To Investigate The Mechanism Of Atherosclerosis Formation Induced By High Fat And Cholesterol Diet In Tibetan Miniature Pigs Based On Metagenomics

Objective Metagenomics was used to analyze the changes of intestinal flora composition,gene function and metabolic pathways during the formation of atherosclerosis(AS)in Tibetan miniature pigs induced by high fat and cholesterol(HFC).To explore the correlation between different flora and gene function,inflammation and lipid metabolism during the formation of AS in Tibetan miniature pigs,and reveal the mechanism of AS formation in Tibetan miniature pigs induced by HFC diet.Methods Ten male Tibetan miniature pigs,aged 3-4 months and weighing 8-10 kg,were randomly divided into normal control group(NC group)and model group(AS group)according to their weight after 8 weeks of adaptive feeding,in which NC group was fed with basic feed and AS group was fed with HFC feed for 24 weeks.At the 0 th,4 th,8 th,16 th and 24 th week of modeling,the fasting weight was weighed after fasting for 16 hours,and the anterior vena cava blood was taken,and the serum was separated.The levels of TC,TG,HDL-C and LDL-C were detected,and the atherosclerosis index(AI)was calculated.At 24 weeks,serum CRP,TNF-α,IL-6,IL-1-β and ox-LDL levels were detected by ELISA,and the abdominal aorta tissue was taken for histopathological observation.The feces of each mini-pig in NC group at week 4 and AS group at weeks 4,8,16 and 24 were collected by rectum.The fecal DNA was extracted by metagenomic technology,and a metagenomic library was constructed.The species and functions of the flora were annotated by means of sequencing,quality control and information analysis.After that,the changes of the composition,structure and gene function of the intestinal flora in mini-pigs at different stages of modeling were analyzed,and the correlation analysis AS carried out with phenotypic data such as blood lipid and inflammation to explore the influence mechanism of intestinal flora in the formation of AS in Tibetan miniature pigs.Results(1)During the modeling period,the weight gain of AS group was greater than that of NC group,and the difference between the two groups was significant(P<0.01).At the same time,the serum TC,HDL-C,LDL-C and AI indexes in AS group were significantly higher than those in NC group(P<0.01),but there was no significant difference in TG level(P>0.05).At 24 weeks,the levels of CRP,TNF-α,IL-6,IL-1-β and ox-LDL in AS group were significantly increased(P<0.05),and obvious plaques were formed in abdominal aorta.(2)Four weeks after modeling,PCoA analysis showed that there was a significant difference in β diversity between the two groups,and Venn analysis found that there were 420 endemic strains in AS04W group.Through database annotation,it was found that the structure of intestinal flora changed significantly.Compared with NC group,at the phylum level,six different phylum were found in AS04W group,in which the relative abundance of Proteobacteria,Bacteroidetes,Spirochaetes and Synergistetes phylum increased significantly,while the relative abundance of Firmicutes and Tenericutes decreased significantly(P<0.01,LDA>2).At the family level,38 different bacterial families were found in AS04W group,and the relative abundance of Streptococcaceae,Peptostreptococcaceae and Turicibacteraceae decreased significantly in top 20.However,the relative abundance of Enterobacteriaceae,Bacteroidaceae and Treponemataceae increased significantly(P<0.05,LDA>2).At the species level,88 different strains were found in AS04W group,and the relative abundance of Lactobacillus amylovorus and Vescimonas fastidiosa decreased significantly in top 20.The relative abundance of Escherichia coli,Phocaeicola vulgatus,Treponema succinifaciens increased significantly(P<0.05,LDA>2).In addition,after using KEGG database to annotate the function of flora,115 different ways were obtained.According to the LDA value,among the top 20 pathways,the relative abundance of ribosomes,secondary bile acid biosynthesis and primary bile acid biosynthesis in AS04W group decreased significantly.The relative abundance of lipopolysaccharide biosynthesis,ubiquinone and other terpenoid quinones biosynthesis and folic acid biosynthesis all increased significantly(P<0.05,LDA>2).(3)At 8 weeks after modeling,530 endemic species were found in AS08W group,and the changes of their flora structure were similar to those at 4 weeks.At the phylum level,10 significantly different phylum were found,and the number of Fibrobacteres phylum decreased significantly,Actinobacteria and Fusobacteria increased significantly(P<0.05,LDA>2).At the family level,39 different families were found in AS08W group,and the relative abundance of digestive Streptococcaceae,Clostridiaceae and Turicibacteraceae in top 20 decreased significantly,and Selenomonadaceae and Tannerellaceae increased significantly(P<0.05,LDA>2).At the species level,115 different strains were found in AS08W group,among which the relative abundance of Escherichia coli,Phocaeicola vulgatus,Treponema succinifaciens,Cloacibacillus porcorum and Megasphaera elsdenii increased significantly,while the relative abundance of Lactobacillus amylovorus,Vescimonas fastidiosa and Enterococcaceae bacterium decreased significantly(P<0.05,LDA>2).In terms of the function of intestinal bacteria,120 significantly different pathways were obtained.AS group ribosomes,secondary bile acid biosynthesis,primary bile acid biosynthesis,folic acid carbon pool and other pathways were significantly reduced.The biosynthesis of lipopolysaccharide,ubiquinone and other terpenoids,folic acid and phenylalanine increased significantly(P<0.05,LDA>2).