IL-18激活NF-κB细胞信号通路对人脐静脉内皮细胞损伤作用的体外研究

In vitro study on injury effect of IL-18 activating NF-κB cell signal pathway on human umbilical vein endothelial cells

目的探讨促炎因子白细胞介素-18(IL-18)通过激活核因子-κB(NF-κB)介导的细胞信号通路,对静脉内皮细胞功能的影响及其与深静脉血栓形成(DVT)的联系。方法利用重组人IL-18作用体外培养的人脐静脉内皮细胞(HUVECs),并以NF-κB激活抑制剂进行干预,通过实时荧光定量PCR、Western blot、免疫荧光、流式细胞仪等检测手段,验证IL-18是否通过激活NF-κB介导的细胞信号转导通路,影响HUVECs正常状态及血管性血友病因子(vWF)、P-选择素(P-selectin)、组织型纤溶酶原激活物(t-PA)等内皮细胞功能标记物的表达,结合既往研究对IL-18参与DVT的机制进行综合分析。结果 IL-18可激活内皮细胞内NF-κB,使细胞核内p65表达增高、细胞内IκBα表达降低,并使HUVECs早期凋亡细胞明显增多;添加QNZ(EVP4593)可使IL-18对NF-κB的激活作用明显抑制,细胞损伤、凋亡的发生显著减少;IL-18可促使vWF、P-selectin和t-PA等DVT相关的内皮细胞标志物发生表达异常(P<0.05),而各标志物可在NF-κB激活抑制后恢复常规表达。结论 IL-18及NF-κB间的相互作用导致HUVECs生长状态和功能异常,可能是与DVT发病相关的疾病机制。

Objective To investigate the influence of proinflammatory factor interleukin-18（IL-18）on vein endothelial cell function by activating NF-κB mediated cell signal pathway and its association with deep vein thrombosis（DVT）.Methods Recombinant human IL-18 was used to act on in vitro cultured human umbilical vein endothelial cell（HUVECs）.The NF-κB activation inhibitor was used to conduct interference.The detection measures of real time fluorescence quantitative PCR,Western blot,immunofluorescence and flow cytometry were used to verify whether IL-18 affect the expression of endothelial cellular function markers such as HUVECs normal statusand vWF,P-selectin and tissue plasminogen activator（t-PA）by activating NF-κB mediated cell signal pathway.Moreover the mechanism of IL-18 participating in the DVT was performed the comprehensive analysis by combining with previous study.Results IL-18 could activate NF-κB in endothelial cell,increased the p65 expression in nucleus,decreased the intracellular IκBαexpression and significantly increased early apoptosis cells in HUVECs;adding QNZ（EVP4593）could significantly inhibit the activation effect of IL-18 on NF-κB,the occurrence of cellular injury and apoptosis was significantly reduced;IL-18 could promote the abnormal expression of DVT related endothelial cell markers vWF,P-selectin and t-PA（P〈0.05）.But various markers could recover conventional expression after inhibiting NF-κB activation.Conclusion The interaction between Il-18 and NF-κB causes the abnormality of HUVECs growth status and function,which may be the DVT onset related pathogenic mechanism.