| ObjectiveUlcerative colitis(UC)is a primary type of inflammatory bowel disease(IBD)characterized by inflammation and ulcers of the colon and rectum.The incidence and prevalence of UC are increasing with time and in different regions around the world,but the exact etiology of UC still remains unknown.There is increasing evidence highlighting the role of microbial dysbiosis in the pathogenesis of UC.In addition to the changes of intestinal flora,the changes of some metabolites and metabolic pathways were also observed in UC patients.There are two formulas named Zuo-Jin-Wan(ZJW)and retro Zuo-Jin-Wan(FZJW)proved to be effective on alleviating UC.However,most studies about the ZJW and FZJW related treatments in UC focused on their immunoregulatory effects.An in vitro study indicated that both ZJW and FZJW could inhibit the growth of gut microbiota from mice.Our team also discovered that excessive and long-term dministration of Coptis influenced the composition of intestinal bacteria and some metabolic pathways.The effects of the intestinal microbiota and metabonomics responsed to the ZJW and FZJW treatments in UC are remain unknown.It is necessary to investigate the relationships between the gut microbiota and ZJW or FZJW treatments in UC for further understanding their mechanisms in the treatments of UC.Methods1.We first evaluated the effect of ZJW and FZJW treatments in a rat model with 4%DSS-induced colitis by assessing the severity of clinical signs,including body weiht loss,the stool consistency and occult/gross rectal bleeding.The body weight,water and food intakes was measured daily.2.At the end of the animal experiment,to further assess the effect of ZJW and FZJW treatments in UC rats,serum sample was collected for determination of inflammatory cytokines,and the colon length was measure before histological observation;the expression of Claudin-1 on colon was detected by immunofluorescence assay for the assessment of the integrity of colonic mucosal barrier.3.The V4 region of the 16S r RNA gene of microbiota in fecal extracts were amplified and then sequenced on the Illumina Mi Seq platform.Subsequent bioinformatics analysis was used to investigate the shifts in the composition and metabolic function of microbiota after ZJW and FZJW treatments.4.~1H-NMR profiles were applied for the profiling of unriary and faecal metabolites in UC rats with ZJW and FZJW treatments.Then the relevant biomarkers were screened through multivariate statistical analysis.Results1.UC had been successfully induced by 4%DSS in the rats.Both ZJW and FZJW treatments could significantly alleviate clinical signs in rats with UC,decrease DAI score and pro-inflammatory cytokines level in serum,improve the colonic damages and protect the mucosal barrier.2.Obvious changes of the gut microbiome composition and function were observed in the rats with UC,which manifest as decrease in biodiversity and abnormal ratio of the harmful and beneficial bacteria.Both ZJW and FZJW treatments could reduce the abundance of pathogenic bacteria(mucus-degrading bacteria)and increase amount of probiotics(SCFAs-producing bacteria increased by ZJW;lactic acid-producing bacteria increased by FZJW)3.ZJW could modulate the dysregulated microbial functions of the colitis to resemble that of the control group,including an increased capacity for basic metabolism(energy and amino acid metabolism)and a decrease in the lipid metabolism,material absorption and transportation,and diseases related pathways,whereas FZJW only presented a weak modulation effect on the metabolic functions.4.A total of 106 metabolites were detected in the ~1H-NMR spectra of the urine(71metabolites)and faecal extracts(54 metabolites).26 metabolic biomarkers were found in comparison between control group and DSS group.9 of them were up-regulated,including amines,while 17 metabolites were down-regulated.Both ZJW and FZJW treatments could improve the proportion of certain metabolites down-regulated in urine of DSS group,for example 1,3-dihydroxyacetone,acetamide,cis-aconitate,glycolate and.N6,N6,N6-trimethyllysine.Meanwhile,both treatments could also reduce the concentration of adenine,dimethylamine and dimethyl sulfoxide in urine.The decreased SCFAs(eg.propionate)and energy related metabolites(such as succinate and pyruvate)in the faeces of DSS group could be improved by ZJW treatment as well.FZJW treatment presented a poor modulation effect on the metabolites in feaces.5.The results of Metabolic Pathway Analysis(Met PA)demonstrated that the significantly changed metabolic pathways among groups are carbohydrate metabolism or energy metabolism(including pyruvate metabolism,citric acid cycle(TCA)and glycolysis or gluconeogenesis),lipid metabolism(eg.glycerolipid metabolism,butanoate metabolism and synthesis and degradation of ketone bodies)and synthesis or metabolism of amino acid(including phenylalanine,tyrosine,tryptophan,valine and many other kinds of amino acid)ConclusionIn this study,administrations of ZJW and FZJW have been proved to act beneficial effects on UC therapy.Combined with the analysis of metabolomics and 16S r RNA gene sequencing,we’ve investigated the shifts in the metabonomics and the gut microbiom composition and function in rats with ZJW and FZJW treatments,in order to further explain the mechanism of the treatments with ZJW and FZJW in UC through the perspective of the intestinal microbiota and metabolomics. |
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