加味补阳还五汤对防治动脉粥样硬化的Toll样受体7及其下游信号转导通路的影响

Effects of modified Buyang Huanwu Decoction on expression of Toll like receptor 7 and its downstream signal transduction pathway in preventing and treating atherosclerosis

目的:探究加味补阳还五汤对于载脂蛋白E基因敲除小鼠的Toll样受体7(TLR7)及其下游信号转导通路的影响,从而探讨加味补阳还五汤对动脉粥样硬化的防治机制。方法:将40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、加味补阳还五汤组,每组10只,10只C57BL/6J小鼠作为空白组。空白组正常饮食饮水,其余组给予高脂饲料喂养8周。空白组和模型组每日用0.9%氯化钠溶液灌胃,阿托伐他汀组、补阳还五汤组及加味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(LDL-C)。采用苏木素-伊红(HE)染色方法观察,并测量斑块所占管腔面积之比。以实时定量荧光PCR检测TLR7及MyD88、TRAF-6、NF-κB、IRF7 mRNA表达;蛋白质印迹法测定其蛋白表达。结果:与空白组比较,模型组TC、TG、LDL-C水平显著升高(P<0.01),HDL-C水平显著降低(P<0.01);与模型组比较,阿托伐他汀组、补阳还五汤组、加味补阳还五汤组TC、TG、LDL-C水平显著降低,同时HDL-C水平升高(P<0.01);病理学观察,与空白组比较,模型组主动脉内膜增厚、隆起、内皮细胞部分脱落,有大量斑块形成,中膜平滑肌细胞增生;与模型组比较,阿托伐他汀组、补阳还五汤组、加味补阳还五汤组均能有效地改善主动脉细胞的炎性浸润程度和斑块管腔占比(P<0.01)。与空白组比较,模型组TLR7、MyD88、TRAF-6、NF-κB mRNA与蛋白的表达显著增高(P<0.01);与模型组比较,阿托伐他汀组、补阳还五汤组、加味补阳还五汤组TLR7、MyD88、TRAF-6、NF-κB mRNA与蛋白的表达显著降低(P<0.05,P<0.01),但3组之间的m R NA与蛋白表达差异无统计学意义。结论:加味补阳还五汤对于动脉粥样硬化的发生发展有防治作用,其机制可能与抑制TLR7及其下游信号转导通路有一定相关性。

Objective:To explore the effects of modified Buyang Huanwu Decoction on the expression of Toll like receptor 7 and its downstream signal transduction pathway,in order to discuss the mechanism of modified Buyang Huanwu Decoction for the prevention and treatment of atherosclerosis.Methods:Forty male ApoE-/-mice were randomly divided into four groups:model group,atorvastatin group,Buyang Huanwu Decoction group and modified Buyang Huanwu Decoction group,with 10 mice in each group,and 10 C57BL/6J mice were used as the control group.The control group was given normal diet and drinking water,and the other groups were given high-fat diet for 8 weeks.The control group and the model group were orally administered with 0.9%NaCl solution daily.The atorvastatin group,Buyang Huanwu Decoction group and modified Buyang Huanwu Decoction group were intragastrical administrated with the corresponding drugs daily.After 9 weeks,cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)were detected by biochemical detection method.In addition,aorta segment taken from the experiment animals was used to observe morphological changes by hematoxylineosin(HE)staining method and measured the ratio of the plaque area to the area of intravascular space.Real-time PCR and Western Blot were used to determine the mRNA and protein expression of TLR7,MyD88,TRAF-6,NF-κB,IRF7.Results:In terms of blood lipid levels,compared with the control group,the levels of TC,TG and LDL-C in the model group were significantly higher,while HDL-C level was significantly lower(P<0.01).Compared with the model group,the levels of TC,TG and LDL-C in the atorvastatin group,Buyang Huanwu Decoction group and modified Buyang Huanwu Decoction group were significantly lower,while the level of HDL-C was significantly higher(P<0.01).Pathological observation showed that compared with the control group,the aortic intima thickened and bulged in the model group,and the endothelial cells were partially shed,and a large number of plaques were formed,and the median smooth muscle cells were proliferated.Compared with the model group,inflammatory cells infiltration was reduced and the proportion of aortic plaque in blood vessels were reduced to varying degrees among atorvastatin group,Buyang Huanwu Decoction group and modified Buyang Huanwu Decoction group(P<0.01).In terms of mRNA and protein expression,compared with the control group,mRNA and protein expressions of TLR7,MyD88,TRAF-6 and NF-κB in aortic were obviously increased in the model group(P<0.01).Compared with the model group,the atorvastatin group,Buyang Huanwu Decoction group and modified Buyang Huanwu Decoction group could effectively reduce the mRNA and protein expressions of TLR7,MyD88,TRAF-6 and NF-κB(P<0.05,P<0.01).The difference of the expression level of mRNA and protein among atorvastatin group,Buyang Huanwu Decoction group and modified Buyang Huanwu Decoction group also didn’t have statistically significance.Conclusion:Modified Buyang Huanwu Decoction can effectively prevent the development of atherosclerosis.The mechanism may be related to the suppression of TLR7 and its downstream signal transduction pathway.