川芎嗪抑制肿瘤坏死因子α诱导人脐静脉血管内皮细胞组织因子表达的机制研究

Mechanism for the inhibitory effect of tetramethylpyrazine on the expression of tissue factor induced by tumor necrosis factor in human umbilical vein endothelial cells

目的探讨川芎嗪(TMP)抑制肿瘤坏死因子α(TNFα)诱导人脐静脉血管内皮细胞组织因子(TF)表达的胞内信号转导机制。方法胰酶消化法分离培养人脐静脉血管内皮细胞,以一期凝固法、ELISA、RT-PCR分别测总的细胞促凝活性、TF抗原和mRNA;放射免疫法测PKC(蛋白激酶C)活性;免疫组织化学染色观察NF-κB的变化。结果TMP和Staurosporine(Sta)可显著减少PMA与TNFα刺激的TF活性、抗原及mRNA的表达(P<0.01);PMA、TNFα使胞浆中PKC活性明显降低(P<0.05),胞膜PKC活性显著增高(P<0.01),而TMP与Sta均能降低胞膜PKC活性(P>0.05);TNFα引起内皮细胞NF-κB从胞浆移入核内,TMP及吡咯二硫氨基甲酸酯(PDTC)均可抑制NF-κB的活化。结论TNFα诱导HUVEC TF的表达中,PKC及NF-κB的活化发挥着重要的作用;TMP通过影响PKC途径及NF-κB的活化而抑制TNFα诱导TF的表达。

Objective To investigate the role of PKC and transcription factor NF - kB in the regulation of TF expression induced by TNFα in human umbilical vein endothelium （HUVEC）. Methods HUVEC were cultured in RPMI - 1640. TF activity was determined with one - stage clotting assay measuring total cellular pro - coagulant activity. TF anti- gen was measured by ELISA. TF mRNA was examined by RT - PCR. Liquid scintillation count was used to check PKC activity by radioimmunoassay, lmmunohistochemical analysis was performed to evaluate the activation of NF - KB, Results The addition of TMP or Staurosporin, significantly inhibited the stimulative effects of TNFα as well as PMA on TF activ- ity, antigen and mRNA expression （P 〈 0.01 ）. Addition of PMA or TNFα resulted in a sharp decrease in protein kinase C activity in the cytosolic fraction （P 〈0.05 ）, a great increase in the activity in the membrane fraction （P 〈 0.01 ） and NF - KB translocation from cytoplasm to the nucleus. Pretreatment with TMP, however, significantly decreased the PKC activity in the membrane fraction and inhibited TNFα - induced NF - KB translocation. Condusion PKC activation and NF - KB translocation play an important role in TNFα - induced TF expression. The inhibitory effect of TMP on TF expression in HUVEC is involved in these signal transduction pathways.