四妙勇安汤对动脉粥样硬化巨噬细胞泡沫化的影响

Effect of Simiao Yongan Decoction on Macrophage Foam Cell Formation in Atherosclerosis

目的探讨四妙勇安汤对于载脂蛋白E敲除(ApoE^(-/-))小鼠动脉粥样硬化(AS)病变及J774A.1小鼠单核巨噬细胞泡沫化的影响。方法将30只ApoE^(-/-)小鼠按随机数字表法分为模型组、阿托伐他汀组、低剂量组、中剂量组和高剂量组,每组6只,给予西方饮食饲料喂养8周后,连续给药8周。阿托伐他汀组给予3.64×10^(-5) g/d阿托伐他汀(蒸馏水溶解)灌胃,低、中、高剂量组分别给予四妙勇安汤0.164、0.328、0.655 g/d灌胃;另将6只C57BL/6J小鼠作为对照组,给予普通饮食喂养16周,对照组和模型组每日予相同体积的蒸馏水灌胃。采用Movat法观察小鼠主动脉窦形态改变及泡沫细胞含量;免疫荧光染色观察主动脉窦F4/80表达情况。制备四妙勇安汤含药血清,CCK-8法筛选给药浓度;建立氧化低密度脂蛋白(ox-LDL)诱导的单核巨噬细胞株J774A.1泡沫化模型,分为模型组、四妙勇安汤组,另设对照组;采用油红O染色检测细胞脂质吸收;Western Blot法检测各组细胞清道夫受体分子蛋白表达水平。结果动物实验证明:与对照组比较,模型组主动脉窦病理形态改变明显,斑块内泡沫细胞含量和F4/80阳性表达的细胞数量均明显增加(P<0.01);与模型组比较,低、中、高剂量组主动脉窦病理形态变化改善,泡沫细胞生成减少(P<0.05,P<0.01),主动脉窦F4/80蛋白表达明显减少(P<0.01),且与阿托伐他汀组比较差异无统计学意义(P>0.05)。细胞实验证明:与对照组比较,模型组脂滴面积明显增加(P<0.01),血凝素样氧化低密度脂蛋白受体-1(LOX-1)、CD36、清道夫受体A1(SRA1)蛋白表达明显增加(P<0.01);与模型组比较,四妙勇安汤组细胞脂质吸收明显减少(P<0.01);LOX-1、CD36、SRA1蛋白表达减少(P<0.05,P<0.01)。结论四妙勇安汤通过抑制巨噬细胞中LOX-1、CD36、SRA1分子表达,降低胆固醇吸收,减少巨噬细胞泡沫化,从而改善AS。

Objective To investigate the effects of Simiao Yongan Decoction(SMYAD)on atherosclerosis(AS)in apolipoprotein E^(-/-)knockout(ApoE^(-/-))mice and macrophage foam cell formation in J774A.1 cell.Methods A total of 30 ApoE^(-/-)mice were randomly divided into the model group,atorvastatin group,low-dose,medium-dose,and high-dose groups,6 in each group.All subjects were fed a Western diet for 8 consecutive weeks,and then continuously administered for 8 weeks.The atorvastatin group was given 3.64×10^(-5)g·d^(-1)atorvastatin(dissolved in distilled water)by gavage,the low-dose,medium-dose and high-dose groups were given SMYAD(0.164,0.328,0.655 g·d^(-1))by gavage,respectively.Meanwhile,6 C57BL/6J mice were recruited in a control group and fed with a normal diet for 16 weeks.The control group and model group were administered with equal volume of distilled water by gavage.The morphological changes of the aortic sinus and the content of foam cells in mice were observed by the Movat method,while the expression of F4/80 in the aortic sinus was detected by immunofluorescence staining.SMYAD-medicated serum had been prepared,and the drug concentration was screened by the CCK-8 method.A model of oxidized low-density lipoprotein(ox-LDL)-induced foaming of monocyte-macrophage cell line J774A.1 was established and divided into the model group,SMYAD group,and another control group was set.Lipid absorption was detected using the Oil-red O staining,while the molecular expression levels of scavenger receptors were detected by the Western Blot.Results In vivo,compared with the control group,the pathological changes of the aortic sinus in the model group were obvious,while the content of foam cells and the number of F4/80 positive cells in the plaque were significantly increased(P<0.01).Compared with the model group,the pathological changes of the aortic sinus in the low,medium,and high-dose group were improved,and the formation of foam cells was decreased(P<0.05,P<0.01),while the expression of F4/80 protein in aortic sinus was significantly decreased(P<0.01).There was no statistical difference in foam cell content and F4/80 protein expression between the intervention groups(P>0.05).In vitro results displayed that compared with the control group,the area of lipid droplets in the model group was significantly increased(P<0.01),and the expressions of lectin like oxidized low density lipoprotein receptor-1(LOX-1),CD36,and scavenger receptor A1(SRA1)proteins were significantly increased(P<0.01).Compared with the model group,the SMYAD group's lipid absorption decreased(P<0.01);while the protein expression of LOX-1,CD36,and SRA1 decreased(P<0.05,P<0.01).Conclusion SMYAD can inhibit the expression of LOX-1,CD36,and SRA1 in macrophages,reducing cholesterol absorption and macrophage foam formation,thus ameliorating AS.