The Underlying Mechanism Of Evodiamine On DSS-induced Ulcerative Colitis By Regulate The Gut Microbiota And Metabolism

ObjectivesEvodia officinalis,a dry nearly ripe fruit of Evodia rutaecarpa（Juss.）Benth.、E.rutaecarpa（Juss.）Benth.var.officinalis（Dode）Huang and E.rutaecarpa（Juss.）Benth.var.bodinieri（Dode）Huang,the fruit is used in traditional Chinese medicine for treating digestive system disease.Its major chemical constituents,including some active components alkaloids,volatile oil,bitter principles and so on,has reported.Evodiamine is one of the most important alkaloids in Evodia officinalis,which is a kind of indoles alkaloid,and has the pharmacological activity of dilating blood vessels、constricting trachea,anti-tumor and anti-inflammatory,and so on.According to the literature investigation,the pharmacological of Evodiamine studies mainly focus on its anti-tumor activity,while the anti-inflammatory activity has not been fully studied,and most of the researches are mainly focused on the inflammatory pathways（NF-κB）or NLRP.Since intestinal microbiota is considered as an important organ of the human body in recent times,an increasing number of studies have linked this microenvironment to gastrointestinal diseases.Many studies inferred the gut microbiota as a potential predictor of health status and a target for therapeutic intervention.However,studied on the relationship between the anti-ulcerative colitis of Evodiamine and gut microbiota and metabolism has not been reported so far.Then,whether the anti-ulcerative colitis effect of Evodiamine is related to the changes of gut microbiota and metabolism,and if so,which specific bacteria and metabolites are playing a role.Therefore,the purpose of this study was to explore the mechanism of Evodiamine on DSS-induced ulcerative colitis based on gut microbiota and metabolomics.In this research,DSS-induced Ulcerative colitis model was established to investigate the effect of Evodiamine on UC,and changes in gut microbiota were observed using 16S rRNA sequencing,and changes in metabolites were analyzed using non-targeted metabolomics（1H-NMR and UPLC-Q/TOF-MS/MS）.Then,the effect of Evodiamine Fecal Microbiota Transplantation on DSS-induced Ulcerative colitis was observed by 16S rRNA sequencing,and the difference microbiota was selected.The content of short-chain fatty acids（SCFAs）was determined by GC,and the effect on intestinal mucosal barrier was evaluated.From the gut microbiota,metabolism and transcriptome to elucidate the potential underlying mechanism of Lactobacillus acidophilus on UC.Methods1.Protective effect of Evodiamine against ulcerative colitisThe effect of Evodiamine on DSS-induced ulcerative colitis was evaluated by the physical sign,length of colon,DAI score and the pathological morphology of colon.Additionally,in order to elucidate the possible protective mechanism of Evodiamine against DSS-induced ulcerative colitis,the level of TNF-α、IL-6、IL-10 and IL-1β were measured by ELISA kit,the Claudin-1 protein was determined using immunofluorescence.The effect of Evodiamine on the composition of gut microbiota and metabolism in DSS rats by 16S rRNA sequencing and 1H-NMR and UPLC-Q/TOF-MS/MS.2.Protective effect of Evodiamine Fecal Microbiota Transplantation against ulcerative colitisThe effect of Evodiamine Fecal Microbiota Transplantation on DSS-induced ulcerative colitis was evaluated by the physical sign,length of colon,DAI score,the pathological morphology of colon and spleen weight.The level of TNF-α、IL-6、IL-1β、IFN-γ and IL-17 were measured by ELIS A.The mRNA expression of Reg3γ、Reg3β、Muc1、Muc2、Tff3、Claudin-1、Claudin-2、Occludin and ZO-1 were detected using qRT-PCR.The ultrastructure of colonic epithelium was observed by transmission electron microscopy.The composition of gut microbiota was observed using 16S rRNA sequencing,and the content of short-chain fatty acids was determined by Gas Chromatography（GC）.3.Protective effect of Lactobacillus acidophilus against ulcerative colitisThe effect of Lactobacillus acidophilus on DSS-induced ulcerative colitis was evaluated by the physical sign,length of colon,DAI score,the pathological morphology of colon and AP-PAS staining.The level of TNF-α and IL-1β were measured by ELISA.The mRNA expression of IL-1β、IL-6、IL-10、IL-22 and TGF-β,Reg3γ、Reg3β、Muc1、Muc2、Tff3,Claudin-1、Claudin-2、Occludin and ZO-1 were detected using qRT-PCR,the apoptosis of intestinal mucosa was detected using TUNEL,the level of Ki-67 and Muc2 