The Role And Mechanism Of Leucine In Mucosal Healing Of Ulcerative Colitis

Objective:To explore the relationship between Leucine(Leu),gut microbiota and mucosal healing in ulcerative colitis(UC),the changes of serum,fecal amino acids and gut microbiota before and after mucosal healing in UC were analyzed.To elucidate the mechanism of Leu in mucosal healing of UC from the perspective of amino acid metabolomics and metagenomics,aimed at providing a new biomarker for predicting mucosal healing of UC and new theoretical basis with dietary therapy.Methods:This study is divided into three parts:Part I(Clinical level):A total of 15 UC patients with infliximab induced mucosal healing were enrolled.Serum and fecal amino acid concentrations before and after mucosal healing were detected by targeted metabolomics.Receiver operator characteristic curve(ROC)was plotted to evaluate the value of different amino acid in predicting mucosal healing of UC,combined with Fecal calprotectin(FC).The changes of fecal gut microbiota was analyzed by metagenomics,and bioinformatics was used to analyze the functional genes and metabolic pathways associated with different species.Part Ⅱ(Animal level):Mice with colitis induced by Dextran sulfate sodium(DSS)were fed leucine-deficient and complete amino acid diet.Inflammation was evaluated through the DAI score,colon,spleen and H&E.The transcription and expression levels of Claudin-4,Occludin and ZO-1 were detected by RT-PCR,Western blot and immunohistochemistry,and the expression of TNF-α and IL-1β in serum of mice were detected by ELISA.Part Ⅲ(Cellular level)1.Caco-2 cells were cultured by Leu-/-and 250μmol/L Leu.The inflammatory conditions were constructed with lipopolysaccharide(LPS).2.Cell proliferation,migration and apoptosis were respectively detected by CCK8,Transwell assay and Flow cytometry.The expression of,TLR4,MyD88,P65,p-P65,Claudin-4,Occludin,ZO-1,mTORC1,S6K,4E-BP1,p-mTORC1,p-S6K and p-4E-BP1 were detected by Western blot.The expression of TNF-α and IL-1β in supernatants were detected by ELISA.3.mTORC1 was inhibited,the same methods were used to analyze the above indicators.Results:Part Ⅰ(Clinical level):1.The targeted metabolomic results show that the fecal concentrations of Alanine,Aspartic acid,Glutamic acid,Glutamine,Glycine,Isoleucine,Leu,Lysine,Methionine,Phenylalanine,Proline,Serine,Threonine,Tryptophan,Tyrosine and Valine after MH were significantly lower than UC(P<0.05).The serum concentrations of Alanine,Cysteine and Valine were significantly increased,while Aspartic acid was significantly decreased(P<0.05).2.ROC curves showed that 16 AA had certain value in diagnosing mucosal healing of UC,the Area under curve(AUC)were>0.7,among which Glutamic acid,Leu,Lysine,Methionine and Threonine had high accuracy in diagnosing mucosal healing of UC,the AUC were>0.9.Fecal concentration of Leu was positively correlated with FC,R=0.831.The AUC of Leu in predicting of MH was 0.9289,the sensitivity and specificity were 86.7%and 93.3%when the cut-off value was 347.35μg/g.3.Metagenomic sequencing results showed that Fitmicutes,Bacteroidetes,Proteobacteria and Ascomycota were dominant in UC,while the proportion of Firmicutes and Bacteroidetes increased significantly after MH.At the level of genus,Bacteroides,Enterococcus,Saccharomyces and Lactobacillus were dominant in UC,while Bacteroides,Faecalibacterium,Roseburia and Clostridium were dominant after MH.At the level of species,Enterococcus faecium and Escherichia coli were dominant in UC,while Faecalibacterium prausnitzii was dominant after MH.4.Comparison of microbiota before and after mucosal healing of UC:at the level of phylum,55 phyla was higher after mucosal healing of UC,especially bacteroidetes,while proteobacteria was lower.At the level of genus,535 bacteria was higher after mucosal healing of UC,including Bacteroidetes,Brucella,Clostridium,Eubacterium,Ruminococcus,Blautia and Parabacteroides,while 41 bacteria was’ significantly lower.At the level of species,2495 species increased after mucosal healing of UC,such as Bacteroides Fragilis and Bacteroides vulgatus,while 329 species decreased,such as Enterococcus faecalis.5.PC A,NMDS and PCoA showed that there were significant differences in the structure of microbiota before and after mucosal healing of UC at different levels.6.LEfSe analysis showed:A total of 89 differences were detected.Among them,14 were enriched in UC and 75 were enriched in MH.At the level of genus,15 bacteria were found to be potential markers that could distinguish UC from MH,of which 1 was enriched in UC and 14 were enriched in MH.At the level of species,57 species were found to be potential markers that could distinguish UC from MH,of which 10 were enriched in UC and 47 were enriched in MH.The following species were significantly elevated after MH and clinically significant:p_Firmicutes→c_Clostridia→o_Clostridiales→f_Ruminococcaceae→g_Faecal