Dextran Sulfate Sodium Treatment Research Articles

Anti-Inflammatory Effect and Signaling Mechanism of Glycine max Hydrolyzed with Enzymes from Bacillus velezensis KMU01 in a Dextran-Sulfate-Sodium-Induced Colitis Mouse Model.

The purpose of this study was to investigate the effect that Glycine max hydrolyzed with enzymes from Bacillus velezensis KMU01 has on dextran-sulfate-sodium (DSS)-induced colitis in mice. Hydrolysis improves functional health through the bioconversion of raw materials and increase in intestinal absorption rate and antioxidants. Therefore, G. max was hydrolyzed in this study using a food-derived microorganism, and its anti-inflammatory effect was observed. Enzymatically hydrolyzed G. max (EHG) was orally administered once daily for four weeks before DSS treatment. Colitis was induced in mice through the consumption of 5% (w/v) DSS in drinking water for eight days. The results showed that EHG treatment significantly alleviated DSS-induced body weight loss and decreased the disease activity index and colon length. In addition, EHG markedly reduced tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 production, and increased that of IL-10. EHG improved DSS-induced histological changes and intestinal epithelial barrier integrity in mice. Moreover, we found that the abundance of 15 microorganisms changed significantly; that of Proteobacteria and Escherichia coli, which are upregulated in patients with Crohn's disease and ulcerative colitis, decreased after EHG treatment. These results suggest that EHG has a protective effect against DSS-induced colitis and is a potential candidate for colitis treatment.

Lactobacillus casei ATCC 393 combined with vasoactive intestinal peptide alleviates dextran sodium sulfate-induced ulcerative colitis in C57BL/6 mice via NF-κB and Nrf2 signaling pathways

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) which is related to an immunological imbalance of the intestinal mucosa. Many clinical evidences indicate probiotics supplementation appears to be effective and safe in patients with UC. Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with multiple physiological and pathological effects. In this study, we investigated the protective effect of the combination of Lactobacillus casei ATCC 393 (L. casei ATCC 393) with VIP on dextran sodium sulfate (DSS)-induced UC in mice and the potential mechanism. The results showed that, compared with the control group, DSS treatment significantly shortened the colon length, caused inflammation and oxidative stress, and further resulted in the intestinal barrier dysfunction and gut microbiota dysbiosis. In addition, intervention with L. casei ATCC 393, VIP or L. casei ATCC 393 combined with VIP significantly reduced UC disease activity index. However, compared with L. casei ATCC 393 or VIP, L. casei ATCC 393 combined with VIP effectively relieved symptoms of UC by regulating immune response, enhancing antioxidant capacity, and regulating nuclear factor kappa-B (NF-κB) and nuclear factor erythroid-derived-2-like 2 (Nrf2) signaling pathways. In conclusion, this study suggests that L. casei ATCC 393 combined with VIP can effectively relieve DSS-induced UC, which is a promising treatment strategy for UC.

Limosilactobacillus mucosae and Lactobacillus amylovorus Protect Against Experimental Colitis via Upregulation of Colonic 5-Hydroxytryptamine Receptor 4 and Transforming Growth Factor-β2

BackgroundLimosilactobacillusmucosae (LM) exerts anti-inflammatory and health-promoting effects. However, its role in the modulation of gut serotonin or 5-hydroxytryptamine (5-HT) metabolism and 5-HT receptors (HTRs) in inflammation requires further investigation. ObjectivesWe compared LM with Lactobacillus amylovorus (LA) for the regulation of 5-HT, HTRs, inflammatory mediators, and their correlations in the colon of mice with experimental colitis. MethodsMale C57BL/6 mice were randomly assigned to 6 groups: control (Con), LM, LA, dextran sodium sulfate (DSS), and DSS with pre-administration of LM (+LM) or LA (+LA). After 7 d of DSS treatment, mice were killed to analyze the expression of inflammatory mediators, HTRs, and concentrations of 5-HT and microbial metabolites in the colon. ResultsLM was more effective than LA in alleviating DSS-induced colonic inflammation. Compared with mice in the DSS group, mice receiving DSS + LM or DSS + LA treatment had lower (P < 0.05) colonic mRNA expression of proinflammatory cytokines. DSS + LM treatment had lower mRNA expression of Il1b, Tnfa, and Ccl3, an abundance of p-STAT3, and greater expression of Tgfb2 and Htr4 in the colon (P < 0.05). The expression of inflammatory mediators (including Tgfb-1) was positively correlated (P < 0.05) with 5-HT and Htr2a and negatively correlated (P < 0.05) with Htr4. However, the expression of Tgfb-2 showed reversed correlations with the 5-HT and HTRs described above. Patterns for these correlations were different for LM and LA. Mice receiving the DSS + LM treatment had greater (P < 0.05) concentrations of acetate and valerate and lower (P < 0.05) concentrations of indole-3-acetic acid in the cecal and colonic contents. ConclusionsLM showed greater efficacy than LA in alleviating DSS-induced colonic inflammation. The coordinated regulation of transforming growth factor-β subtypes and serotonin receptors in the colon may be one of the most important mechanisms underlying the probiotic effects of lactobacilli in gut inflammation.

