NF-κB在抗β_2GPⅠ/β_2GPⅠ复合物诱导THP-1细胞表达组织因子中的作用探讨

The role of NF-κB in anti-β_2GPⅠ/β_2GPⅠ complex-induced tissue factor expression in THP-1 cells

目的:探讨核因子κB(NF-κB)在抗β2GPⅠ/β2GPⅠ复合物诱导单核细胞株THP-1表达组织因子(TF)中的作用。方法:使用一定剂量的抗β2GPⅠ/β2GPⅠ复合物处理THP-1细胞一定时间,收集细胞总RNA和总蛋白,实时定量PCR(RT-qPCR)检测细胞TF mRNA水平,TF活性试剂盒检测TF活性;Western blot检测细胞NF-κB p65、磷酸化-NF-κB p65及NF-κB抑制蛋白IκB-α的表达情况;进一步采用NF-κB抑制剂(PDTC)观察是否能干预抗β2GPⅠ/β2GPⅠ复合物对细胞的刺激效应,并利用上游信号分子MAPKs的抑制剂观察抗β2GPⅠ/β2GPⅠ复合物对NF-κB p65磷酸化的影响。结果:抗β2GPⅠ/β2GPⅠ复合物(100μg/ml)能够诱导THP-1细胞表达TF mRNA及活性,与对照相比差异显著(P<0.05);能够增强细胞内NF-κB p65磷酸化(P<0.05 vs media),降低IκB-α水平(P<0.05 vs media);NF-κB抑制剂PDTC(20μmol/L)能够抑制抗β2GPⅠ/β2GPⅠ复合物(100μg/ml)诱导THP-1细胞表达TF及NF-κB磷酸化的效应;MAPKs抑制剂均能影响抗β2GPⅠ/β2GPⅠ复合物(100μg/ml)诱导THP-1细胞NF-κB p65磷酸化。结论:在抗β2GPⅠ/β2GPⅠ复合物诱导THP-1细胞表达TF过程中,NF-κB被激活并发挥重要作用,MAPKs为NF-κB的关键上游分子。

Objective：To investigate whether nuclear factor kappa B（NF-κB） is involved in anti-β2GPⅠ/β2GPⅠ-induced tissue factor（TF） expression in monocytic cell line THP-1.Methods：The total RNA was extracted and the protein lysates were collected from THP-1 cells treated with anti-β2GPⅠ/β2GPⅠ complex.And the TF mRNA expression was detected by real time quantitative PCR（RT-qPCR） and TF activity was measured by TF activity kit.The activation of NF-κB p65 and level of NF-κB inhibitory protein IκB-α was determined by Western blot using the specific antibodies.The NF-κB inhibitor,PDTC,was used for inhibitory assays in order to demonstrate the activation and effect of NF-κB in anti-β2GPⅠ/β2GPⅠ complex induced TF expression in cells.And the inhibitors of MAPKs（the upstream signaling molecules） were applied to observe whether the effect of anti-β2GPⅠ/β2GPⅠ complex on NF-κB p65 phosphorylation could be affected.Results：The TF mRNA expression and TF activity in THP-1 cells was significantly up-regulated with the treatment of anti-β2GPⅠ/β2GPⅠ complex（100 μg/ml）,compared with the untreated cells（P0.05）.The level of phospho-NF-κB p65 in cells was also significantly increased,but IκB-α level was markedly decreased.PDTC（20 μmol/L） could decrease the TF expression and NF-κB p65 phosphorylation.MAPKs inhibitors could reduce the phosphorylation of NF-κB p65 in THP-1 cells treated with anti-β2GPⅠ/β2GPⅠ complex.Conclusion：NF-κB is activated and plays an important role in the process of anti-β2GPⅠ/β2GPⅠ complex-induced TF expression in THP-1 cells.MAPKs are key upstream signaling molecules of NF-κB in this process.