依折麦布对高迁移率族蛋白1诱导血管内皮细胞激活的影响机制研究

目的探讨依折麦布对高迁移率族蛋白1(HMGB1)诱导血管内皮细胞激活影响的分子机制.方法分离、培养雄性SD大鼠胸主动脉内皮细胞,分为空白对照组、HMGB1刺激组、依折麦布组(HMGB1刺激前10μmol/L依折麦布预处理)、CLI-095组(HMGB1刺激前1μmol/L CLI-095预处理).荧光定量分析中性粒细胞与内皮细胞的黏附能力;RT-PCR和Western blot检测内皮细胞中TLR4,ICAM-1、可溶性E选择素mRNA和蛋白表达水平;EMSA法检测NF-κb-p65的DNA结合活性.结果 HMGB1活化的内皮细胞与中性粒细胞黏附活力均明显高于空白对照组,差异具有统计学意义(P0.05);依折麦布组、CLI-095组内皮细胞与中性粒细胞黏附活力均明显低于HMGB1诱导组,差异具有统计学意义(P0.05).HMGB1刺激组的内皮细胞TLR4,mRNA和蛋白表达量明显高于空白对照组,差异具有统计学意义(P0.05);依折麦布组和CLI-095组mRNA和蛋白表达量均明显低于HMGB1组,差异具有统计学意义(P0.05).HMGB1组内皮细胞NF-κb p65亚单位核位移明显强于空白对照组,差异具有统计学意义(P0.05);依折麦布组和CLI-095组较HMGB1组核位移明显减弱,差异具有统计学意义(P0.05).HMGB1组ICAM-1、可溶性E选择素表达水平明显高于空白对照组,差异具有统计学意义(P0.05).而经依折麦布、CLI-095干预后可明显降低ICAM-1、可溶性E选择素表达水平,与HMGB1组之间差异具有统计学意义(P0.05).结论依折麦布可通过调节黏附分子(ICAM-1、可溶性E选择素)的表达而有效抑制HMGB1诱导的血管内皮细胞活化效应,其机制与抑制TLR4的表达和NF-κb激活有关.

Effect Mechanism of Ezetimibe on High Mobility Group Box-1 Protein-Induced Activation of Vascular Endothelial Cell

Objective To investigate the molecular effect mechanism of ezetimibe on high mobility group box-1 protein ( HMGB1 )-induced activation of vascular endothelial cells. Method Male SD rats ’ thoracic aorta endothelial cells were separated and cultured, which were randomly divided into blank control group, HMGB1 stimulation group,ezetimibe group ( pre-treated with 10 μmol/L ezetimibe before HMGB1 stimulation) ,and CLI-095 group ( pre-treated with 1 μmol/L CLI-095 before HMGB1 stimulation ) . Fluorescence quantitative analysis was applied to analyze the adhesion of neutrophils to endothelial cells;RT-PCR and Western blot were used to detect TLR4 , ICAM-1, soluble E-selectin mRNA and protein expression levels in endothelial cells;the DNA binding activity of NF-κb p65 was tested by EMSA assay. Results Adhesion activities of HMGB1-activated endothelial cells to the polymorphonuclear cells were significantly higher than those in the blank control group ( P<0 . 05 );adhesion activities of CLI-095 group endothelial cells to the polymorphonuclear cells in ezetimibe group were significantly lower than those in HMGB1-induced group (P<0. 05). The expression of TLR4,mRNA and protein in endothelial cells in HMGB1 stimulation group was significantly higher than that in the control group ( P<0 . 05 );mRNA and protein expressions in endothelial cells in ezetimibe group and CLI-095 group were significantly lower than those in HMGB1 group ( P<0 . 05 ) . The nuclear shift of NF-κb p65 subunit of endothelial cells in HMGB1 group was greater than that in the blank control group ( P<0 . 05 );the nuclear shift in ezetimibe group and CLI-095 group was significantly weakened compared with that in HMGB1 group (P<0. 05). The expression levels of ICAM-1 and soluble E-select in HMGB1 group were significantly higher than those in the blank control group (P<0. 05),and the ezetimibe and CLI-095 intervention could significantly reduce the ICAM-1 and soluble E-selectin mRNA expression compared with HMGB1 group ( P<0. 05 ). Conclusion Ezetimibe can effectively inhibit the activation of HMGB-induced vascular endothelial cells by regulating the expression of adhesion molecules (ICAM-1 and soluble E-selectin),and the mechanism may be related to its inhibition on the expression of TLR4 and the activation of NF-κb.