Résumé
Objective To investigate the effect of 1α, 25-dihydroxyvitamin D3 [1α,25-(OH)2 D3] 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) 2 D3 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) 2 D3 down-regulated the expression of VCAM and E-selectin induced by TNF-α. ( 2 ) Western blotting showed that TNF-α induces I-κBαphosphorylation, thereby activating NF-κB p65 subunit. Immunofluorescence showed that 1α, 25-( OH ) 2 D3 significantly inhibited the nuclear translocation of NF-κB p65 subunit. ChIP analysis revealed that 1α,25-( OH) 2 D3 inhibited the binding of NF-κB p65 to VCAM and E-selectin promoters and thus affected gene expression. Conclusions TNF-αenhanced the expression of E-selectin and VCAM in HUVECs via NF-κB signaling pathway. 1α,25-( OH) 2 D3 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.
Résumé
AIM:To investigate the role of p38 MAPK/ATF-2 pathway in C-relative protein ( CRP)-induced endothelial cell activation.METHODS:Human coronary artery endothelial cells ( HCAEC) were cultured and were used between passages 3 and 7.CRP served as a stimulus for endothelial cell activation.Western blotting was performed to de-termine the expression and phosphorylation of eNOS, p38 and ATF2.ELISA was carried out to detect the levels of ICAM-1, VCAM-1 and MCP-1 released from HCAEC.Pharmacological p38 inhibitors SB203580 and SB202190 were used to de-termine the effect of p38/ATF-2 pathway.RESULTS:CRP reduced the p-eNOS level in a concentration-dependent man-ner and induced the release of ICAM-1, VCAM-1 and MCP-1.The p38/ATF-2 pathway was activated by CRP treatment. SB203580 and SB202190 partially rescued p-eNOS level and suppressed the secretion of ICAM-1, VCAM-1 and MCP-1. CONCLUSION:p38MAPK/ATF-2 pathway participates in CRP-induced endothelial activation.
Résumé
Objective:The pathogenesis of placental vascular disease(PVD) was further investigated. Methode:PVD group (study group) and normal pregnancy (control group) were recruited. Two-channel mi-croarray technique was applied to screen the most differently expressive genes of human genome, and veri-fied the different expressions in mRNA and protein levels by quantitative PCR and Westem-blot, also local-ized the expression of different genes in placental tissue by immunohistochemistry. Reeults:HspA6 and Hs-pA1A had extremely higher expression in placentas with PVD than normal pregnancy. These genes both en-coded Hsp70. Hsp70 was mostly expressed in endothelial and smooth muscle cells of placental microves-ssls. The expression of Hsp70 mRNA and protein were up-regulated in placental tissue and microvascular endothelial cells from PVD group compared with normal pregnancy ( P <0.05) ;The expression of Hsp70 mR-NA and protein had negative correlation with infant birth weight ( P < 0.05). Conclueione:Hsp70 was associ-ated with endothelial activation in the pathogenesis of PVD by initiating an innate immune response and in-flammatory reaction.