乌司他丁对炎症状态下血管内皮屏障功能的影响及机制

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

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VEcadherin)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。

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-αrespectively,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-αtreatment,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-αthrough Rho/ROCK signaling pathway,and improve vascular endothelial barrier dysfunction.