(4)After 16 weeks of modeling,529 endemic species were found in AS16W group,and 10 different phylum were found,among which the relative abundance of newly emerging phylum Lentisphaerae and Verrucomicrobia increased significantly,while Actinobacteria did not change significantly.At the family level,41 different families were found,including digestive Peptostreptococcaceae,Enterobacteriaceae and Erysipelotrichaceae.At the species level,there were 204 different bacteria,among which the relative abundance of Escherichia coli,Escherichia fergusonii and Alistipes finegoldii increased significantly,while the relative abundance of Lactobacillus amylovorus,Vescimonas fastidiosa decreased significantly(P<0.05,LDA>2).231 significantly different pathways were found,among which ribosomes,secondary bile acid biosynthesis,primary bile acid biosynthesis,glycolysis/gluconeogenesis and other pathways were significantly reduced.However,the pathways of lipopolysaccharide biosynthesis,folic acid biosynthesis and phenylalanine metabolism all increased significantly(P<0.05,LDA>2).(5)At the 24th week of modeling,there were 661 endemic species in AS24W group,and 10 different phylum were also found,among which the newly-emerged phylum Elusimicrobia decreased significantly.There are 42 different bacteria in the family level,among which the relative abundance of Enterobacteriaceae,Bacteroidaceae,Synergistaceae and Odoribacteraceae increased significantly.137 different bacteria were found at the species level,and the relative abundance of Escherichia coli,Cloacibacillus porcorum and Phocaeicola vulgatus increased significantly,while the relative abundance of Vescimonas fastidiosa decreased significantly.In addition,111 pathways with significant differences were found,among which ribosomes and other pathways decreased significantly,while lipopolysaccharide biosynthesis,ubiquinone and other terpenoid quinones biosynthesis,folic acid biosynthesis,phenylalanine metabolism,nitrotoluene degradation and β-alanine metabolism increased significantly(P<0.01,LDA>2).(6)Venn analysis of bacteria and functional pathways significantly changed at each stage showed that there were 6 phylum with common changes,among which the phylum Firmicutes and Tenericutes were significantly reduced.However,Proteobacteria,Bacteroidetes,Spirochaetes,Synergistetes all increased significantly(P<0.05,LDA>2).There are 17 families with common changes,among which 5 families are significantly reduced:Actinomycetaceaem,Paenibacillaceae,Eggerthellaceae,Aerococcaceae,Streptomycetaceae;The number of 12 families increased significantly:Enterobacteriaceae,Bacteroidaceae,Treponemataceae,Synergistaceae,Rikenellaceae,Coriobacteriaceae,Desulfovibrionaceae,Odoribacteraceae,Selenomonadaceae,Pseudonocardiaceae,Vibrionaceae,Spirosomaceae(P<0.05,LDA>2).There are 25 strains whose commonness has changed,among which 18 strains have increased significantly:Escherichia coli,Phocaeicola vulgatus,Treponema succinifaciens,Cloacibacillus porcorum,Escherichia fergusonii,Desulfovibrio piger,Saccharomonospora viridis,Alistipes finegoldii,Alistipes shahii,etc.Seven strains significantly reduced Vescimonas fastidiosa and Treponema ruminis(P<0.05,LDA>2).There are 93 ways to change commonness,mainly at the metabolic level,including lipid metabolism(arachidonic acid metabolism,α-linolenic acid metabolism,linoleic acid metabolism,ether lipid metabolism),lipopolysaccharide synthesis,carbohydrate and carbohydrate metabolism(citric acid cycle,glyoxylic acid and dicarboxylic acid metabolism,sphingolipid biosynthesis-ganglion series);The pathways related to amino acid and vitamin metabolism(ubiquinone and other terpenoid quinones biosynthesis,glutathione metabolism,phenylalanine metabolism,folic acid biosynthesis,β-alanine metabolism,arginine biosynthesis)have all increased significantly.In addition,the ribosome function of genetic information processing level is the largest in NC group,and it is significantly reduced in AS group.In addition,most related pathways including organic system,environmental information processing,human diseases and cell process level have changed significantly(P<0.05,LDA>2).(7)Focusing on lipid metabolism,lipopolysaccharide synthesis,citric acid cycle,etc.,the bacteria producing KO in the pathway were analyzed and found to be mainly from Escherichia coli,Treponema succinifaciens,Escherichia fergusonii.Correlation analysis showed that all these were positively correlated with IL-1β,LDL-C,TC and Al(P<0.05).Conclusion Lipid disorder,inflammation and AS plaque formation were observed in 24-week Tibetan miniature pigs induced by HFC diet.The structure and function of intestinal flora changed significantly,and the endemic species increased in the later stage of the disease.At the same time,a total of 6 phyla,17 bacterial families and 25 bacterial species were found to show consistent changes during the formation of AS,suggesting that these bacteria play a core role in the occurrence and development of AS disease.In addition,it was also found that the functional pathways of intestinal flora with common changes in AS formation,such as ribosome function decreased significantly,lipopolysaccharide synthesis,lipid metabolism-related pathways,carbohydrate and carbohydrate metabolism-related pathways increased.It was further revealed that the KO gene families with significant changes in these pathways were mainly from the common altering bacteria of different stages--Escherichia coli,Escherichia fergusonii,Treponema succinifaciens.At the same time,these three bacteria were positively correlated with lipid,inflammatory factors and other indicators,suggesting that Escherichia coli,Escherichia fergusonii,Treponema succinifaciens may be the key bacteria groups in the occurrence and development of AS in Tibetan miniature pigs.The intestinal bacteria and their functional characteristics related to the occurrence and development of AS in Tibetan miniature pigs were revealed,and the potential mechanism of intestinal flora and its metabolic pathway affecting the progression of AS disease in Tibetan miniature pigs was clarified.