| Background:Coronary artery disease（CAD）is a cardiovascular disease with high morbidity and mortality worldwide.CAD is a heart disease that forms plaque of atherosclerosis by oxidizing lipids inside the coronary artery,causes arterial stenosis and occlusion,and brings about myocardial ischemic-anoxic injury or necrosis.Correcting dyslipidemia is the recognized prevention and treatment target in preventing and reversing atherosclerotic lesions,and reducing the morbidity and mortality of CAD.Despite great progress in treatments,the incidence and mortality of CAD are still increasing year by year.Therefore,at present,the control and treatment rate of dyslipidemia and CAD still need to be improved,and strengthening prevention and treatment work is urgent.In-depth research on gut microbiota provides new opportunities for the prevention or treatment of cardiovascular diseases.Gut microbiota may directly or indirectly regulate lipid metabolism,immune,and CAD.According to our previous metagenomic study,Bacteroides cellulosilyticus,Faecalibacterium prausnitzii and Roseburia intestinalis were significantly lacking in CAD patients.Previous clinical studies also found that B.cellulosilyticus and F.prausnitzii were related to a healthy diet and lipid profile in other populations besides CAD.However,the beneficial role of B.cellulosilyticus in various diseases is still little known at present.Although F.prausnitzii treatment has been shown to improve liver health and reduce adipose tissue inflammation in high-fat-fed mice,its role in atherosclerosis is unclear.Treatment with R.intestinalis combined with a high-fiber diet also reduced the size of aortic atherosclerotic plaques in germ-free mouse models specifically clothed with eight butyrate-producing core communities.However,the beneficial effects of supplementing R.intestinalis alone on conventional mice of the atherosclerosis model and the possible molecular mechanisms in addition to butyric acid production are still unknown.Moreover,previous study has reported that Bacteroidetes can promote the growth of F.prausnitzii,but whether the combined supplement of Bacteroidetes and Firmicutes has better efficacy than a single supplement has not been reported.Therefore,it is of great clinical significance to explore whether these candidate intestinal strains and their combinations can effectively prevent and/or treat dyslipidemia and atherosclerosis in atherosclerotic model mice and to elucidate their possible mechanisms of action from multiple perspectives.It is well known that metabolites are important messengers for intestinal bacteria to affect the balance of lipid metabolism and the occurrence and development of cardiovascular diseases.Studies have shown that these three candidate bacteria can play a beneficial role by producing short-chain fatty acids.However,the metabolites derived from intestinal microbial symbionts are extremely rich and diverse,and many circulating intestinal microbiota-dependent metabolites are still unknown under different disease conditions or the actions of intestinal flora.Intestinal flora can also participate in the pathogenesis of CAD by regulating the bile acid signal pathway,such as activating the farnesoid X receptor（FXR）by regulating the bile acid ratio,thus influencing the lipid metabolism of liver and promoting the development of CAD.Therefore,based on the advances in sequencing technology and multi-omics methods,to reveal the complex regulatory network changes of host-microbe-metabolite-gene expression levels related to lipid dysregulation and atherosclerosis of CAD that caused by strains interventions at different molecular levels based on the specific disease environments and tissues.It will further expand our understanding of the role of intestinal flora in CAD.This study is a comprehensive study combining clinical data and animal experiments.Firstly,the relationships between B.cellulosilyticus,F.prausnitzii and R.intestinalis and CAD development and cardiovascular metabolic factors such as blood lipid,were confirmed by using population cohort.Apoe-/-mouse model was used to verify the effect of supplementing the three candidate intestinal strains alone or in combination to improve blood lipid and atherosclerotic lesions.Then,combined with liver transcriptome analysis and plasma widely-targeted metabolomics analysis,we elucidated the important metabolites and signal pathways that may be regulated by supplementing candidate strains to improve lipid metabolism balance and