Research On The Correlation Between Intestinal Flora And Hyperlipidemia

Research purposesThe clinical biochemical information and fecal biological samples of chronic disease examination personnel of the Central Nationalities University Hospital from January to October 2015 were collected,and the abundance expression of the ampoule sequence variant table?ASV?was detected by second-generation sequencing technology.Propensity Score Matching method controls the influence of confounding factors such as gender,age,hypertension,diabetes,etc.,and screens the differential ASV between patients with hyperlipidemia and non-hyperlipidemia,and uses the probability map model?Bayesian network?to simulate The causal relationship between differential ASV and hyperlipidemia provides a scientific basis for the key prevention and treatment of intestinal flora in hyperlipidemiaResearch metholdIntestinal flora sequencing and structural analysis.The total DNA of the intestinal flora of each fecal sample was extracted,and the primers were designed according to the V3-V4 region of the bacteria?the upstream primer was 5'-ACTCCTACGGGAGGCAGCA-3'and the downstream primer was 5'-GGACTACHVGGGTWTCTAAT-3'?,and the 16S rRNA gene was amplified.High-throughput sequencing on the illumina Hiseq2500 platform to generate raw data in FASTQ format,and then splicing and filtering the data using R software DADA2 package to generate amplicon sequences?ASV?and species annotations;analysis and amplification using R software vegan package Subsequence?ASV?,sample alpha diversity index,and Beta diversity index were used to compare whether the flora between the two groups had significant differencesDifferential screening and enrichment analysis.In order to control the influence of confounding factors on the experimental results,the R software MatchIT package was used to match the propensity scores of the hyperlipidemia group and the non-hyperlipidemia group according to the ratio of 1:1.The R software DESeq package was used to screen the differential ASV between the hyperlipidemia group and the non-hyperlipidemia group after the preference score matching;the differential ASV was drawn according to the classification structure of boundaries,gates,classes,orders,families,genera and species.Classify trees and perforam enrichment analysis on each classification node.Functional clustering and causal simulation.According to the principle of“similar expression,similar function",the difference between the hyperlipidemia group and the non-hyperlipidemia group was selected.The R language WGCNA package was used for co-expression analysis of differential ASV,and to find the relationship between different functional clustering modules of ASV and hyperlipidemia and its biochemical indicators?CHO,TG,HDL-C,LDL-C?;Bayesian network analysis of differential ASVs that are significantly associated with clinical indicators of hyperlipidemia,speculating between ASVs in the module Interactions,as well as predicting core flora.Research resultA total of 133 clinical biochemical information and fecal biological samples were collected from the study.Among them,87 cases of hyperlipidemia and 46 cases of non-hyperlipidemia.Intestinal flora sequencing and structural analysis.Pair-end obtained the data format in FASTQ format,using Dada2 package of R.3.51 software to quality control the effective sequence of 133 columns of samples,denoising?correcting the sequence of sequencing errors?,splicing the sequence and removing the chimera to form 20295 ASV.At the level of the phylum,87 non-hyperlipidemia fecal samples have a total of 19 phyla.The four highest bacterial phyla were Bacteroidetes?46.7%?,Firmicute?44.2%?,Actimobacteria?8.0%?and Proteobacteria?0.3%?.Hyperlipidemia fecal samples have 368 genera at the genus level,and 18 relative abundances greater than 1.0%.Having a relative abundance of more than 5%is Bacteroides?33.32%?,Prevotella 9?7.68%?,Bifidobacterium?6.74%?and Faecalibacteriu?5.77%?.Among the bacterial genera with relative abundance greater than 1.0%,Bacteroides and Prevotella₉ were Bacteroides.It belongs to the bacterium Bifidobacterium.Belonging to Firmicutes were Lachnospira?3.64%?,Agathobacter?3.46%?,Lachnoclostridium?3.39%?,Blautia?1.93%?,etc.At the level of the phylum,46 non-hyperlipidemia fecal samples have a total of 19 phyla.The four highest bacterial phyla are Firmicute?50.67%?,Bacteroidetes?42.88%?,Actinobacteria?5.84%?,and Verrucomicrobia?0.26%?.At the genus