补肾降脂方调控自噬与胆固醇流出干预ApoE-/-小鼠动脉粥样硬化的机制研究

Study on the mechanism of Bushen Jiangzhi Formula regulating autophagy and cholesterol efflux on the intervention of atherosclerosis in ApoE-/-mice

目的探讨补肾降脂方调控自噬与胆固醇流出干预动脉粥样硬化(AS)的作用机制。方法高脂饮食诱导100只ApoE-/-小鼠复制AS模型,采用随机数字表法分为模型组、补肾组、他汀组、3-甲基腺嘌呤(3-MA)组,每组25只;另选25只C57BL/6J小鼠为正常对照。苏木精-伊红(HE)、油红O及FILIPIN染色检测主动脉窦病理形态、脂质蓄积及游离胆固醇(FC)染色情况;电镜观察自噬小体、脂滴;比色法检测血清总胆固醇(TC)、FC含量;Western blot检测主动脉肝X受体α(LXRα)、ATP结合膜盒转运蛋白A1(ABCA1)、微管相关蛋白1轻链3(LC3)及p62蛋白表达。结果模型组主动脉窦AS斑块、脂质蓄积及FC阳性染色面积多于正常组,差异有高度统计学意义(P<0.01);正常组主动脉内皮细胞结构完整,可见自噬小体;模型组主动脉内皮细胞肿胀,可见脂滴,未见自噬小体;模型组TC、FC、胆固醇酯(CE)含量高于正常组,差异有高度统计学意义(P<0.01);模型组主动脉LXRα、ABCA1、LC3Ⅱ/Ⅰ表达低于正常组,p62表达高于正常组,差异有高度统计学意义(P<0.01);补肾组主动脉窦AS斑块、脂质蓄积及FC阳性染色面积少于模型组,差异有高度统计学意义(P<0.01);补肾组主动脉内皮细胞可见自噬小体;补肾组TC、FC、CE含量低于模型组,差异有统计学意义(P<0.05或P<0.01);补肾组LXRα、ABCA1、LC3Ⅱ/Ⅰ表达高于模型组,p62表达低于模型组,差异有统计学意义(P<0.05或P<0.01)。结论补肾降脂方可能通过调控自噬与胆固醇流出进而有效干预治疗AS。

Objective To explore the mechanism of Bushen Jiangzhi Formula in regulating autophagy and cholesterol efflux in the intervention of atherosclerosis(AS).Methods One hundred ApoE-/-mice induced by high fat diet were divided into model group,kidney tonifying group,statin group and 3-methyladenine(3-MA)group by using random number table method,with 25 mice in each group.Twenty-five C57BL/6J mice were normal controls.Hematoxylin-eosin(HE),oil red O and FILIPIN staining were used to detect the pathological morphology,lipid accumulation and free cholesterol(FC)staining of the aortic sinus.Autophagosomes and lipid droplets were observed by electron microscopy.The content of serum total cholesterol(TC)and FC were detected by colorimetry.The expression of liver X receptor alpha(LXRα),ATP-binding membrane cassette transport protein A1(ABCA1),microtubule-associated protein 1 light chain 3(LC3)and p62 protein were detected by Western blot.Results The model group had more aortic sinus AS plaque,lipid accumulation and FC positive staining area than the normal group,and the differences were highly statistically significant(P<0.01).The normal group had intact aortic endothelial cell structure and autophagosome.The endothelial cells of aorta in model group were swollen with lipid droplets,but no autophagosomes were observed.The contents of TC,FC,and cholesterol ester(CE)in model group were higher than those in normal group,and the differences were highly statistically significant(P<0.01).The expression of LXRα,ABCA1,and LC3Ⅱ/Ⅰin aorta of model group was lower than that of normal group,and the expression of p62 was higher than that of normal group,the differences were highly statistically significant(P<0.01).AS plaque,lipid accumulation,and FC positive staining area of aortic sinus in the kidney tonifying group were less than those in the model group,and the differences were highly statistically significant(P<0.01).In the kidney tonifying group,autophagosomes were found in the endothelial cells of aorta.The contents of TC,FC,and CE in kidney tonifying group were lower than those in model group,and the differences were statistically significant(P<0.05 or P<0.01).The expression of LXRα,ABCA1,LC3Ⅱ/Ⅰin kidney tonifying group was higher than that in model group,while the expression of p62 was lower than that in model group,the differences were statistically significant(P<0.05 or P<0.01).Conclusion Bushen Jiangzhi Formula may effectively intervene in the treatment of AS by regulating autophagy and cholesterol efflux.