atherosclerosis in Apoe-/-mice.Further,metagenomic sequencing was used to reveal whether the supplement of candidate strains plays a lipid-lowering role by regulating the distribution of intestinal flora and to explore the potential protective effects of the three candidate intestinal bacteria on clinical prognosis in patients with CAD.Thus,these study results would deepen the knowledge of the complex regulatory network between intestinal microbiota,metabolite,and the host,and then provide new evidence to facilitate the illustration of the potential mechanism of cardiovascular protection of the three candidate strains,which is expected to provide potential biomarkers and new methods for the prevention and treatment of CAD.Chapter I The effect of three candidate intestinal bacteria on improving lipids and anti-atherosclerosis was confirmed in the human cohort and Apoe-/-miceObjective:1.To clarify the effects of candidate intestinal bacteria B.cellulosilyticus,F.prausnitzii and R.intestinalis on atherosclerosis and cardiovascular metabolic factors such as blood lipid in the population cohort.2.To elucidate the protective effect of three candidate strains alone or in combination on improving blood lipid levels and anti-atherosclerosis in Apoe-/-mice using an animal model.Methods:1.R software was used to re-analyze metagenomic data from 218 patients with coronary atherosclerotic heart disease and 187 healthy people.Wilcoxon rank sum-test was used to compare the relative abundance of B.cellulosilyticus,F.prausnitzii and R.intestinalis between the two groups.2.The metagenomic sequencing data of 2183 healthy adults were used to analyze the correlation between intestinal flora and cardiovascular metabolic factors such as blood lipids.3.Three strains of bacteria（B.cellulosilyticus AF17-25,F.Longum CM04-06,and R.Intestinalis AM43-11）were isolated from young healthy individuals and given to Apoe-/-mouse model alone or in combination for 18 weeks by gavage.Grouping:normal high-fat control group,Apoe-/-model control group,B.cellulosilyticus group,F.Longum group,R.Intestinalis group,B,F,R consortium group.4.Plasma glucose（GLUC）,triglyceride（TG）,low-density lipoprotein cholesterol（LDLC）,total cholesterol（CHOL）,and high-density lipoprotein cholesterol（HDLC）of mice after intervention were measured by an automatic chemical analyzer.5.Oil-red O staining of the intima of longitudinally opened aortas,Oil-red O,Masson,and HE staining of frozen sections of aortic root were used to evaluate the atherosclerotic lesions of the aorta,and cardiac ultrasound of small animal was used to evaluate the cardiac structure and function of mice.Results:1.Compared with healthy individuals,B.cellulosilyticus(P=6.06×10-3),F.prausnitzii(P=8.83×10-5)and R.intestinalis(P=1.17×10-4)were significantly lower in patients with coronary atherosclerosis.2.In 2183 healthy young individuals,B.cellulosilyticus was negatively correlated with blood pressure,serum TG level,and waist to hip ratio,but positively correlated with HDLC,F.prausnitzii is negatively correlated with increased TG,CHOL,and LDLC levels,and body fat ratio.R.intestinalis was negatively correlated with waist-hip ratio and body fat（FDR<0.05）.3.Compared with the mice of Apoe-/-control group using vehicle intervention（P<0.05）,GLUC,TG,and LDLC of mice of B.cellulosilyticus group were significantly reduced,TG and LDLC of mice of F.Longum group were significantly reduced.The mice of the R.intestinalis group were significantly reduced in TG and increased in HDLC,while the mice of the B,F,R consortium group were also significantly reduced in GLUC and LDLC levels.4.Compared with the mice of Apoe-/-control group using vehicle intervention（P<0.05）,oil-red O staining of the longitudinally opened aortas showed that the accumulation of lipid in the aortic intima of the mice of the group of B.cellulosilyticus and B,F,R consortium significantly reduced;oil-red O staining and HE staining on the cross-section of the aortic root showed that the lipid deposition and aortic atherosclerotic plaque formation of the mice of the group of B.cellulosilyticus and B,F,R consortium were significantly reduced.Masson staining showed that the collagen deposition in the aorta of the mice treated with the R.intestinalis and B,F,R consortium was significantly reduced.Cardiac ultrasound showed that the left ventricular ejection fraction and the shortened fraction of mice treated with the B,F,R consortium were higher.Conclusion:The reduction of B.cellulosilyticus,F.prausnitzii and