化瘀祛痰方对ApoE^(-/-)动脉粥样硬化模型小鼠肠道菌群驱动TMA/FMO3/TMAO通路的影响

Effect of Huayu Qutan Formula (化瘀祛痰方) on Gut Microbiota-driven TMA/ FMO3/ TMAO Pathway in ApoE^(-/-) Atherosclerosis Model Mice

目的探讨化瘀祛痰方抗动脉粥样硬化(AS)的可能作用机制。方法24只ApoE-/-小鼠随机分为模型组、辛伐他汀组和化瘀祛痰组,每组8只。8只C57BL/6J小鼠作为空白对照组。除空白对照组外,其余各组给予高脂饲料喂养制备AS模型。造模后辛伐他汀组给予辛伐他汀2.275mg/(kg·d)灌胃,化瘀祛痰组给予化瘀祛痰方20g/(kg·d)灌胃,空白对照组和模型组给予等体积(0.5ml)生理盐水灌胃。每日1次,灌胃4周。观察肝脏病理组织形态,检测盲肠内容物肠道菌群,血浆三甲胺(TMA)、氧化三甲胺(TMAO)含量,肝脏黄素单加氧酶3(FMO3)mRNA和蛋白表达。结果与模型组比较,化瘀祛痰组细胞肿胀明显减轻,脂肪空泡显著减少,脂滴沉积减少。与空白对照组比较,模型组血浆TMA、TMAO含量,肝脏FMO3蛋白及mRNA水平升高(P<0.05);与模型组比较,辛伐他汀组和化瘀祛痰组血浆TMA、TMAO含量,肝脏FMO3蛋白及mRNA水平降低P<0.05);辛伐他汀组和化瘀祛痰组各指标比较差异无统计学意义(P>0.05)。肠道菌群Beta多样性分析,各组样本能够显著区分。基于属水平,与空白对照组比较,模型组拟普雷沃菌属、拟杆菌属、大肠杆菌科志贺菌、副拟杆菌属相对丰度下降,gLachnospiraceaeUCG-006、罗氏菌属相对丰度升高(P<0.05);与模型组比较,化瘀祛痰组厌氧棍状菌属、gEubacteriumxylanophilumgroup、gLachnospiraceaeUCG-006、gRikenellaceaeRC9gutgroup、罗氏菌属相对丰度下降,拟杆菌属、柠檬酸杆菌属、大肠杆菌科志贺菌属、副拟杆菌属、葡萄球菌属相对丰度升高(P<0.05)。拟杆菌属、副拟杆菌属与TMA呈负相关,拟普雷沃菌属、拟杆菌属、副拟杆菌属与TMAO呈负相关(P<0.05)。结论化瘀祛痰方可能通过改变肠道菌群结构,减少TMA生成,降低肝FMO3表达,抑制TMAO合成,从而抑制AS发生发展。

Objective To explore the mechanism underlying the antiatherosclerotic effect of Huayu Qutan Formula(HQF,化瘀祛痰方).Methods Twenty-four ApoE-/-mice were randomly divided into model group,simvastatin group and HQF group with eight mice in each group,fed with high fat diet to prepare AS models.Eight C57 BL/6 J mice as the blank group were fed with normal diet.After modelling,2.275 mg/(kg·d)simvastatin and 20 g/(kg·d)HQF were administered by gavage in simvastatin group and HQF group,respectively;equal volume normal saline(0.5 ml)was given by gavage in blank group and model group,once daily for four weeks.The hepatic pathological histology,gut microbiota of cecal contents,plasma trimethylamine(TMA)and trimethylamine oxide(TMAO)contents,and liver flavin monooxygenase 3(FMO3)mRNA and protein expression were assessed.Results Compared to those in the model group,the swelling of cells,the fat vacuole and lipid droplet deposition were significantly reduced in HQF group.Compared to blank group,the model group had significantly higher plasma TMA and TMAO contents,as well as higher FMO3 mRNA and protein expression(P<0.05).Compared to those in the model group,TMA and TMAO contents and FMO3 mRNA and protein expression decreased in simvastatin group and HQF group(P<0.05).There were no significantly statistical differences between simvastatin group and HQF group in all outcomes(P>0.05).Beta-diversity analysis of the intestinal flora indicated that different group samples can be significantly distinguished.In terms of the genus level,model group had decreased relative abundance of Prevotella,Bacteroides,Shigella of Escherichia and Parabacteroides,as well as increased gLachnospiraceaeUGG-006 and Rosella compared to the blank group(P<0.05);relative abundance of anaerobic bacillus,gEubacteriumxylanophilumgroup,gLachnospiraceaeUCG-006,gRikenellaceaeRC9gutgroup,and Rosella decreased,while Bacteroides,Citrobacter,Shigella of Escherichia,Parabacteroides,and Staphylococcus increased in HQF group,compared to those in model group(P<0.05);Bacteroides and Parabacteroides were negatively correlated with TMA,while Prevotella,Bacteroides and Parabacteroides were negatively correlated with TMAO(P<0.05).Conclusion HQF can change gut microbiota structure,decrease TMA,reduce FMO3 expression,and limit TMAO synthesis,thereby inhibit the development of atherosclerosis.