ibacterium→s_Faecalibacterium_prausnitzi p_Bacteroidetes→c_Bacteroidia→o_Bacteroidales→f_Bacteroidaceae→g_Bac teroides→s_Bacteroides_fragilis;p_Firmicutes→c_Clostridia→o_Clostridiales→f_Eubacteriaceae→g_Eubacteri um→s_Eubacterium_hallii.7.KEEG showed that carbohydrate and AA metabolism were the most abundant functional genes in intestinal microorganisms.A total of 1273 KO functional groups showed significant differences in abundance between UC and MH.Among them,135 KO were enriched in UC,which was related to inflammation and immunity,1138 KO were increased after MH,which was related to metabolism and genetic information processing.8.In KEGG,KO pathway level 3 showed that 59 metabolic pathways were enriched in UC,mainly in carbohydrate metabolism and membrane transport,82 metabolic pathways were enriched after MH,mainly in carbohydrate metabolism,glycan biosynthesis and metabolism,amino acid metabolism,energy metabolism,biosynthesis of fatty acids,nucleotide metabolism,cofactor and vitamin metabolism.9.KEGG showed that the biosynthesis of Leu was significantly increased after MH,represented by ketol-acid reductoisomerase,which was closely related to Bacteroidetes,Ruminococcus,Eubacterium,Acidaminococcus,Alistipes and Faecalibacterium prausnitzii.Part Ⅱ(Animal level):1.In animal experiments,it was found that the DAI score,spleen,the macroscopic observation colon damage score and histology score increased in mice fed Leu-deficient diet,while colon shortened.The changes of above indexes in mice fed Leu-deficient diet were more obvious after DSS induction.After supplementation of Leu,DAI score decreased progressively,colon length recovered,spleen retracted,the macroscopic observation colon damage score and histology score decreased significantly(P<0.01).2.The expressions of TNF-α and IL-1β were increased in mice fed Leu-deficient diet,and significantly increased after DSS induction,decreased after supplementation of Leu(P<0.01).3.The expression of Claudin-4,Occludin and ZO-1 decreased in mice fed Leu-deficient diet.After DSS induction,the expression further decreased,and significantly increased after supplementation of Leu(P<0.01).Part III(Cellular level)1.The proliferation and migration activity of cells in Leu deficient cultured cells decreased slightly,and there was a little apoptosis.After LPS stimulation,the proliferation and migration activity of cells decreased,the apoptosis rate increased significantly(P<0.05).2.The expressions of TLR4,MyD88,p-P65,TNF-α and IL-1β were increased in Leu deficient cultured cells.After LPS stimulation,the expressions of TLR4,MyD88,p-P65,TNF-α and IL-1β were significantly upregulated(P<0.05).3.The expression of Claudin-4,Occludin and ZO-1 decreased in Leu deficient cultured cells.After LPS stimulation,the expression of Claudin-4,Occludin and ZO-1 was significantly decreased(P<0.05).4.The expression of p-mTORC1,p-S6K and p-4E-BP1 were decreased in Leu deficient cultured cells.After LPS stimulation,the expression of p-mTORC1,p-S6K and p-4E-BP1 were significantly decreased(P<0.05).5.After mTORC1 inhibition,proliferation and migration activity of cells decreased,apoptosis increased,and the expressions of TLR4,MyD88,p-P65,TNF-α and IL-1βwere upregulated,while the expressions of p-S6K,p-4E-BP1,Claudin-4,Occludin and ZO-1 were down-regulated(P<0.05).Conclusion:1.There were significant differences in fecal amino acids before and after mucosal healing of UC,among which Leucine had high sensitivity and specificity in predicting MH,which had the potential to become a novel adjuvant biomarker for mucosal healing of UC.2.There were significant differences in gut microbiota before and after mucosal healing of UC.After mucosal healing,the diversity of gut microbiota increased and the structure changed.Species of producing SCFA increase obviously,represented by Faecalibacterium prausnitzii,Bacteroides fragilis,Eubacterium hallii.3.MH was enriched in amino acid metabolism,such as the biosynthesis of Leu,which may affect gut microbiota,such as Ruminococcus,Acidaminococcus,Alistipes,Faecalibacterium prausnitzii,to increase carbohydrate metabolism,nucleotide metabolism,fatty acid biosynthesis and other metabolic pathways,produce SCFA and Branched chain fatty acid,participated in intestinal epithelial metabolism to promote mucosal healing of UC.4.Mice fed Leu-deficient diet showed inflammation and intestinal barrier damage,which was aggravated after DSS induction and relieved after supplementation of Leu,suggested that Leu could participate in mucosal healing of UC through anti-inflammatory and enhancing intestinal barrier.5.Leu promoted cell proliferation,migration,anti-apoptosis,inhibited NF-κB pathway to anti-inflammatory and stimulated protein synthesis to enhance intestinal barrier function by activating mTORC1 signaling pathway,participated in mucosal healing of UC.