R.intestinalis in human is related to the occurrence of coronary atherosclerosis and unhealthy lipid metabolism parameters.Adding three candidate strains（B.cellulosilyticus AF17-25,F.Longum CM04-06,and R.intestinalis AM43-11）,especially B.cellulosilyticus and a consortium of the three strains,has good effects on alleviating lipid imbalance and/or atherosclerotic lesions in atherosclerotic model mice,and the consortium supplement also has a moderately protective effect on the heart function of mice.These evidences will provide important evidence of their effectiveness as probiotics and intervention targets for the prevention and treatment of potential lipid imbalances and/or atherosclerosis.Chapter 2 Effects of candidate intestinal strains on the liver transcriptome and plasma widely-targeted metabolome of Apoe-/-miceObjective:To explore the potential regulation of major signal pathways and key metabolites associated with the intervention of candidate strains,we hope to deepen the understanding of host-microbe-metabolite regulatory networks involved in lipid metabolism disorders and atherosclerosis and provide important insights into the molecular mechanism of the beneficial effect of these candidate strains.Methods:1.The liver of the Apoe-/-mice separated after the intervention in chapter 1 were taken as the research object,gene expression levels were detected in the liver by transcriptome sequencing,and Gene Ontology（GO）enrichment analysis was carried out using“cluster Profiler”package."Edge R"package was used to analyze the differentially expressed genes associated with strain intervention.2.The plasma of Apoe-/-mice collected after the intervention in chapter 1 were used as the research object,and the endogenous metabolites in the plasma were detected by a widely-targeted metabolome.The differential metabolites associated with strains intervention were analyzed by orthogonal partial least squares discriminant analysis（OPLS-DA）and t-test.Results:1.GO enrichment analysis showed that,compared with the Apoe-/-control group,the group of B.cellulosilyticus,F.longum and R.intestinalis intervention alone and in combination,genes of lipid synthesis and metabolism pathway were significantly down-regulated,while genes related to circadian rhythm were significantly up-regulated.2.Transcriptome analysis of the differentially expressed genes showed that,compared with the Apoe-/-control group,candidate strains intervention could significantly regulate the expression of genes related to lipid metabolism in the liver of Apoe-/-mice,including reduced synthesis genes of cholesterol（such as Hmgcs1,Pmvk,and Dhcr7）,fatty acids（such as Acc,Fasn,Acly,and Elovl6）and TG（Agpat2）,increased TG and fatty acid decomposition（such as Pnpla2,Cpt1a）and bile acid synthesis（Cyp8b1,Cyp7a1）genes.Candidate strain intervention also activated the expression of nuclear receptors（such as Rev-erb,Pparα,FXR,Nr4a1）and circadian genes（Rev-erb,Dbp,Ciart,Per3）to promote lipid balance.3.Compared with the Apoe-/-control group,the supplement of candidate strains was accompanied by an increase in plasma levels of metabolites such as lithocholic acid,3-hydroxy-3-methylbutyric acid,indoleacetic acid,and xanthosine,and a decrease of 20,26-dihydroxyecdysone,glycerophospholipid and fatty acids.The changes in these differential metabolites were significantly associated with healthy lipid balance.Conclusion:1.B.cellulosilyticus,F.longum,and R.intestinalis and their combination supplements can improve the imbalance of blood lipids in atherosclerotic model mice by inhibiting the liver expression of genes related to the production of fatty acids,TG,and CHOL,and promoting the genes expression of fatty acid and TG decomposition and bile acid formation,and further prevent the formation of atherosclerosis.2.The intervention of candidate strains may also promote lipid balance by regulating the plasma metabolites of hosts,increasing the levels of lithocholic acid and 3-hydroxy-3-methylbutyric acid,and decreasing the levels of 20,26-dihydroxy-ecdysone,and glycerophospholipid.3.On the one hand,candidate strains supplement may regulate the balance of lipid metabolism pathway through up-regulation of circadian genes such as Rev-erb,Dbp,Ciart,and Per3.On the other hand,it may regulate lipid metabolism through the activation of nuclear receptor genes FXR and Pparɑby lithocholic acid,thus improving the lipid metabolism disorders and/or atherosclerosis induced by a high-fat diet.Chapter 3 The effect of candidate intestinal