性的影响（n=3）

组别	相对通透性（吸光值A值）
1 U/ml UTI＋TNF-α组	1.854±0.064
10 U/ml UTI＋TNF-α组	1.500±0.081^b
100 U/ml UTI＋TNF-α组	1.234±0.013^b
1000 U/ml UTI＋TNF-α组	1.654±0.155
TNF-α组	2.097±0.308^a
正常对照组	1.346±0.038

图1 UTI对TNF-α作用下EA.hy926细胞p-MYPT1表达水平的影响。以UTI（100 U/ml）预处理EA.hy926细胞1 h，然后加入TNF-α作用24 h，于激光共聚焦显微镜下观察p-MYPT1的表达情况，其中图a为正常对照组；图b为TNF-α组；图c为UTI组；图d为UTI＋TNF-α组；箭头示TNF-α所引起的p-MYPT1高表达；UTI为乌司他丁；TNF-α为肿瘤坏死因子α

图2 UTI对TNF-α作用下EA.hy926细胞RhoA及ROCK2表达的影响（×200）。以UTI（100 U/ml）预处理EA.hy926细胞1 h，然后加入TNF-α作用24 h，于200倍显微镜下观察RhoA及ROCK2的表达情况，其中图a/e为正常对照组；图b/f为TNF-α组；图c/g为UTI组；图d/h为UTI＋TNF-α组；UTI为乌司他丁；TNF-α为肿瘤坏死因子α

图3 UTI对TNF-α作用下EA.hy926细胞Rho/ROCK信号通路关键分子表达的影响。以UTI（100 U/ml）预处理EA.hy926细胞1 h，然后加入TNF-α作用24 h，用Western-blot法检测细胞RhoA、ROCK2、VE-cadherin、MYPT1及p-MYPT1蛋白的表达

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

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