level,there were 297 genera and 18 bacterial genera with relative abundance greater than 1.0%.Having a relative abundance of more than 5%,the species includes Bacteroides?33.75%?,Faecalibacteriu?9.40%?and Prevotella₉?5.23%?.Among the bacterial genera with relative abundance greater than 1.0%,Bacteroides?33.75%?,Prevotella 9?5.23%?,Alistipes?1.51%?and Parabacteroide?1.17%?were Bacteroidetes It is Bifidobacterium?4.77%?.The bacteria belonging to Firmicutes were Lachnospira?3.54%?,Agathobacter?3.77%?,Lachnoclostridium?2.78%?,Blautia?1.66%?,Lachnoclostridium?9.40%?,etc.Alpha diversity?Chao 1 p=0.64,Observed species p=0.15,Simpson p=0.6,Shannon p=0.71?,Beta diversity?bray-curtis distance?R2=0.007,p=0.52?,jaccard distance?R2=0.07,p=0.48??were not significantly different between the hyperlipidemia group and the non-hyperlipidemia group?p>0.05?.Differential screening and enrichment analysis.Then,the DESeq package of R software was used to screen the ASV differences between the hyperlipidemia group and the non-hyperlipidemia group after propensity score matching.A total of 359 ASV differences were screened?|logFC|>2 and ad.P.Val<0.01?.In comparison with the annotated list of ASV species,the differences in ASV were divided into Bacteroidetes,Firmicutes,Proteobacteria,Fusobacteria,Actinobactena and Verrucomicrobia at the phylum level,which were a total of 6 phyla.At the genus level,they were divided into Bacteroides,ruminococcaceae u.cg-014,Paraprevotella,Asteroleplasma,Prevotella₉,etc.,a total of 42 genera,of which 35 ASV could only be annotated to the family level.From the perspective of taxonomy,the key flora of hyperlipidemia and non-hyperlipidemia are Bacteroides,Prevotella 9,Parabacteroides and Paraprevotella,all of which belong to the choroid of Bacteroideaceae to Bacteroidetes.Functional clustering and causal simulation.By analyzing the indicators of hyperlipidemia in 88 samples and correlating the disease with each module,the target modules significantly related to clinical information were screened out.According to the results of weighted gene co-expression network and hyperlipidemia clinical information,there is no module related to CHO.The module with significant positive correlation with TG is blue module?cor=0.22,p=0.44?,red module?cor=0.26,p=0.01?,where in the ASV contained in the module belongs to Finnicutes and the Bacteroides,and the main genus is Bact.eroides,Lachnospiraceae,Erysipelatoclostridium,etc.;Modules significantly negatively correlated with HDL-C,for the cyan module?cor=-0.25,p=0.02?,the ASV in the module belongs to the Bacteroidetes and both are Bacteroides;the module with significant negative correlation with LDL-C is the magenta module?cor=-0.23,p=0.03?ASV belongs to the group of Firmicutes and Actinomycetes.There are 3 genera,Bifidobacterium,Ruminococcus₂,Rothia,salmon module?cor=-0.22,p=0.04?,and pink module?cor=0.23,p=0.03?,green module?cor=0.26,p=0.02?was significantly associated with hyperlipidemia?HL?,but the salmon module was significantly negatively correlated with HL,and the pink module and green module HL were significantly positively correlated.Among them,the Prevotella₉ of ASV,which has a significant negative correlation with HL salmon module,belongs to Bacteroidetes.The ASV of pink and green modules with significant positive correlation with HL belongs to Bacteroidetes and Firmicutes,mainly Lachnospiraceae.In the differential function module,according to the Bayesian network analysis,ASV151 directly affects HDL-C,which is Bacteroides.ASV 1756 directly affects LDL-C and it is Bifidobacteriu.m.ASV360 directly affects hyperlipidemia,and hyperlipidemia is negatively correlated.The bacterium Prevotella₉ belongs to bacteroidetes.ASV428 and ASV658 directly affect hyperlipidemia and are positively correlated with hyperlipidemia.This bacterium is Bacteroides.Analysis conclusionIt is suggested that the core flora with causality with hyperlipidemia is ASV 1756,ASV 151,ASV360,ASV428,ASV658,respectivelyTaxonomically,ASV 151,ASV360,ASV428 and ASV658 belong to bacteroidetes,while ASV1756 belongs to actinomycetes.ASV151,ASV428 and ASV658 belong to Bacteroides,ASV360 is Prevotella 9 and ASV 1756 is Bifidobacterium.Functionally,bacteroides?ASV151?was positively correlated with HDLC level.Bifidobacterium ASV1756 was negatively correlated with LDLC level.Bacteroides?ASV428,ASV658?was positively correlated with hyperlipidemia,and Prevotella₉?ASV360?was negatively correlated with hyperlipidemia.