MIA mice exhibit enteric nerve defects and are more susceptible to dextran sulfate sodium-induced colitis

Maternal immune activation (MIA) during pregnancy impairs the development of the central nervous system as well as the peripheral nervous system. Emerging evidence indicates that individuals with MIA suffer more from gastrointestinal disorders. The present study aims to test the hypothesis that MIA-induced susceptibility to inflammatory bowel disease is due to defects in the innervation of mucosal sensory nerves. Acute dextran sulfate sodium (DSS) colitis was induced in MIA and control adult mice. Body weight loss, disease activity index and colonic histological changes were measured during colitis. The study found that MIA mice were hypersusceptible to DSS-induced colitis and that macrophage infiltration and cytokine production were elevated in the colon of MIA mice. In vitro experiments also demonstrated that colonic macrophages from MIA mice presented hyperinflammatory responses to LPS stimulation. Sensory nerve-secreted calcitonin gene–related peptide (CGRP) is an important neuropeptide in modulating enteric inflammation. Intriguingly, we found that CGRP-positive nerves were sparsely distributed in the colon of MIA mice regardless of DSS treatment. And the protein level of CGRP was significantly reduced in colon of MIA mice. However, there was no decrease in the number of CGRP-positive cell bodies in either the DRG or vagal ganglion, suggesting that innervation defects of CGRP mucosal sensory nerves exist in the colon of MIA mice. Critically, administration of recombinant CGRP to MIA mice during DSS colitis significantly reversed their hyperinflammatory pathology. Additionally, the hyperinflammatory phenotype of colonic macrophages of MIA mice could also be reversed by CGRP treatment in vitro. Collectively, these findings suggested that the sensor nerve innervation defect-induced CGRP deficiency in MIA mice participates in their increased susceptibility to colitis. Thus, sensor nerve-secreted CGRP may be a new therapeutic target for autism combined with inflammatory bowel disease.

Grimace scale assessment during Citrobacter rodentium inflammation and colitis development in laboratory mice.

Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium. In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation (n = 9) and 2. acute intestinal inflammation (with (n = 23) and without (n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours. The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6-Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable. In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation.

Fucose ameliorates the proinflammatory property of Fusobacterium nucleatum in colitis via altering its metabolism.

Previous studies reported that fucose plays a protective role in inhibiting pathogens. Fusobacterium nucleatum (Fn) was recently found to promote the progression of colitis. However, the effects of fucose on Fn are poorly understood. This study aimed to explore whether fucose could ameliorate the proinflammatory property of Fn in colitis and the underlying mechanisms. To validate our hypothesis, mice were administrated with Fn and fucose-treated Fn (Fnf) before dextran sulfate sodium (DSS) treatment to establish Fn related colitis model. The metabolism variation of Fn was detected by metabolomic analysis. To verify the effects of bacterial metabolites on intestinal epithelial cells (IECs), Caco-2 cells were treated with bacterial supernatant. More severe inflammation, intestinal barrier damage, autophagy block, and apoptosis in the colon were noted in DSS mice that were administrated with Fn or Fnf. However, the severity degree in Fnf+DSS group was less compared to Fn+DSS group. Metabolic pathways of Fn were altered after fucose treatment and proinflammatory metabolites were decreased. The supernatant of Fnf induced a lower level of inflammation than Fn in Caco-2 cells. One of the decreased metabolites, homocysteine thiolactone (HT), was proven to induce inflammatory effects in Caco-2 cells. In conclusion, fucose ameliorates the proinflammatory property of Fn via altering its metabolism and these findings provide evidence for the application of fucose as functional food or prebiotic in the treatment of Fn related colitis.

