| ObjectiveSyndrome of spleen-qi deficiency is prevalent in diseases of digestive system in TCM accompanied with gastrointestinal dysfunction,muscle weakness and other symptoms.More and more studies show that the syndrome of spleen-qi deficiency is not only closely related to internal metabolism but the gut microbiota.It reveals that the gut microflora dysbiosis may affect the normal metabolism.Nowsday,syndrome of spleen-qi deficiency has been studied on gut microecology and metabonomics respectively.However,exploring spleen-qi deficiency essentially by integrating gut microecology with metabonomics has not been tried out so far.With the quick development of technologies of molecular biology and metabonomics,especially application of high-through test techonology,the study on exploring the essence is given the technical support fully.Therefore,to access the impact of gut microbiota on metabolism in the condition of spleen-qi deficiency,an intergrated approach of polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)combined with nuclear magnetic resonance(NMR)based metabonomics was performed in multifactor-induced spleen-qi deficiency rat model for studying the coeerlation between the gut microbial cpmposition and host metabolites.And then the high-throughput sequencing technology was used to reveal the unexplored "rare biosphere"of gut microbiota in spleen-qi deficiency rat.Methods1.The spleen-qi deficiency rat model was established by the method of "catharsis with bitter-cold purgatives+excessive fatigue+hunger disorder".The rats'changes of weight,food and water intake,stool,spirit condition and behavior were observed and recorded and organic index was ciphered.And then the spleen-qi deficiency rat model was evaluated by incorporating above features with the Chinese medicine symptom standards.2.With the successful establishment of spleen-qi deficiency rat model,D-xylose excretion rate in urine was measured after the preparation of urine sample with the D-xylose test kit to investigate the effect of syndrome of spleen-qi deficiency on the changes of D-xylose excretion rate in urine in model rats.3.The activity of creatine phosphate kinase(CPK)and the content of motilin(MTL)and gastrin(GAS)were measured after the preparation of blood sample based on the spleen-qi deficiency rats with the correlative ELISA kits to investigate the effect of syndrome of spleen-qi deficiency on the changes of activity of creatine phosphate kinase and the content of motilin and gastrin in blood in model rats.4.The metabolites in urine and faeces were profiled with the method of 1H-NMR after the preparation of urine and faeces samples.The peaks of metabolites were ascribed according to the chemical shift values,peak splitting case,the coupling constant and othe references.The data were analyzed by using SIMCA-P 13.0 with the method of principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Then the relevant the biomakers were screened and trace back to the metabolic pathways.5.The V3 region of the 16S ribosomal ribonucleic acid(16S rRNA)sequences are detected by polymerase chain reaction(PCR)and denaturing gradient gel electrophoresis(DGGE)for descripting the diversity of the gut microbiome after spleen-qi deficiency rat model building.Then the correlation between the data of PCR-DGGE and NMR was analyzed with a view to probe the function of gut microbiota that affect the normal metabonomics.6.The species abundance and alpha diversity of rats'faeces samples were analyzed by Illumina MiSeq high-throughput sequencing of the 16S rDNA-V4 region.Furthermore,LEfSe(version 1.0)was used to detect differentially abundant genera in the two groups for biomarker discovery using the online Galaxy workflow framework.the threshold on the logarithmic linear discriminant analysis(LDA)score for discriminative features was set to 2.0.Microbial functions were predicted using PICRUSt(version 1.0.0)and aligned to the Kyoto Encyclopedia of Genes and Genomes(KEGG)database.Results1.The spleen-qi deficiency rat model was successfully established.The model rats appeared symptom such as diarrhea,extreme tiredness and narrowed eyes and so on.Compared with those in control group,the weight of SQD(spleen-qi deficiency)group rats grew significantly slow(P<0.05).The index of spleen in SQD group was lower than control group and index of thymus was significantly lower than control group(P<0.05).The activity of CPK and the content of MTL and GAS in SQD group were significantly lower than control group.2.Analysis of urine and faeces by metabonomics:Totally,73 metabolites were detected in urine samples and 60 in faeces samples.At last,12 metabolic biomakers were screened respectively in urine and faeces samples.In urine of SQD group,7 metabolites were down-regulated and 5 metabolites up-regulated which involve in TCA cycle,bile acid synthesis and purine metabolism.6 metabolites down-regulated and 6 metabolites were up-regulated in faeces of SQD group which involve in purine metabolism,valine,leucine and isoleucine synthesis and so on.3.After 14-day spleen-qi deficiency rat model building,the bacterial diversity of SQD group was found to be impacted on the abundance level and diversity of gut microbiota compared with the control group.Levels of and Proteobacteria change sharply.Interestingly,Proteobacteria excrementihominis increased slightly in SQD group.Twelve differential bands were identified by cloning and sequencing of 16S rRNA gene V3 regions.The sequences are deposited in the GenBank with access numbers of KY 792535-792546.It showed that ?-diversity index such as chao1 index,observe species index,PD whole tree index and Shannon index and the corresponding rarefaction curves in SQD group were significantly lower than those in control group(P<0.05).The unweighted principal co-ordinates analysis(PCoA)in P-diversity showed that the principal flora in faeces of SQD group completely separate from that in control group.The major bacterial phyla were Bacteroidetes,Proteobacteria and Firmicutes in both groups.However,Deferribacteres only appeared at control group and Verrucomicrobia only appeared at SQD group.And we found that the phenomenon above forecast that the energy metabolism,cancer pathway,amino acid metabolism and so on were changed apparently by PICRUSt analysis.ConclusionWe developed a global analysis to study the correlation between the gut microbiota and host metabolism,showing that the symptom of spleen-qi deficiency not only changes the composition and abundance of gut microflora but also change the host metabolism.In addition,the key functional members of gut microbiome can modulate the host specific metabolic pathways.Advances in technology in both metabolic phenotyping and microbial profiling methods have improved our ability to derive correlations between microbial and metabolic phenotypes,which are of particular important for understanding the function of gut micobiota in human health and disease. |
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