高脂高糖饮食诱导五指山小型猪动脉粥样硬化模型肠道菌群的变化

Changes in intestinal flora in a Wuzhishan minipig atherosclerosis model induced by high-fat and high-sugar diet

目的观察高脂高糖饮食致五指山小型猪动脉粥样硬化(atherosclerosis,AS)模型肠道菌群的变化。方法雄性五指山小型猪12只,按体重分为两组,即正常对照(NC)组和模型(AS)组,每组6只。NC组饲喂正常饲料,AS组饲喂高脂高糖饲料,连续饮食24周后,前腔静脉采血测定糖脂代谢等生化指标,同时进行葡萄糖耐量试验。实验结束后行安死术,观察体内脂肪沉积,并取主动脉血管行苏丹IV染色和腹主动脉血管行HE染色分别观察脂质沉积和组织病理学变化。取盲肠内容物进行16S rDNA测序分析肠道菌群的变化。结果与NC组比较,AS模型组血清总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、血糖(GLU)、果糖胺(FMN)菌明显升高(P<0.05,P<0.01),体重和体内脂肪沉积亦显著增加(P<0.01),同时AS模型组主动脉血管明显脂质沉积和AS病变。肠道菌群测序分析显示,与NC组相比,AS模型组肠道菌群α-多样性显著降低(P<0.05,P<0.01)。在门水平上,AS模型组拟杆菌门和放线菌门明显下降(P<0.05,P<0.01),变形菌门和厚壁菌门/拟杆菌门比值均显著升高(P<0.05)。在属水平上,AS模型组黑斯伯利亚菌属(Hespellia)、瘤胃球菌属(Ruminococcus)、毛螺菌属(Lachnospiracea_incertae_sedis)、假丁酸弧菌属(Pseudobutyrivibrio)、巴恩斯氏菌属(Barnesiella)等17个菌属的相对丰度显著下降(P<0.05,P<0.01),沙壤土杆菌属(Ramlibacter)、鞘氨醇杆菌属(Sphingorhabdus)、污水管杆菌属(Cloacibacillus)、草酸杆菌属(Oxalobacter)、鞘脂单胞菌属(Sphingomonas)、新鞘脂菌属(Novosphingobium)等11个菌属的相对丰度显著上升(P<0.05)。功能预测分析显示,脂多糖生物合成、脂多糖生物合成蛋白途径、初级胆汁酸的生物合成、花生四烯酸代谢等多条途径可能参与AS的形成。相关分析显示,TC、内中膜厚度、脂质沉积、果糖胺与草酸杆菌属、鞘氨醇单胞菌属、污水管杆菌属、梭菌属、瘤胃球菌属等14个菌属存在关联。结论高脂高糖饮食诱导的五指山小型猪AS模型具有肥胖、糖脂代谢紊乱和明显的AS病变的特点,其发生可能与肠道菌群失调有关。

Objective To observe changes in the intestinal flora in a Wuzhishan minipig model of atherosclerosis(AS)induced by a high-fat/high-sugar diet.Methods Twelve male Wuzhishan minipigs were divided into normal control(NC)and AS model groups according to body weight(n=6 per group).The NC group was fed a normal diet and the AS model group was fed a high-fat/high-sugar diet.After 24 weeks of continuous diet,blood samples were taken from the anterior vena cava to determine biochemical indexes,including glucolipid metabolism,and a glucose to tolerance test was also performed.At the end of the experiment,the minipigs were euthanized and body fat deposition was observed.The aortic vessels were stained with Sudan IV and the abdominal aortic vessels were stained with hematoxylin and eosin to observe lipid deposition and histopathological changes,respectively.The cecal contents were used for 16S rDNA sequencing to analyze changes in the intestinal flora.Results Serum total cholesterol,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,blood glucose,and fructosamine levels were all significantly higher in the AS model group compared with the NC group(P<0.05,P<0.01).Body weight and body fat deposition were also significantly higher in the AS model group(P<0.01).The aortic vessels showed increased lipid deposition and AS lesions.DNA sequencing analysis showed significantly lower intestinal floraα-diversity in the AS model group compared with the NC group(P<0.05,P<0.01).At the phylum level,the phyla Bacteroidetes and Actinomycetes were significantly reduced in the AS model group(P<0.05,P<0.01),while the phylum Proteobacteria and the Firmicutes/Bacteroidetes ratio were significantly increased(P<0.05).At the genus level,the relative abundances of 17 genera,including Hespellia,Ruminococcus,Lachnospiracea_incertae_sedis,Pseudobutyrivibrio,and Barnesiella,were significantly decreased(P<0.05,P<0.01),and the relative abundances of 11 genera,including Ramlibacter,Sphingorhabdus,Cloacibacillus,Oxalobacter,Sphingomonas,and Novosphingobium,were significantly increased(P<0.05).Functional prediction analysis showed that multiple pathways such as lipopolysaccharide biosynthesis,lipopolysaccharide biosynthesis proteins,primary bile acid biosynthesis,and arachidonic acid metabolism may participate in the formation of AS.Correlation analysis showed that total cholesterol,intima media thickness,lipid deposition,and fructosamine were associated with 14 genera,including Oxalobacter,Sphingomonas,Cloacibacillus,Clostridialse_unclassified,and Ruminococcus.Conclusions High-fat/high sugar diet-induced AS in Wuzhishan minipigs is characterized by obesity,disturbed glucolipid metabolism,and obvious AS lesions,possibly related to dysbiosis of the intestinal flora.