补阳还五汤含药血清对Toll样受体4信号转导通路及氧化低密度脂蛋白受体-1表达的影响

Effect of Serim Contained Buyang Huanwu Decoction on the Expression of Toll-like Receptor 4 and Downstream Signal Transduction Pathway and LOX-1 in Human Vein Endothelial Cells

目的:从补阳还五汤含药血清对脂多糖(lipopolysaccharide,LPS)诱导的人脐静脉内皮细胞(human umbilical veinendothelial cells,HUVECs)Toll样受体4(toll-like receptor 4,TLR4)信号转导通路及血凝素样氧化低密度脂蛋白受体-1(LOX-1)的影响,研究补阳还五汤防治动脉粥样硬化(atherosclerosis,AS)的机制。方法:含药血清的制备:选择新西兰大耳白兔20只,随机分为正常组、补阳还五汤高、中、低浓度组,每组5只。中药组分别以13.2,6.6,3.3 g.kg-1补阳还五汤ig,正常组以同等量的生理盐水ig,连续ig 7 d。在末次ig给药2 h后,心脏采血,离心后分离血清,获得3种不同浓度药物血清;体外培养人脐静脉内皮细胞,用脂多糖(LPS)(1 mg.L-1)刺激后,分别加入含10%高、中、低浓度补阳还五汤药物血清干预,24 h后收集细胞,用荧光定量PCR方法测定TLR4,MyD88,TRAF-6,TRAM,TRIF,NF-κB及LOX-1的基因表达,用Western blotting法测定TLR4,MyD88,TRAF-6,LOX-1的蛋白表达。结果:用LPS刺激人脐静脉内皮细胞后,引起TLR4,MyD88,TRAF-6,TRAM,TRIF,NF-κB及LOX-1的高表达(与空白对照组比较P<0.01),用补阳还五汤含药血清干预后显著抑制TLR4,MyD88,TRAF-6,NF-κB及LOX-1的高表达(与模型组比较P<0.05),而对TRAM和TRIF与模型组比较无明显抑制作用。结论:补阳还五汤可抑制TLR4和MYD88依赖性信号转导通路及LOX-1的表达,这可能是其治疗各种免疫炎症性疾病及防治动脉粥样硬化作用的机制之一。

Objective： To investigate the influence and possible anti-atherosclerotie mechanism, of serum contained Buyang Huanwu decoction on the expression of toll-like receptor 4 （TLR4） and its downstream signal transduction pathway and LOX-1 in human umbilical vein enddhelial cells （HUVECs）. Method： Twenty New Zealand rabbits were divided into 4 groups in random： the normal control group, the high dosage group, the middle dosage group, and the low dosage group of Buyang Huanwu decoction, with 5 rabbits in each group. Each group was ig given normal saline and Buyang Huanwu decoction at dosage of 13.2, 6.6, 3, 3 g .kg-1, respectively everyday. In the 7Lh day, blood was collected from lefe ventricle in 2 h after administration, and then the serum was separated. HUVECs was cultured in vitro and stimulated with lipopolysaccharide （LPS） 1 mg . L-1 for 2 h, then treated separately with the serum contained different concentrations of Buyang Huanwu decoction for 24 h, finally the expression of TLR4, MyD88, TRAF-6, TRAM, TRIF, NF-κB and LOX-1 mRNA was measuredwith real-time PCR. Western blotting method was used to analyze the expression of TLR4, MyD88, TRAF-6 and LOX-1 protein. Result： LPS enhanced the expression of TLR4, MyD88, TRAF-6, TRAM, TRIF, NF-κB and LOX-1 （vs normal control group, P 〈 0.01）. The serum contained Buyang Huanwu decoction decreased the high expression of TLR4, MyD88, TRAF-6, NF-κB and LOX-1 （vs model group, P 〈 0.05） , which was stimulated by LPS. Conclusion： The serum contained Buyang Huanwu decoction can block the high expression of TLR4 and its downstream signal transduction pathway and the high expression of LOX-1, which may be the mechanism of anti- artherosclerosis and inhibition of inflammatory reaction.