The Role And Mechanism Of Butyric Acid In The Treatment Of Ulcerative Colitis By Fecal Microbiota Transplantation

Objective: In this study,based on clinical samples and colitis mouse model,we found that butyric acid(an important bacterial metabolite)increased significantly in ulcerative colitis(UC)following Fecal microbiota transplantation(FMT).Multi-omics analysis was used to explore the therapeutic effect of butyric acid on experimental colitis.Microbial culture in vitro and autophagy activated / inhibited colitis mouse model were used to verify whether FMT improving experimental colitis through butyric acid mediated gut microbial regulation and autophagy activation.Methods:(1)Fecal samples from 12 UC patients and healthy donors were analyzed by 16 S r RNA sequencing,and the contents of short chain fatty acids(SCFAs)in feces from 5 patients with recurrent symptoms were analyzed by liquid chromatography-mass spectrometry to verify the relative abundance of butyric acid producing bacteria and butyric acid in UC mice treated with FMT.(2)The effects of butyric acid on disease activity index(DAI),colon shortening,colonic HE,expression of colonic barrier protein and serum inflammatory factors in DSS induced colitis mice were observed.The fecal 16 S r RNA sequencing,serum non targeted metabonomics,colon tissue RNA-seq analysis and in vitro selective culture of colitis were used for multi-omics analysis.(3)IHC,WB and PCR were used to detect the content and transcription level of autophagy protein in colon of colitis mice treated with sodium butyrate.Autophagy activated / inhibited colitis mice were constructed to determine whether FMT could improve experimental colitis by upregulating autophagy through butyric acid.(4)The clinical data of 45 UC treated with FMT were reviewed,and the effect of combined use of butyric acid producing probiotics on long-term remission was observed.Results:(1)The relative abundance of butyric acid producing bacteria(Ruminococcaceae,Lachnospiraceae,Ruminococus,Alistipes,Akkermansia,Roseburia,Faecalibacterium)in responsed UC patients was increased,and the butyric acid content was significantly increased(1 mo post-FMT vs.pre-FMT,P=0.028)after FMT.The content of butyric acid in feces was negatively correlated with the severity of symptoms(r=0.5321,P=0.0412);DAI,colon shortening,colonic HE,as well as serum IL-6 and TNF-α were significantly improved(P<0.05),while barrier protein in colonic IHC(ZO-1,P=0.0236,0.0186;Occludin,P=0.0106,0.0319)was significantly increased after FMT.Relative abundance of butyric acid producing bacteria such as Ruminocaceae,Rikenellaceae,Ruminiclustriaum,Alisties,Rosebria,Faecalibacterium＿prausnitzii was upregulated while the relative abundance of pro-inflammatary pathogens such as Bacteroidaceae,Streptococcaceae,Corynebacterium,Eschericia-Shigella was down regulated,while fecal butyric acid was significantly increased after FMT(P=0.0126).(2)DAI,colon shortening,colonic HE,as well as serum IL-6,IL-1β and TNF-α of colitis mice were significantly improved after sodium butyrate intervention(P<0.05).Barrier protein in IHC(ZO-1,P=0.0075;Occludin,P=0.0113)and WB(ZO-1,P=0.004;Occuldin,P=0.0009)of colitis mice were significantly increased after sodium butyrate intervention;In vivo and in vitro experiments,diversity of gut microbiota in colitis mice was increased and microbial structure was adjusted to healthy mice,which shown as increased relative abundance of butyric acid producing bacteria such as Firmicutes,Lachnospiraceae,Ruminococcaceae,Rikenellaceae,Ruminiclostridium,Butyricicoccus,Roseburia,Alistipes while decreased relative abundance of pro-inflammatary pathogens such as Bacteroidetes,Bacteroidaceae,Enterobacteriaceae,Helicobacteraceae,Enterococcaceae,Bacteroides,Enterococcus,Escherichia-Shigella,Klebsiella,Helicobacter.Non targeted metabonomics analysis,a total of 342 kinds of differential metabolites were found in the positive/negative ion state,106 differentially expressed genes were detected,and 4 target signaling pathways were identified by KEGG enrichment analysis: Autophagy(ko04140),Regulation of lipolysis in adipocyte(ko04923),Ovarian steroidogenesis(ko04913),Biosynthesis of amino acids(ko01230).(3)In the experiment of sodium butyrate treating colitis mice,the expression of P62(P=0.0164)and p-MTOR(P=0.0325)were significantly decreased in IHC,while P62(P=0.0184),p-MTOR(P=0.0024)and LC3-II/I(P=0.0024)were significantly decreased in WB,and the expression of P62(P=0.0162)and TFEB(P=0.0007)were significantly increased in PCR;Activated autophagy by rapamycin improved the index of experimental colitis: DAI(P=0.0128),colon shortening(P=0.043),colonic HE(P=0.0392),serum inflammatory factors(IL-6,P=0.0396;TNF-α,P=0.0164).On the contrary,inhibited autophagy by hydroxychloroquine aggravated some indexes of experimental colitis:DAI(P=0.0027),colon shortening(P=0.0393),colonic HE(P=0.765)and serum inflammatory factors(IL-6,P=0.205;TNF-α,P=0.7863);After the intervention of sodium butyrate or FMT,some indexes of autophagy inhibited colitis mice were improved: DAI(P=0.5130;P=0.6926),colon shortening(P=0.009;P=0.0008),colonic HE(P=0.0028;P=0.0015),serum inflammatory factors(IL-6,P=0.0159,P=0.0037;TNF-α,P=0.0714;P=0.0623);IHC(P62,P=0.0468,P=0.1183;LC3B,P=0.0118,P=0.0117;MTOR,P=0.1083,P=0.0134;p-MTOR,P=0.0618,P=0.2508;ATG16L1,P=0.0203,P=0.0176),WB(P62,P=0.0472,P=0.3440;LC3-II/I,P=0.0808,P=0.0287;MTOR,P=0.2696,P=0.3103;p-MTOR,P=0.9668,P=0.6356;ATG16L1,P=0.5863,P=0.0499)and PCR(P62,P=0.0202,P=0.006;LC3,P=0.0113,P=0.0033;TFEB,P=0.0003,P=0.0039;ATG16L1,P=0.0401,P=0.001)showed that autophagy expression was up regulated by sodium butyrate and FMT.(4)The addition of Shiyi(C.butyricum-Containing Probiotics)to the standard therapies in 11 out of 45 UC patients were significantly enhanced the therapeutic efficacy of FMT by increasing the therapeutic response rate(9/11 vs.15/34 in the FMT alone group)at 3 months post-FMT(χ~2=3.9477,P=0.0469).Conclusion: Fecal butyric acid may be an important therapeutic marker of FMT for UC;FMT may improve experimental colitis through butyric acid mediated microbial regulation;FMT may improve experimental colitis through butyric acid mediated autophagy activation;Combined with butyric acid related probiotics may prolong the long-term remission of FMT for UC.