1α,25-二羟维生素D3通过NF-κB信号通路抑制肿瘤坏死因子-α诱导的人脐静脉内皮细胞活化

1α, 25-dihydroxyvitamin D3 inhibits tumor necrosis factor-α induced activation of human umbilical vein endothelial cells via NF-κB signaling pathway

目的体外观察1α,25-二羟维生素D3[1α,25-(OH)2D3]对肿瘤坏死因子-α(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)活化的影响,并探讨其机制。方法体外培养HUVECs,采用血管细胞黏附分子(VCAM)、E选择素(E-selectin)作为内皮细胞活化标志物,以TNF-α(40 ng/ml),1α,25-(OH)2D3(10-8 mol/L)以及特异性NF-κB阻断剂SN50干预,Western印迹法、实时定量PCR(RT-PCR)法检测VCAM、E-selectin表达水平。在机制研究中,以NF-κB信号通路为切入点,分别采用Western印迹、RT-PCR、免疫荧光法、染色质免疫共沉淀(ChIP)法观察1α,25-(OH)2D3对其早期活化、核转运及与VCAM、E-selectin启动子结合的影响。结果(1)Western印迹及RT-PCR显示TNF-α可明显上调HUVECs VCAM及E-selectin的表达,该效应可被特异性NF-κB阻断剂SN50抑制,1α,25-(OH)2D3可下调TNF-α诱导的VCAM及E-selectin的表达。(2)Western印迹显示TNF-α可诱导I-κBα磷酸化,从而活化NF-κB p65亚基。免疫荧光显示1α,25-(OH)2D3对NF-κB p65亚基核转运有明显抑制作用。ChIP法分析显示1α,25-(OH)2D3可抑制NF-κB p65与VCAM及E-selectin启动子的结合从而影响基因表达。结论TNF-α通过NF-κB信号通路上调HUVECs VCAM和E-selectin表达,1α,25-(OH)2D3可抑制NF-κB早期活化、核转运及NF-κB p65与VCAM及E-selectin启动子的结合,进而抑制TNF-α诱导的内皮细胞活化。

Objective To investigate the effect of 1α, 25-dihydroxyvitamin D3[1α, 25-(OH)2D3] on tumor necrosis factor-α(TNF-α) induced activation of human umbilical vein endothelial cells (HUVECs). The mechanism involved in this process was also studied. Methods HUVECs were cultured and treated with TNF-α(40 ng/ml), 1α, 25-(OH)2D3(10-8 mol/L), and SN50 as indicated. Vascular cell adhesion molecule (VCAM) and E-selectin were used as markers of endothelial activation, which were detected by Western blotting and realtime PCR (RT-PCR). NF-κB signaling pathway was investigated to study the mechanism. Western blotting, RT-PCR, immunofluorescence assay, and chromatin immunoprecipitation (ChIP) method were used to evaluate the effects of 1α, 25-(OH)2D3 on its early activation, nuclear transport, and binding to VCAM and E-selectin promoters. Results (1)Western blotting and RT-PCR showed that TNF-α could significantly up-regulate the expression of VCAM and E-selectin in HUVECs, which can be inhibited by specific NF-κB blocker SN50. 1α, 25-(OH)2D3 down-regulated the expression of VCAM and E-selectin induced by TNF-α.(2) Western blotting showed that TNF-α induces I-κBα phosphorylation, thereby activating NF-κB p65 subunit. Immunofluorescence showed that 1α, 25-(OH)2D3 significantly inhibited the nuclear translocation of NF-κB p65 subunit. ChIP analysis revealed that 1α, 25-(OH)2D3 inhibited the binding of NF-κB p65 to VCAM and E-selectin promoters and thus affected gene expression. Conclusions TNF-α enhanced the expression of E-selectin and VCAM in HUVECs via NF-κB signaling pathway. 1α, 25-(OH)2D3 may inhibit NF-κB early activation, nuclear transport and the binding of NF-κB p65 to VCAM and E-selectin promoters, thereby inhibiting TNF-α-induced endothelial cell activation.