Qingxin Jieyu Decoction Reshapes The Intestinal Flora And Regulates The Lipid Metabolism Of Atherosclerosis And Its Mechanism

In our previous multi-center,randomized placebo controlled,double blinded clinical study,we found that the addition of Qingxin Jieyu Granule(QXJYG)to standard medical therapy of coronary heart disease(CHD)could safely reduce the incidence of non-fatal myocardial infarction and cardiogenic death as well as the composite endpoint events of CHD.Meanwhile,QXJYG could reduce the inflammatory factors of patients with enhanced inflammatory response.Therefore,we conducted related studies to reveal the potential mechanism of effects of QXJYG.Part 1:Studies on human serum samples metabolome of patients with stable CHD based on a randomized,double-blinded,placebo controlled clinical trial.Objective:We studied the differences of serum metabolome between QXJYG treatment and placebo treatment to reveal the metabolites and their relative pathways changes after 6-months QXJYG treatment.Methods:We randomized selected 93 serum samples from our previous multi-central,randomized placebo controlled,double blind clinical study,in which 51 samples were from QXJYG treatment group and the others were from placebo group.UPLC/MS analysis was used to detected the serum metabolites.Progenesis QI(Waters,Milford,USA)was used for data analysis.Then Principal Component Analysis(PCA)and Partial Least Squares Discriminant Analysis(PLS-DA)were conducted by EZinfo program.Metabolites abundance more than 1000,max fold change more than 2 and P value of student T test less than 0.05 were selected as differential compounds based on PLS-DA.Then we conducted compounds identification,content analysis and metabolic pathway analysis.Results:PCA did not showed significant differences between QXJYG treatment and placebo treatment because of the individual differences of clinical serum samples.PLS-DA showed significant difference between two groups.Therefore,17 different compounds were selected after analysis.Compared with placebo group,10 compounds were increased and 8 compounds were decreased in QXJYG group.These different compounds were from glycerophospholipid metabolism.Conclusion:The major impact metabolic pathway of QXJYG was glycerophospholipid metabolic pathway.Part 2:Studies on the lipid metabolism_changes and potential mechanisms of QXJYG-treated high-fat fed ApoE-/-mice.Objective:Based on the results from Part One,we designed animal experiments to reveal the changes of lipid metabolites and potential mechanisms of QXJYG treated high-fat fed ApoE-/-mice.Methods:40 male ApoE-/-mice randomized divided into 4 groups of 10,including QXJYG low dosage(Low)group,QXJYG high dosage(High)group,chow diet group(Chow)and AS group(Mod).Chow group fed with non-cholesterol,low fat diet(10%fat(wt/wt),0%cholesterol),the other three groups were fed with high fat and high cholesterol diet.After 12-week of relevant intervention,we collected serum samples,metabolites,ileum,ileocecal valve contents,liver and aorta.Then we evaluated the aortic plaque areas,the quantity of monocyte/macrophage in aortic valve plaque,hepatocyte appearance and lipid contents,urine metabolism of short-chain fatty acids(SCFAs),serum lipid profiles and lipid metabolites,serum IL-1? level,lipid metabolites gene mRNA expression,cholesterol metabolism and reverse cholesterol transport gene expression.Results:QXJYG dosage-dependently exhibited effects in alleviating the aortic plaque areas and their monocyte/macrophage level,improving serum lipid profile,fatty degeneration and lipid deposition in liver tissue and serum IL-1? level.After serum lipid metabolites analysis,we found 22 different compounds most of which were productions in glycerophospholipid metabolism pathway.Compared with Mod group,there were 18 compounds decreased and 4 compounds increased in High group.QXJYG changed he mRNA levels of key enzymes and proteins in glycerophospholipid metabolism and cholesterol metabolism and reverse cholesterol transport.In addition,QXJYG significantly increased butyrate acid,ISO-VA and 4-MEVA levels compared with Mod and Chow groups.Conclusion:QXJYG exhibited anti-AS effects,improving lipid metabolism,SCFAs metabolism and serum IL-1? level.The major impact metabolic pathway of QXJYG was glycerophospholipid metabolic pathway,which was related to the relative mRNA expression changes of key enzymes in this pathway.Part 3:QXJYG changed gut microbiota composition of high-fat fed ApoE-/-mice.Objective:To evaluate the effects of QXJYG on modulating gut microbiota composition of high-fat fed AopE-/-mice.Methods:Microbial DNA was extracted from ileocecal valve contents samples,the V3-V4 hypervariable regions of the bacteria 16S rRNA gene thermocycler PCR system.Operational taxonomic units(OTUs)were clustered with 97%similarity cutoff using UPARSE with a novel 'greedy' algorithm that performs chimera filtering and OTU clustering simultaneously.Based on these analysis,alpha-diversity,beta-diversity and differential species analysis.Results:QXJYG affected gut microbiota abundance but not the diversity of species.There were 449 OTUs in our sequencing samples,represented for 449 gut bacteria,45%of which were shared by all samples,12%OTUs were specific to each group.We detected 130 gut microbiota genus,71 genus were shared by all samples,19 genus were specific to each group.The highest abundance of the top five phylum were firmicutes,Proteobacteria,Actinobacteria,Bacteroidetes and Verucomicrobia.The highest abundance of the top 15 genus were Lactobacillus?Lachnospiraceae?Faecalibaculum?Lachnospiraceae?Lachnoclostridium?Bifidobacterium?Blautia?Erysipelatoclostridium?Akkermansia?Clostridium?Ruminococcus?Lachnospiraceae NK4A136_group?Enterorhabdus?Helicobacter?g_unclassified_p_Firmicutes.Compared with Chow and Mod groups,Low and High groups increased the abundance of Akkermansia(Phylum Verucomicrobia)(P<0.05).In spearman correlation analysis,the 15 different genes showed correlation to the serum IL-1? levels and SCFAs levels.The network interaction community analysis of gut microbiota showed positive relations between genus Lachnoclostridium,Erysipelatoclostridium,Clostridium and Blautia(community1)as well as Bifidobacterium,Lactobacillu and Faecalibaculum(community 2).However,negative correlation detected between community 1 and community 2,which may showe agonist mechanism between the two communities.In conclusion,QXJYG may increase the abundance of Akkermansia genus as well as its coexisted species which showed anti-AS effects.Conclusion:QXJYG modulated the composition of gut microbiota,which showed was correlated with lipid metabolism and anti-AS effects.