补阳还五汤对防治动脉粥样硬化的ApoE-/-小鼠Toll样受体4及其下游主要元件的影响

Effects of Buyang Huanwu Decoction on the expression of Toll like receptor 4 and its main components of downstream signal transduction pathway in apolipoprotein E gene knockout mice

目的:探究补阳还五汤对于载脂蛋白E基因敲除小鼠的Toll样受体4(TLR4)及髓样分化因子88(My D88)、肿瘤坏死因子受体相关因子6(TRAF-6)、核因子κB(NF-κB)表达的影响,讨论补阳还五汤对于动脉粥样硬化的防治机制。方法:将30只雄性载脂蛋白E基因敲除小鼠随机分为模型组,阿托伐他汀组,补阳还五汤组,每组10只,以10只C57BL/6J小鼠设空白对照组。除空白对照组正常饮食饮水外,每组给予高脂饲料喂养8周。空白对照组及模型组每日给予0.9%氯化钠溶液灌胃,阿托伐他汀组及加为补阳还五汤组每日以相应药物灌胃。第9周后采用生化检测方法检测血清总胆固醇(TC),甘油三酯(TG),高密度脂蛋白(HDL-C)和低密度脂蛋白(LDC-C)值。采用苏木素-伊红染色方法观察,并测量斑块所占管腔面积之比。以荧光定量PCR检测TLR4、My D88、TRAF-6、NF-κB的mRNA表达;蛋白质印迹法测定其蛋白表达的变化。结果:血脂水平方面,与模型组相比,补阳还五汤组与阿托伐他汀组均能降低Apo E-/-小鼠TC、TG、LDL-C水平,同时升高HDL-C水平(P<0.01);镜下观察补阳还五汤组与阿托伐他汀组均能较模型组斑块有所减少,主动脉血管细胞排列较整齐,炎性细胞浸润减轻,小鼠的主动脉斑块在血管的占比均有不同程度的降低(P<0.01)。mRNA与蛋白水平上与模型组相比,补阳还五汤与阿托伐他汀组均能有效降低TLR4、My D88、TRAF-6、NF-κB的mRNA基因与蛋白的表达(P<0.05,P<0.01)。结论:补阳还五汤对于AS的发生发展有预防作用,其机制可能与抑制TLR4及其下游信号转导通路主要元件有关。

Objective： To explore the effects of Buyang Huanwu Decoction on the expression of Toll like receptor 4 （TLR4） and myeloid differentiation factor 88 （MyD88） and tumor necrosis factor receptor associated factor 6 （TRAF-6）, nuclear factor kappa B （NF-KB） in the apolipoprotein E gene knockout mice （ApoE-/- mice）, and discuss the mechanism of Buyang Huanwu Decoction for the prevention and treatment of atherosclerosis （AS）. Methods： Thirty male ApoE-/- mice were randomly divided into three groups： model group, Buyang Huanwu Decoction group and atorvastatin group, and C57BL/6J mice was as a control group. In addition to the normal diet and drinking water in the control group, each group was fed a high-fat diet for 8 weeks. At the same time, Buyang Huanwu Decoction group and simvastatin group were intragastrically administrated with Buyang Huanwu Decoction （18.69g/kg） and atorvastatin （1.3mg/kg）, while control and model group were administrated with 0.9% saline. After 9 weeks of treatment, cholesterol （TC）, triglyceride （TG）, high density lipoprotein cholesterol （HDL-C） and low density lipoprotein cholesterol （LDL-C） were detected by the biochemical method. In addition, the morphological changes of aorta segment was observed by HE, and the ratio of the area of the plaque to the intravascular space was measured. PCR and Western Blot was used to determine the expression of aortic Toll like receptor 4 and myeloid differentiation factor 88 and tumor necrosis factor receptor associated factor 6 and nuclear factor kappa B. Results： In blood lipid levels, compared with the normal group, the TC, TG and LDL-C of model group were significantly higher, while HDL-C levels was significantly lower （P〈0.01）. Compared with the model group, TC,TG and LDL-C in the Buyang Huanwu Decoction group and atorvastatin group were significantly lower, while HDL-C were significantly higher （P〈0.01）. HE staining showed no aortic plaque formation in C57BL/6J mice control group, but there were significantly aortic plaque formation and increased inflammatory cells in model group. Compared with the model group, both in Buyang Huanwu Decoction group and atorvastatin group, aortic vascular cells arranged more orderly, inflammatory cell infiltration was reduced, the proportion of aortic plaques in blood vessels was decreased （P〈0.01）. To compared with C57BL/6J mice control group, the mRNA and protein expression of aortic TLR4, MyD88, TRAF-6 and NF-xB obviously increased in model group （P〈0.01）. The mRNA and protein expression of aortic TLR4, MyD88, TRAF-6 and NF-xB significantly decreased in both Buyang Huanwu Decoction group and atorvastatin group, compared with model group （P〈0.05, P〈0.01）. Conclusion： Both Buyang Huanwu Decoction and atorvastatin have an effect in preventing the development of atherosclerosis. The mechanism could be related to intervene of TLR4 and its main components of downstream signal lxansduction pathway.