| Objective:1)To analyze the distribution characteristics of DN intestinal flora based on metagenome sequencing technology and bioinformatics theory;2)To look for new biomarkers that can predict DN early;3)To explore the pathophysiological mechanism of this marker and DN.Methods:1)The feces samples of 20 patients with diabetic nephropathy and 20 healthy people were collected from each group,and the data of clinical indicators such as blood biochemistry and urine analysis of the population in each group were collected and counted;2)After fecal DNA was extracted from each group,an appropriate database construction scheme was selected for database construction,and metagenomic sequencing was performed on the Illumina-Navo6000 high-throughput sequencing platform;3)For off-machine data,based on Linux platform,bcl2 fastq was used to split the data,Fast QC was used to carry out quality control on the original data,knoaddata was used to remove the host,HUMANN2 was used to carry out microbial taxonomy units,gene families,and pathway annotation,Metaphlan2 calculates species abundance and diversity index,and R was used to standardize and merge to obtain diversity,species,and functional abundance expression profiles to describe the distribution characteristics of diabetic nephropathy intestinal flora and its overall function;4)The differential flora among the groups was found by Lefse and Stamp based on the expression spectrum,and the Randomforest model was constructed using R’s random forest package to find the key flora.5)wilcox method was used to find the difference of functional composition between two groups.6)Clustered flora by using R with a difference module as a standard to find a core flora of a corresponding module;7)The use of Spar CC,a novel approach suitable for sequencing data features,inferred the correlation between species and UACR based on the estimation of correlation values from component data and constructed an interaction network.Results:1)The composition and diversity of intestinal flora in the patients with diabetic nephropathy had changed significantly.The Shannon diversity index of the intestinal flora in the DN group was 0.37±0.13,while that in the Control group was 0.25±0.15.The diversity in the DN group was significantly higher than that in the Control group and was statistically significant.PCo A combined with PERMANOVA was used to calculate the Beta diversity index,and the difference was significant(P=0.004),with statistical significance.2)Genus level: the three dominant genera in the DN group were Bacteroides,Parabacteria and Klebsiella,and the three dominant genera in the Control group were Bacteroides,Prevotella and Lachnospira.The relative abundance of Citrobacter,Escherichia,Hungatella,Erysipellatoclastic,Klebsiella,Akkermansia and Lachnoclastic in the DN group was high and statistically significant.The number of Prevotella and Rothia in the DN group was significantly decreased and statistically significant.At species level,B.vulgatus,B.uniformis,B.stercoris were dominant species in healthy subjects in DN,K.pneumoniae was dominant species in DN,E.coli,C.innocuum,E.ramosum,A.muciniphila,C.clostridioforme,E.faecium,B.Intentionalis,Lachnospiraceae-＿spp,K.pneumoniae,in the DN group showed significantly increased abundance of intestinal flora(P<0.05),with statistical significance.P.copri,R.faecis,L.pectinoschiza,R.intestinalis and Eubacterium＿spp in human body were decreased significantly with statistical significance.The results of the analysis showed that there were significant differences in the composition and abundance of the intestinal flora between the Control group and the DN group.The relative abundance of pathogenic bacteria in the DN group was significantly increased,and the probiotics was significantly reduced.3)Through KEGG pathway enrichment analysis,we obtained metabolic pathways with significant differences in the DN group: main metabolic pathways such as amino acids(mainly including tryptophan and tyrosine),and fatty acid modules(mainly including short-chain fatty acids)showed significant differences,the metabolic pathways of tyrosine and tryptophan were significantly increased,while the metabolic pathways of short-chain fatty acids were significantly decreased,with statistical significance.4)Citrobacter,the key flora in the tyrosine metabolism module of patients with diabetic nephropathy,was significantly positively correlated with UACR of DN patients,while Faecalibacterium,the key flora in the short-chain fatty acid metabolism module,was significantly negatively correlated with UACR of DN patients,with statistical significance.Conclusion:There is an imbalance of intestinal flora in DN.The imbalance intestinal flora may enter tyrosine metabolism through Citrobacter to synthesize excessive PS,and the SCFAs synthesized by Faecalibacterium in fatty acid metabolites are reduced,which accelerates the intestinal leakage of PS,and then damages the injured podocytes,accelerates the thickening of GBM,induces proteinuria,and causes inflammation caused by perivascular fibrosis,thus promoting early renal injury in DN. |
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