益气活血复方含药血清对人脐静脉内皮细胞TLR4及其下游髓样分化因子88、肿瘤坏死因子受体相关因子-6表达的影响

Effect of Yiqi Huoxue Recipe Containing Drug-serum on the Toll-like Receptor-4 and Its Downstream Signaling Components MyD88 as well as the Tumor Necrosis Factor Receptor Related Factor-6 in Human Vein Endothelial Cells

目的研究益气活血复方含药血清对脂多糖(lipopolysaccharide,LPS)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)Toll样受体4(TLR4)及其下游信号转导通路主要元件髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子-6(TRAF-6)表达的影响,从mRNA和蛋白水平探讨益气活血复方含药血清防治动脉粥样硬化的机制。方法选择新西兰大耳白兔20只,随机分为4组,即正常组、中药高、中、低浓度(1.6、0.8、0.4g/mL)组,每组5只。各组白兔分别以生理盐水和高、中、低浓度益气活血复方连续灌胃7天。末次灌胃给药2h后,心脏采血,离心后分离血清。体外培养HUVECs,随机分为6组,即空白对照组、模型组、西药对照组、中药高、中、低浓度组,用LPS刺激后,分别加入高、中、低浓度益气活血复方含药血清干预24h,收集细胞,用荧光定量PCR方法和Western blotting法分别测定TLR4、MyD88及TRAF-6 mRNA和蛋白的表达。结果用LPS刺激HUVECs后,引起TLR4、MyD88及TRAF-6 mRNA和蛋白的高表达(与空白对照组比较,P<0.01),用益气活血复方含药血清干预以后显著抑制TLR4、MyD88及TRAF-6 mRNA和蛋白的高表达(与模型组比较,P<0.01,P<0.05)。结论益气活血复方可阻断TLR4高表达,同时阻断TLR4胞内信号转导的MyD88依赖性途径,抑制下游NF-κB以及各种相关基因表达,这可能是其抗动脉粥样硬化的作用机制之一。

Objective To investigate the influence of Yiqi Huoxue Recipe （YHR） containing drug-serum on the expression of Toll-like receptor-4 （TLR4） and its downstream signaling components MyD88, as well as the tumor necrosis factor receptor related factor-6 （TRAF-6） in human vein endothelial cells （HUVECs） induced by lipopolysaccharide （LPS）, and to study its possible anti-atherosclerotic mechanism from the gene and protein levels. Methods Twenty New Zealand male rabbits were equally divided into four groups in random： the normal control group and the three YHR groups, 5 in each group. They were gastric perfused daily with normal saline and YHR in low, moderate and high concentration respectively. Blood drawn from rabbits′ heart 2 h after ending perfusion on the 7th day, and the serum separated （that is the drug-serum） was taken for testing. HUVECs were cultured in vitro and equally divided into six groups in random： the normal control group, the model group, the Western medicine group and the three YHR groups. HUVECs were stimulated with LPS, then treated separately with the drug-serum containing different concentrations of YHR for 24 h. Then the mRNA expressions of TLR4, MyD88 and TRAF-6 were measured with Real-time PCR, and their protein expressions were analyzed using Western blotting. Results Protein and mRNA expressions of TLR4, MyD88 and TRAF-6 increased significantly after LPS stimulation （P〈0.01 ）, but the changes in the drug-serum treated groups were significantly lower than those in the saline control group respectively （P〈0.01 or P〈0.05 ）. Conclusion YHR can block the high expression of TLR4, and also influence the MyD88-dependent signaling pathway of TLR4, suppress the downstream expression of NF-κB and various related gene expressions, which may be one of its mechanisms of action for anti-atherosclerosis.