strains on intestinal microflora distribution and plasma lipids of Apoe-/-miceObjective:To observe the colonization effect of the candidate strains in the intestinal microbiota of mice after candidate strains intervention and the dynamic changes in the distribution of each intestinal microbiota,to reveal the influence of the difference in the distribution of intestinal microbiota caused by intervention on the levels of blood lipids and metabolites of the host,and then to analyze the potential function of the synthesis and metabolism of key microbial metabolites in each group of mice.Methods:1.Quantitative analysis of fecal microbiota of Apoe-/-mice at 6-25 week-olds during the intervention was performed by Shotgun metagenome sequencing on the BGISEQ-500 platform.Differences in microbial composition between groups were compared by Per MANOVA test,and the growth and overall structure of microbiota of mice in each group were analyzed by DMM clustering model.2.Linear regression was used to analyze the correlation between lipid levels and the relative abundance of intestinal flora in mice at 25 week-olds after the intervention of candidate strains,as well as the effect of the intervention of candidate strains on the relative abundance of intestinal flora after the intervention and the growth rate of intestinal flora during the intervention.3.The metabolic changes in 7α-dehydroxylation pathway of lithocholic acid（LCA）synthesis,synthetic and metabolic pathways of short-chain fatty acids（SCFA）,and branched-chain amino acids（BCAA）of each group of mice at 25 week-olds after intervention were analyzed by functional annotation of the metagenome.4.Linear regression analysis was used to analyze the correlation between the relative abundance of fecal intestinal flora and plasma metabolite levels of mice after the intervention of candidate strains.Results:1.After the intervention,the intestinal microbial composition of Apoe-/-mice in each group were significantly different(R2=0.285,P=1×10-4),and the corresponding abundance of the candidate bacteria in B.cellulosilyticus and F.longum groups increased significantly.The relative abundance of the bacteria corresponding to the R.intestinalis group was slightly increased,and only the abundance of B.cellulosilyticus and R.intestinalis in mice of the combined intervention group was significantly increased.DMM cluster analysis showed that the candidate strains differently affected the growth and overall structure of the intestinal microbiome in mice.2.There 11 taxa were correlated with blood lipid levels（FDR<0.05）,and seven were negatively correlated,including Lactobacillus genus（e.g.L.johnsonii and L.taiwanensis）,Bacteroides phylum（Bacteroides caecimuris,B.cellulosilyticus and Alistipes inops）,Bilophila wadsworthia and Staphylococcus nepalensis.Four taxa were positively correlated,including Olsenella profuse,Bordetella pseudohinzii,Oscillibacter sp 13,and Turicimonas muris.The relative abundance and growth rate of the taxa negatively correlated with LDLC level in the intestinal flora of the groups of candidate strain intervention were significantly higher than that of the Apoe-/-control,while the taxa positively correlated with LDLC significantly decreased.3.The relative abundances of the above 11 lipid-related taxa were correlated with the levels of69 plasma metabolites,including Lysopc,Lysope,carnitine,LCA,BCAA,and 20,26-dihydroxyecdyone.4.Functional annotation of metagenome showed that,compared with the Apoe-/-control group,isoleucine synthesis of BCAA synthesis pathway was decreased in the intestinal flora of mice after candidate strain interventions,while phosphopantothenic acid biosynthesis I and tetrapyrrole biosynthesis I（from glutamate）of the BCAA catabolism were significantly increased.SCFA synthesis and metabolism including the pathway of pyruvate fermentation to propanoate I and phytate degradation I to butyric acid increased.5.After the intervention of the candidate strains,the fecal LCA level of mice showed a consistent trend with the bai operon transcription abundance of 7α-dehydroxylation.Compared with the Apoe-/-control group,the bai transcription abundance of mice after the strain interventions showed an overall increasing trend.Thereinto,Lachnospiraceae bacterium,Olsenella profuse,Roseburia intestinalis,Faecalibacterium prausnitzii,and Eubacterium plexicaudatum have higher bai abundance.Conclusion:1.The intervention of B.cellulosilyticus,F.longum and R.intestinalis and their combination can effectively remodel