乌司他丁对炎症状态下血管内皮屏障功能的影响及机制

doi:10.3877/cma.j.issn.2096-1537.2020.04.013

目的

阐明乌司他丁（UTI）对炎症状态下血管内皮屏障功能的影响及具体分子机制。

方法

以人脐静脉内皮细胞系EA.hy926为研究对象，建立炎症细胞模型及RhoA过表达细胞模型，分别用UTI和TNF-α处理细胞，采用Transwell法、TEER法检测细胞通透性；Western Blot法及RT-PCR法检测Rho/ROCK信号通路相关关键分子（RhoA、ROCK2、MYPT1、p-MYPT1及VE-cadherin）的表达变化情况。

结果

与正常对照组相比，TNF-α作用后EA.hy926细胞电阻值明显降低，细胞通透性显著升高，差异具有统计学意义[（33.67±4.04）Ω/cm² vs（67.17±3.81）Ω/cm²，t=10.435，P<0.01]，细胞内RhoA、ROCK2、p-MYPT1的表达量明显增加，差异具有统计学意义（均P<0.05），VE-cadherin的表达量明显降低，差异具有统计学意义（P<0.05），而UTI可逆转上述变化情况；与UTI处理组相比，RhoA过表达细胞模型中RhoA、ROCK2及p-MYPT1的表达显著增高，VE-cadherin表达降低，细胞通透性增加，差异具有统计学意义（均P<0.05），而空载病毒组中上述分子的表达水平以及细胞通透性无明显变化，差异无统计学意义（均P>0.05）。

结论

UTI能通过Rho/ROCK信号通路抑制TNF-α引起的血管内皮细胞通透性增加，改善血管内皮屏障功能障碍。

关键词: 乌司他丁, 炎症, 内皮屏障功能

Objective

To clarify the effect of Ulinastatin (UTI) on vascular endothelial barrier function under inflammatory state and its molecular mechanism.

Methods

Human umbilical vein endothelial cell line (EA.hy926) was used as the research object to established inflammatory cell model and RhoA overexpressing cell model. Cells were treated with UTI and TNF-α respectively, a transwell chamber system andtransepithelial electric resistance (TEER) method were used to detect cell permeability. The expression of Rho/ROCK signaling pathway-related key molecules (RhoA, ROCK2, MYPT1, p-MYPT1 and VE-cadherin) were measured by Western blot and quantitative real time polymerase chain reaction.

Results

Compared with the normal control group, after TNF-α treatment, the resistance of EA.hy926 cell was significantly decreased, cell permeability was significantly increased (P<0.01), and the expression of RhoA, ROCK2, p-MYPT1 was increased and VE-cadherin was decreased (P<0.05), while UTI could reverse the above changes. Compared with UTI treatment group, the expression of RhoA, ROCK2, p-MYPT1 in RhoA overexpressing cell model were significantly increased, VE-cadherin was decreased, and cell permeability was increased (P<0.05). However, the expression of the above molecules and cell permeability in the empty virus group did not change significantly (P>0.05).

Conclusion

Ulinastatin can inhibit the increase of vascular endothelial cell permeability induced by TNF-α through Rho/ROCK signaling pathway, and improve vascular endothelial barrier dysfunction.

Key words: Ulinastatin, Inflammtion, Endothelial barrier function

表1 不同浓度UTI对TNF-α诱导的血管内皮细胞电阻值变化的影响（n=3）

组别	单层细胞电阻（Ω/cm²）
1 U/ml UTI＋TNF-α组	40.67±5.03
10 U/ml UTI＋TNF-α组	50.33±3.21^a
100 U/ml UTI＋TNF-α组	63.67±6.51^a
1000 U/ml UTI＋TNF-α组	48.33±2.08
TNF-α组	33.67±4.04^b
正常对照组	67.17±3.81

表1 不同浓度UTI对TNF-α诱导的血管内皮细胞电阻值变化的影响（n=3）

组别	单层细胞电阻（Ω/cm²）
1 U/ml UTI＋TNF-α组	40.67±5.03
10 U/ml UTI＋TNF-α组	50.33±3.21^a
100 U/ml UTI＋TNF-α组	63.67±6.51^a
1000 U/ml UTI＋TNF-α组	48.33±2.08
TNF-α组	33.67±4.04^b
正常对照组	67.17±3.81

图1 RhoA过表达慢病毒载体成功转染EA.hy926细胞。RhoA过表达慢病毒载体转染EA.hy926细胞后，观察RhoA蛋白表达量（图a）和mRNA的表达水平（图b）判断是否转染成功

图2 UTI对RhoA过表达慢病毒载体转染后EA.hy926细胞通透性的影响。RhoA过表达慢病毒载体成功转染EA.hy926细胞后，分别用UTI及TNF-α干预RhoA过表达细胞，采用TEER法和Transwell小室法检测单层细胞电阻值（图a）和HRP通过率（图b），观察UTI对细胞通透性的影响

图3 UTI对RhoA过表达慢病毒载体转染后EA.hy926细胞中关键分子的影响。RhoA过表达慢病毒载体成功转染EA.hy926细胞后，分别用UTI及TNF-α干预RhoA过表达细胞，采用western blot法和RT-PCR检测Rho/ROCK信号通路中关键分子的表达水平。图a为p-MYPT1蛋白的表达情况；图b为VE-cadherin蛋白的表达情况；图c为RhoA、ROCK2蛋白的表达情况；图d为RhoA、ROCK2、VE-cadherin mRNA的表达水平

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Effect of ulinastatin on vascular endothelial barrier function under inflammatory state and its mechanism

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Effect of ulinastatin on vascular endothelial barrier function under inflammatory state and its mechanism