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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 effect of Buyang Huanwu Decoction-containing serum on toll-like receptor 4 (TLR4) signaling induced by lipopolysaccharide (LPS) in human umbilical veinendothelial cells (HUVECs) Conduction pathway and hemagglutinin-like oxidized low density lipoprotein receptor-1 (LOX-1), and to study the mechanism of Buyang Huanwu Decoction in preventing and treating atherosclerosis (AS). Methods: Preparation of drug-containing serum: 20 New Zealand white rabbits were randomly divided into normal group, Buyang Huanwu decoction high, medium and low concentration group, 5 rats in each group. Chinese medicine group were 13.2,6.6,3.3 g.kg-1 Buyang Huanwu Tang ig, the normal group with the same amount of normal saline ig, continuous ig 7 d. After the last ig administration for 2 h, the blood was collected from the heart and centrifuged to separate the serum. Three different concentrations of serum were obtained. Human umbilical vein endothelial cells were cultured in vitro and stimulated with lipopolysaccharide (LPS) (1 mg.L-1) The serum of Buyang Huanwu Decoction containing 10% high, medium and low concentration was added respectively. After 24 h, the cells were collected and the expression of TLR4, MyD88, TRAF-6, TRAM, TRIF, NF- 1 gene expression, Western blotting assay TLR4, MyD88, TRAF-6, LOX-1 protein expression. Results: The expressions of TLR4, MyD88, TRAF-6, TRAM, TRIF, NF-κB and LOX-1 in LPS-stimulated human umbilical vein endothelial cells were significantly higher than that in the blank control group Wu-Tang containing serum could significantly inhibit the high expression of TLR4, MyD88, TRAF-6, NF-κB and LOX-1 (P <0.05 compared with the model group), but had no obvious inhibitory effect on TRAM and TRIF compared with the model group . Conclusion: Buyanghuanwu Decoction can inhibit TLR4 and MYD88-dependent signal transduction pathway and LOX-1 expression, which may be one of the mechanisms for its treatment of various immune and inflammatory diseases and the prevention and treatment of atherosclerosis.