the species balance of the intestinal flora in Apoe-/-mice,suggesting that the intervention of candidate strains is dependent on the complex interaction network between intestinal microbiota,thus conferring health benefits on the host.2.Intervention of candidate strains can transform Apoe-/-mice communities dominated by lipid positively correlated taxa（e.g.Aerococcus viridans and Olsenella profuse）into lipid negatively correlated taxa,such as Lactobacillus（such as L.taiwanensis,L.johnsonii）,Bacteroides（such as B.caecimuris,B.cellulosilyticus,Alistipes inops）.These changes were beneficial to reduce the lipid levels of the host and alleviate the lipid deposition of the aorta.3.The remodeled intestinal flora structure could further regulate the plasma metabolite levels and the corresponding metabolic function of the intestinal flora in the host,including increasing the potential of SCFA production and reducing the potential of BCAA generation,and increasing the transcription abundance of bai of the intestinal flora to promote LCA production,and then play a cardiovascular protective role.Chapter 4 Effects of three candidates’intestinal microbiota on the clinical prognosis of patients with coronary artery diseaseObjective:In patients with existing CAD,we explored which cardiovascular metabolic factors the intestinal microbiota B.cellulosilyticus,F.prausnitzii,and R.intestinalis might regulate,to play a protective role for patients and ultimately reduce the risk of clinical endpoint events.Methods:1.Patients with CAD were prospectively enrolled,and cardiovascular metabolic factors such as blood lipid,blood glucose,blood pressure,and markers of heart failure,myocardial infarction,and immune inflammation were collected at admission.Death and major adverse cardiovascular events（MACE）were regularly followed up after discharge.2.The BGISEQ-500 metagenome sequencing platform was used to detect the composition and relative abundance of B.cellulosilyticus,F.prausnitzii and R.intestinalis in the feces of patients.3.Using R software,linear regression was used to analyze the correlation between the relative abundance of candidate gut microbiota and cardiovascular metabolic factors,and the Cox regression model was used to evaluate the impact of the relative abundance of gut microbiota on the risk of death and MACE.Results:1.Linear regression analysis（P<0.05）showed that the abundances of F.prausnitzii and R.intestinalis in CAD patients were negatively correlated with N-terminal pro-brain natriuretic peptide（pro BNP）,high sensitivity Troponin T（hs-Tn T）and an inflammatory indicator of fibrinogen（FIB）,and were positively correlated with serum-free triiodothyronine（FT3）.R.intestinalis was negatively correlated with glycosylated hemoglobin（Hb A1c）and inflammatory marker of C-reactive protein（CRP）and neutrophil count（NEUT）.2.Cox regression analysis showed that F.prausnitzii（HR[95%CI]:0.80[0.74-0.88],P=0.0067）and R.intestinalis（HR[95%CI]:0.74[0.67-0.86],P=0.0006）were associated with a reduced risk of death.F.prausnitzii was also significantly associated with a reduced risk of MACE（HR[95%CI]:0.84[0.76-0.91],P=0.0004）.Conclusion:F.prausnitzii and R.intestinalis had the potential benefit of reducing the risk of heart failure and clinical endpoints in patients with CAD,and the results further supported the possibility that R.intestinalis could reduce glucose and inflammation.It provides a new direction and theoretical basis for expanding its application in the secondary prevention of CAD.In summary,the results of the cohort study and animal model in this study consistently confirmed the effectiveness of supplementing B.cellulosilyticus,F.prausnitzii,and R.intestinalis alone or in combination,in alleviating lipid imbalance and/or atherosclerosis.This study also provides new insights into the microbial-metabolite-lipid metabolism gene regulatory network in the host during the intervention of candidate strains to improve lipid metabolism imbalance and provides important evidence for elucidating the potential mechanism of candidate strains exerting cardiovascular protection.Therefore,the results of this study will provide important theoretical support for taking the candidate bacteria as probiotics or intervention targets for the prevention and treatment of lipid imbalance and atherosclerosis,and will be hoping to extend the role of F.prausnitzii and R.intestinali to the improvement of clinical prognosis in patients with CAD. |
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