| Recent years,the relationship between intestinal microbiota and human health and disease has become an important area.The critical roles of this micro-world in our bodies have been gradually unveiled by the researches deepened.The intestinal microbiota not only regulate intestinal homeostasis,but also control series of physiological activities of human body through liverintestine axis,intestine-brain axis and etc.The disorder of intestinal microbiota is directly related to the occurrence and development of many human diseases including the inflammatory bowel disease(IBD),such as the Crohn’s disease and ulcerative colitis.However,the pathology of IBD is still unclear and the treatment is limited.Clinical studies have shown that probiotics can significantly relieve the symptoms of IBD.In addition,in recent years,fecal bacteria transplantation of(FMT)is also considered an effective way in IBD treatment.These results further suggest that intestinal microbiota plays an important role maintaining of intestinal micro environment.However,the efficacy of either probiotic and FMT is characterized by individual heterogeneity.The effective probiotic strains or the donor of FMT are completely depends on the clinical experience with no clear principle to follow,strongly limiting the use of these methods in clinics.Intestinal microbiota is usually considered to regulate intestinal homeostasis either through direct interaction with intestinal cells or interaction with intestinal tissue through their metabolites.Compared with intestinal bacteria themselves,their metabolites are simple to identify and analyze,and have more advantages in drug transformation.Thus,the intestinal microbiota metabolites have been focus in this study to screen potential chemicals for IBD treatment.Tryptophan is an essential amino acid for human body.A large number of studies have shown that alteration of tryptophan metabolism is closely related to IBD.There are three pathways of tryptophan metabolism in vivo,which are uric acid metabolic pathway,indole metabolic pathway and 5-hydroxytryptamine pathway,in which the indole metabolic pathway is unique to intestinal microbiota,suggesting that the metabolic changes of indole pathway may be one of the important factors in the development of IBD.This raised the possibility that the products of indole metabolic pathway could be used as clinical drugs for IBD intervention.There are 13 intermediate metabolites involved in indole metabolic pathway of tryptophan metabolism by microbiota.In this study,zebrafish was used as a model animal to analyze the toxicity of 10 intermediates.As well,the effect inflammation has also been evaluated by Ah R expression and neutrophil migration.Firstly,we have accessed the toxicity of these intermedia metabolites on the development of zebrafish embryos.The 24-hour survival curve has been drawn and the 24-hour LC50 value has been calculated.As well,the effect on the 72-hour hatching rate and the 72-hour deformity rate has been assayed.The deformities caused by each tryptophan metabolites treatment have been dissected in details.Secondly,the expression of ahr2 and arnt2 b genes of zebrafish embryos after treatment with tryptophan metabolites at concentration of 50%LC50 has been analyzed.Notably,indole-3-acetic acid,indole-3-formaldehyde,indole-3-acetamide,indole-3-propionic acid and indole-3-pyruvate significantly increased the expression of ahr2,while indole substantially decreased the expression of ahr2 gene.In addition,indole-3-acetic acid also significantly increased the expression of arnt2 b,while indole,indole-3-propionic acid and tryptophol significantly decreased the expression of arnt2 b.Consistently,the effect on ahr2 by indole and indole-3-pyruvate has been further verified by in situ hybridization assay.Finally,the effect on inflammation by different metabolites has been evaluated by assay of neutrophil migration using zebrafish Tg(mpx:e GFP)strain.The zebrafish inflammation larva generated by tail fin clipped were treated by the metabolites at concentration of 50%LC50,10%LC50 and1%LC50,respectively.It has been shown that indole-3-acetamide and indole at 50%LC50 concentration strongly suppressed the migration of the neutrophil to the wound.Taken together,the toxicity of ten tryptophan metabolites of indole pathway by intestinal microbiota has been assessed using zebrafish model.The effects of these metabolites on AHR pathway have been evaluated and the effects on inflammation has been assayed in this study.Our results provided important data for screening the potential drugs for IBD treatment,and gave useful clues for mechanism studies on tryptophan metabolites on homeostasis of intestine. |
PDF Full Text Request