肿瘤坏死因子α通过RhoA-ERK信号通路调控人脐静脉内皮细胞单层通透性的实验研究  

Tumor necrosis factor-α promote permeability of human umbilical vein endothelial cells via activating RhoA-ERK1/2 pathway

作　　者：闫承慧[1] 于海波[1] 黄明方[2] 李杰[1] 张效林[1] 韩雅玲[1] 

机构地区：[1]沈阳军区总医院全军心血管病研究所心内科,110840 [2]南京军区福州总医院心内科

出　　处：《中华心血管病杂志》2011年第6期531-537,共7页Chinese Journal of Cardiology

基　　金：国家自然科学基金（30770793,30971218,81070097）

摘　　要：目的 探讨肿瘤坏死因子α（TNF-α）诱导的人脐静脉内皮细胞（HUVEC）单层通透性改变的效应分子,以寻找内皮细胞损伤修复的有效治疗靶点.方法 应用TNF-α刺激体外培养的HUVEC,观察HUVEC骨架蛋白F-actin和细胞单层通透性的改变.应用荧光免疫组织化学和Western blot方法检测TNF-α刺激前后细胞中RhoA和MAPK信号通路的改变.并通过添加特异性分子抑制剂Y27632和PD98059阻断RhoA和ERK表达,观察细胞F-actin及细胞单层通透性的改变.进一步应用持续活化型RhoA和主导抑制型RhoA逆转录病毒分别感染改变人HUVEC,改变细胞中RhoA的活化状态,观察TNF-α刺激前后细胞骨架蛋白及单层细胞通透性的变化.并确定RhoA与ERK的相互作用.结果 TNF-α刺激引起HUVEC中F-actin快速重构并形成大量应力纤维,细胞单层通透性明显增强,呈现时间和剂量依赖性趋势.其中TNF-α（100 ng/ml）刺激2 h后细胞单层通透性最强,HRP-BSA渗漏最大浓度为40 ng/ml.同时,细胞中活化RhoA的表达明显增加.TNF-α刺激前后细胞中MAPK信号转导蛋白JNK、P38和ERK1/2总蛋白均无明显改变,但p-JNK、p-p38和p-ERK表达均显著增加.其中,应用Y27632抑制RhoA的活化或PD98059阻断p-ERK表达后,TNF-α刺激的HUVEC中F-actin重构现象消失,同时细胞单层通透性增加也受到明显抑制,HRP-BSA渗漏浓度从40 ng/ml下降至12.5 ng/ml（P〈0.05）.进一步应用持续活化型RhoA感染人HUVEC细胞,发现RhoA活化的HUVEC单层通透性明显高于GFP感染组,HRP-BSA在TNF-α（100 ng/ml）刺激2 h后渗漏浓度从达到50 ng/ml（P〈0.05）,同时p-ERK表达也明显增加.而应用TNF-α刺激后,抑制型RhoA感染的HUVEC的骨架蛋白与单层细胞通透性均无明显变化.同时,p-ERK活化也受到明显抑制.结论 RhoA-pERK通路的活化介导了TNF-α诱导的HUVEC通透性增加,特异性抑制RhoA-ERK通路可以阻断TNF-α引起的内皮细胞通透性增加.Objective Tumor necrosis factor-α （TNF-α） is known to induce changes in endothelial cell morphology and permeability. The aim of this study is to determine the underlying signaling mechanisms involved in these responses. Methods Cultured human umbilical vein endothelial cells （HUVECs） were exposed to TNF-α, and HUVEC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of horseradish peroxidase （HRP）-albumin across the EC monolayers. To explore the signaling pathways behind TNF-α-induced changes in HUVEC morphology and permeability, HUVECs were treated with either the Rho GTPase inhibitor Y27632 or the mitogen-activated protein kinases （MAPK） inhibitors PD98059 and SB203580 before TNF-α administration. To further elucidate possible involvement of the RhoA and ERK pathways in TNF-α-induced HUVEC changes, retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA, namely T19NRhoA and Q63LRhoA, were pre-infected into HUVECs prior to TNF-α exposure.Results TNF-α induced F-actin cytoskeleton rearrangement and increased HUVEC permeability in a dose and time-dependent manner. The maximal increase in the HRP-BSA flux （40 ng/ml） was seen in cells exposed to TNF-α at 100 ng/ml after 2 h. Preconditioning of HUVEC monolayer with Y27632 or PD98059 significantly reduced TNF-α induced permeability increase （HRP concentration from 40 ng/ml decreased to 12.5 ng/ml, P〈0.05） and F-actin cytoskeleton rearrangement, HUVEC pre-infection with activated forms of Q63LRhoA increased HUVEC permeability and upregulated pERK compared to GFP infection, while HUVEC pre-infection with inhibited forms of T19NRhoA attenuated the effects of TNF-α on HUVEC permeability.Conclusion These results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.

关 键 词：内皮细胞 通透性 肿瘤坏死因子Α 

分 类 号：R346[医药卫生—基础医学]