肉桂酸对肿瘤坏死因子诱导血管内皮细胞组织因子表达的作用及其机制

The Effects of Cinnamic Acid on Expression of Tissue Factor Induced by TNFα in Endothelial Cells and its Mechanisms

目的:观察肉桂酸(CINN)对TNF-α诱导人脐静脉血管内皮细胞株ECV304表达组织因子(TF)的影响,并探讨其作用的细胞内信号传导机制。方法:内皮细胞培养采用RPMI-1640完全培养基;一期凝固法测总的细胞促凝活性(PCA):TFmRNA采用RT-PCR的方法检测;用免疫组织化学染色法观察NF-κB的变化;Western-blod方法检测胞浆中I-κB的变化。结果:(1)与对照组相比,TNF-α(1000u／ml)引起ECV304的PCA增加(P<0.01),但可以被TF单抗阻断,其阻滞作用随TF单抗的增加而增加(P<0.05),由此证明血管内皮细胞的PCA来自于TF。在给予TNF-α之前30min用不同浓度的CINN预处理ECV304细胞,可以明显抑制TNF-α(1000u／ml)刺激内皮细胞表达TF的作用,具有显著的量效关系(r=-0.953,P<0.05)。在500μg/ml时,CINN有轻度抑制TF基础表达的效应;但以CINN预处理ECV304,可使TNF-α刺激TF的活性下降90％(P<0.001);CINN也可显著降低受刺激的ECV304TFmRNA表达。(2)Western-blot发现TNF-α作用45min胞浆中I-κB减少最明显,CINN预处理后,I-κB的减少不明显;免疫组织化学染色发现TNFα作用1h可引起内皮细胞NF-κB从胞浆移入核内,CINN可抑制NF-κB的活化。结论:CINN抑制血管内皮细胞对TNFα诱导的TF表达,它是通过影响NF-κB的活化发挥作用的。

Objective:To observe the effects and mechanisms of cinnamic acid(CINN)on the expression of tissue factor (TF) induced by tumor necrosis factor(TNF-α) in human umbilical vein endothelium derived cell line ECV 304. Methods: TF activity was determined with one-stage clotting assay measuring total cellular pro-coagulant activity.TF mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemical analysis was performed to evaluate the activation of NF-κB. I-κB was measured by Western-blot. Results: (1)CINN (500μg/ml) decreased the TF activity induced in ECV 304 by TNF-α(1 000 u/ml ) (P < 0.01) , in a dose-dependent fashion (r=-0.953, P<0.05). CINN also inhibited the expression of TF mRNA induced by TNF-α. (2) Immunohistochemical analysis demonstrated that NF-κB translocation from cytoplasm to the nucleus and I-κB decrement in cytoplasm were found by Western-blot after treatment of endothelial cells with TNF-α.CINN inhibited these changes induced by TNF-α. Conclusion: CINN can attenuate TNF-α-induced TF expression of vascular endothelial cells by inhibited NF-κB activation.