Experimental Study On The Effect Of Fecal Microbiota Transplantation (FMT) In Mice With DSS-induced Colitis

Background and purposeUlcerative colitis(UC)is an immune-mediated chronic nonspecific inflammatory disease.At present,the mechanism of UC is unclear.The complex relationship between intestinal microbiota and immunity has attracted extensive attention In recent years.Gut microbiota and immune system mediate UC-related mucosal damage,in which inflammatory factors play an important role.Many studies have suggested that CD30 is related to the differentiation and activation of Th cell subsets such as Th1,Th2 and Th17 cells,which are all involved in the intestinal mucosal injury and play an important role in immune regulation.Th17 cells are a subgroup of T cells that can specifically produce interleukin-17(IL-17),which involved in autoimmunity,inflammation and tumorigenesis.A20 and AP-1 are important regulators of the il-17 signaling pathway,and TNF-α is one of the main effectors downstream of this pathway.The increase of IL-17 and Th17 in patients with UC showed the pathogenicity effects,but the aggravation of enteritis after the blocking of IL-17 indicated that such factors were protective.In addition,Th17 cells induced by different microorganisms have different states and functions in intestinal.Pathogenic bacteria usually induce the generation of pro-inflammatory Th17 cells,which mediate the initiation,differentiation,infiltration and tissue destruction of various pro-inflammatory effector molecules.Therefore,intestinal flora can participate in the occurrence and development of UC by activating the Th17 pathway.At present,the efficacy of Fecal microbiota transplantation(FMT)in the treatment of UC varies greatly among individuals.About0-63% patients with UC have been relieved after FMT intervention in the clinical.The main target of FMT is still unclear,and there is no method to predict the efficacy of FMT.Therefore,this project intends to explore the functional pathway of FMT in the treatment of UC and find the relevant factors that can potentially predict the efficacy.We constructed DSS-induced mice enteritis model,and then transplant the fecal bacteria liquid from patients with UC and healthy people,using Illumina sequencing technology analyzing the change of intestinal flora in mice after transplantation and high-throughput sequencing technology screening differentially expressed genes.We found that IL-17 signal channel showed the greatest change between groups.And then we further verified the signal pathways in mice and patients with UC in the expression of related genes,as well as the relationship between those genes and the curative effect of FMT.We preliminary found that FMT participated in the treatment of UC mainly by IL-17 signaling pathways.CD30 may be the potential predictors of FMT curative effect in UC patients.MethodsAnimal experiment:40 BALB/c mice were divided into Normal control group(Water+PBS group)and Experimental group(DSS model group,DSS+FMT-HC group and DSS+ FMT-UC group).The Normal control group was fed with sterile water or PBS.All of the experimental groups were given 2% DSS solution used to replace drinking water after treated with mixed antibiotics for 1 week and fed with sterile water for 3 days.After 3 days of modeling,DSS model group,DSS+FMT-HC group and DSS+ FMT-UC group were given normal saline,feces of UC patients and the healthy people by gavage respectively.In the course of the experiment,we recorded the occult blood status of the body weight and stools of the mice every day and calculated the DAI score daily.On the last day,we measured the colonic length of mice and stained the colon tissues with HE to observe the inflammation of mice.Before the end of the experiment,we collected the feces of mice.Then observations were made on alteration of fecal microbiota of mice by 16 s high throughput sequencing of microorganism group.And high-throughput sequencing technology was used to screen the differentially expressed genes.RT-PCR and ELISA were used to detect the expression of related genes and related cytokines in blood like TNF-α respectively.What’s more,the expression of AP-1,CD30 and A20 protein were detected by Westernblot and Immunohistochemistry.Clinical study: we collected the colon tissues of UC patients before and after Fecal microbiota tansplantation.All of them were divided into two groups with effective or ineffective treatment.The expression of CD30 protein in the colon tissues of the two groups were detected by Immunohistochemistry before and after treatment.Results1.Disease activity