proteins were detected using immunohistochemistry,the Claudin-1 protein was measure using immunofluorescence,the ultrastructure of colonic epithelium was observed by transmission electron microscopy.Colonic mucosal barrier integrity was detected by Fluorescence in Situ Hybridization.The diversity of gut microbiota and metabolites were analyzed by 16S rRNA sequencing and Gas Chromatography（GC）.Using transcriptome sequencing to analyze the differentially expressed genes in the colon after Lactobacillus acidophilus treatment.Results1.Protective effect of Evodiamine against ulcerative colitisIn the model of DSS-induced ulcerative colitis,the reduced body weights,diarrhea,shortened colon and DAI score were improved,as well as histomorphology of colonic mucosa was improved.Evodiamine could down-regulate the level of pro-inflammatory cytokines（TNF-α、IL-6 and IL-1β）,and up-regulate the level of anti-inflammatory cytokines（IL-10）.The expression of Claudin-1 protein also was increased.Meanwhile,the results of 16S rRNA sequencing showed that the relative abundance of Bifidobacterium,Parabacteroides,Mucispirillum,Turicibacter,Allobaculum and Proteus was decreased,and the relative abundance of Bacteroidetes,Lactobacillus,Ruminococcus,Desulfovibrio and Akkermansia were increased after Evodiamine treatment.Furthermore,Evodiamine could regulate the amino acids metabolism,energy metabolism,nucleic acid metabolism and lipid metabolism.2.Protective effect of Evodiamine Fecal Microbiota Transplantation against ulcerative colitisEvodiamine Fecal Microbiota Transplantation could reduce the body weight loss,increased the colon length,decreased the DAI score and microscopic score,reduced the spleen weight.Regulated the immune-inflammatory cytokines（down-regulate the levels of TNF-α、IL-6、IL-1β、IFN-γ and IL-17）,increased the expression of antimicrobial peptide Reg3y and Reg3β,promoted the secrete of mucin Muc1、Muc2 and Tff3.In addition,the mRNA expression of Claudin-1 and Occludin were increased,and Claudin-2 was decreased,the microvilli on the surface of colon epithelial cells were abundant and well arranged,and intercellular were seen the thin and long tight connections.The results of 16S rRNA sequencing showed that the relative abundance of Ruminococcus_UCG-014 and Ruminococcus₁（Ruminococcaceae）,Lactobacillus（Lactobacillaceae）and the content of acetic acid were significantly increased.3.Protective effect of Lactobacillus acidophilus against ulcerative colitisLactobacillus acidophilus could reduce the body weight loss,increased the colon length,decreased the DAI score and microscopic score.Increased the number of goblet cells and mucus secretion,regulated inflammation cytokines（down-regulate the level of TNF-α、IL-6 and IL-1β,up-regulate the level of IL-10,IL-22 and TGF-β）,promoted antibacterial peptide Reg3γ and Reg3β,inhibited colon epithelial cell apoptosis,promoted the expression of cell proliferation factor Ki-67 and mucins（Muc1、Muc2 and Tff3）,enhanced the tight junction protein Claudin-1 and Occludin and reduced Claudin-2.On the surface of colon epithelial cells,the microvilli were abundant and well arranged,and multiple goblet cells could be seen.There was no obvious expansion of rough endoplasmic reticulum,tight connections between cells could be seen,and the number of bacterial invasion could be reduced.In addition,Lactobacillus acidophilus could increased the relative abundance of Firmicutes,decreased the relative abundance of Bacteroidetes,and promoted the production of short-chain fatty acids.Transcriptome analysis results showed that Lactobacillus acidophilus could change the colon epithelial gene expression,mainly involved in protein digestion and absorption,mucin type O-glycan biosynthesis,metabolism of xenobiotics by cytochrome P450 and glycosaminoglycan biosynthesis-keratan sulfate.The expression of colon epithelial gene may be related to gut microbiota.ConclusionOur findings demonstrated that Evodiamine can attenuates DSS-induced ulcerative colitis,and the underly mechanism of Evodiamine is related with its role in regulating the gut microbiota Lactobacillus acidophilus,metabolites acetate and intestinal mucosal barrier function.The results suggest that Evodiamine has the effect of anti-ulcerative colitis,which is one of the effective constituents of Evodia officinalis in the treatment of digestive diseases.Therefore,Evodia oficinalis may be further developed as a promising drug for the treatment of ulcerative colitis.