severity and histological inflammation in miceDisease activity index(DAI): After the induction of DSS,all of the Experimental groups got higher disease activity score(DAI).The disease activity score of DSS+FMTUC group showed the highest score(P<0.05).Colon length: Colon length of DSS+FMT-UC group was significantly shorter than that of DSS model group and DSS+FMT-HC group(P<0.05).The colon length of DSS model group was slightly shorter than DSS+FMT-HC group’s but with no statistical significance(P>0.05).Histopathology: The colon HE staining score of DSS+FMT-UC group was significantly higher than that of DSS model group(P<0.05),and the score of DSS+FMT-HC group was lower than that of DSS group but with no statistical difference(P>0.05).2.Changes of intestinal flora in mice1)Principal Co-ordinates Analysis(PCoA): Since DSS+FMT-UC group was furthest from DSS model group on the principal component coordinate axes,there is the most obvious difference between the two groups.DSS+FMT-HC group and Water+PBS had the highest similarity.DSS+FMT-UC group had the least overlap with Water+PBS group.2)Species abundance clustering was used to analyze the changes of flora structure:After the intervention of DSS,Fusobacteriaceae,Bacterioidaceae,Enterobacteriaceae,Akkermansiaceae,Parabacteroides-goldsteinii,Bacteroides-Dorei were increased.After the intervention of fecal bacteria solution from healthy human,Firmicutes,Sphingomonadales,Sphingobacteriales,Phasolarctobacterium,Lactobacillus,Sutterella,Blautia,Fusobacterium,Bacteroides-Massiliensis were increased in different degrees.After the intervention of fecal bacteria solution from UC patients,Firmicutes,Clostridiales,Anaeroplasmatales,Ruminoccaceae,Lachnoclostridium,Lachnosiraceae,Unidentified-Ruminoccaceae,Alistipes,Oscillibacter,Bacteroides-Acidifaciens,Clostridium-Papyrosolvens were increased.3.High-throughput sequencing technology screened differentially expressed genes and related pathways1)We screened 3892 differentially expressed genes from the groups(DSS+FMT-HC group,DSS+FMT-UC group and DSS group)by RNAseq analysis.And there were 178 significantly differentially expressed genes between the groups(112 up-regulated and 66 down-regulated).2)GO analysis showed that the differentially expressed genes could be divided into three categories: biological process,cell components and molecular functions.3)KEGG enrichment analysis: Compared with DSS goup,the genes of IL-17 signaling pathway like tumor necrosis factor(TNF),activator protein-1 and A20 in DSS+FMT-UC group raised.In addition,CD30 gene was significantly upregulated in DSS+FMT-UC group and down-regulated in DSS+FMT-HC group(P <0.05).4.The expression of CD30,AP-1,A20 protein and serum TNF-α in mice.1)RT-PCR:The expression of C-FOS gene in intestinal tissues of each group from high to low were as follows: The expression of C-FOS,C-JUN and TNF-a in DSS+FMT-UC group were higher than that of DSS model group(P<0.05).2)Westenblot: After the intervention of DSS,the expression of colonic C-FOS,C-JUN、P-C-FOS and P-C-JUN proteins in mice were up-regulated(P<0.05).The expression of P-C-JUN proteins in DSS+FMT-UC group was higher than that of DSS model group(P<0.05).3)ELISA : The expression levels of serum TNF-α were up-regulated in all experimental groups.Among them,DSS+FMT-UC group showed the most obvious up-regulation,followed by DSS model group and DSS+FMT-HC group(P<0.05).4)Immunohistochemistry(IHC): The expression of CD30 and A20 were up-regulated in DSS model group.Down-regulation of both CD30 and A20 were found after the intervention of fecal bacteria solution from healthy human.While those proteins were up-regulated after the intervention of fecal bacteria solution from UC patients.5.Expression of CD30 in UC patients: CD30 protein in intestinal mucosa of UC patients were up-regulatedthe compare with the healthy one.Effective group showed high expression of CD30 before FMT intervention,and down-regulated after the intervention.However,the expression of CD30 was lower in the ineffective group before and after intervention.Conclusion1.The fecal bacteria of the patients with UC promoted the intestinal flora disorder in DSS-induced mice,which aggravated the intestinal inflammation.However,fecal bacteria of the healthy human helped to restore intestinal flora disorder and intestinal inflammation in DSS-induced mice.2.The fecal bacteria of the patients with UC may aggravate the intestinal inflammation in DSS-induced mice mainly by activating the IL-17 signaling pathway.3.The high expression of CD30 may be a potential predictor of FMT efficacy in UC patients.