Tissue-specific regulatory role of Sorting Nexin 27 in the intestinal inflammation

The sorting nexin (SNX) family involves in endocytosis and protein trafficking. SNX27 specifically binds and directs the sorting transmembrane proteins containing PDZ-binding motif and is critical in protein-protein interactions in various organs. While SNX27 has been studied in neurological disorders, the tissue-specific role of SNX27 in intestinal health and disease remains unexplored. We conditionally deleted the SNX27 gene from intestine epithelial cells (SNX27 ΔIEC ) and myeloid cells (SNX27 ΔLyz ) by crossing the SNX27 Loxp mice with Villin-cre mice and Lyz-cre mice, respectively. The basic phenotypes and functional alterations were evaluated. To investigate their susceptibility to inflammations and colitis-associated cancer, mice were challenged in dextran sulfate sodium (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced cancer models. We validated the loss of SNX27 in these cells by PCR and Western blots. We established the conditional deletion of SNX27 from the IECs and myeloid cells in mice, which is the first report to our knowledge. At the basal level, the SNX27 ΔIEC and SNX27 ΔLyz mice (10 weeks old) showed slightly lower body weight compared to the SNX27 Loxp mice, but no intestinal pathological symptoms, e.g., diarrhea and bloody stools. SNX27 ΔLyz mice (24-week old) had significantly longer colon and small intestine, heavier liver and spleen, compared with the age-matched SNX27 Loxp mice. Abnormal secretory cells, Paneth cells and Goblet cells, were observed in the small intestine of SNX27 ΔIEC mice, indicated by the low expression of Lysozyme and Muc-2. More importantly, the inflammatory responses of two mouse models were different. The SNX27 ΔIEC mice were more susceptible to DSS-induced colitis. DSS-SNX27 ΔIEC mice showed significantly more bodyweight loss, shorter colon and small intestine, and higher intestine permeability. In contrast, SNX27 ΔLyz mice were more resistant to the DSS treatment, compared with the SNX27 ΔIEC and SNX27 Loxp mice. In the AOM/DSS model, more tumors and bodyweight loss were found in SNX27 ΔIEC mice. The experiment ended early (11 weeks post-treatment) due to earlier onset of tumors and death in SNX27 ΔIEC mice (18-19 weeks old). The AOM/DSS-SNX27 ΔLyz mice showed less sensitivity compared to the SNX27 ΔIEC mice. Larger tumors were developed in SNX27 ΔLyz mice, compared to the SNX27 Loxp mice at 16 weeks post-treatment. A significantly larger spleen and liver were found in the SNX27 ΔLyz mice, indicating the importance of SNX27 in the immune system. The susceptibility of SNX27 ΔIEC mice to inflammations and colitis-associated colon may be due to the important role for SNX27 in intestinal barriers through epithelial tight junctions and secretory cells. Here, we highlight the different tissue-specific roles of SNX27 in maintaining intestinal homeostasis and preventing inflammation and colon cancer. Our study will provide new insights into the functions of a sorting nexin protein in intestinal disorders. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FGF21 Depletion Attenuates Colitis through Intestinal Epithelial IL-22-STAT3 Activation in Mice.

Fibroblast growth factor 21 (FGF21) is a glucose and lipid metabolic regulator. Recent research revealed that FGF21 was also induced by inflammatory stimuli. Its role in inflammatory bowel disease (IBD) has not been investigated. In this study, an experimental IBD model was established in FGF21 knockout (KO) and wild-type (WT) mice by adding 2.5% (wt/vol) dextran sodium sulfate (DSS) to their drinking water for 7 days. The severity of the colitis and the inflammation of the mouse colon tissues were analyzed. In WT mice, acute DSS treatment induced an elevation in plasma FGF21 and a significant loss of body weight in a time-dependent manner. Surprisingly, the loss of body weight and the severity of the colitis induced by DSS treatment in WT mice were significantly attenuated in FGF21 KO mice. Colon and circulating pro-inflammatory factors were significantly lower in the FGF21 KO mice compared to the WT mice. As shown by BrdU staining, the FGF21 KO mice demonstrated increased colonic epithelial cell proliferation. DSS treatment reduced intestinal Paneth cell and goblet cell numbers in the WT mice, and this effect was attenuated in the FGF21 KO mice. Mechanistically, FGF21 deficiency significantly increased the signal transducer and activator of transcription (STAT)-3 activation in intestinal epithelial cells and increased the expression of IL-22. Further study showed that the expression of suppressor of cytokine signaling-2/3 (SOCS 2/3), a known feedback inhibitor of STAT3, was significantly inhibited in the DSS-treated FGF2 KO mice compared to the WT mice. We conclude that FGF21 deficiency attenuated the severity of DSS-induced acute colitis, which is likely mediated by enhancing the activation of the IL-22-STAT3 signaling pathway in intestinal epithelial cells.

DNA polymerase κ suppresses inflammation and inflammation-induced mutagenesis and carcinogenic potential in the colon of mice

BackgroundChronic inflammation induces DNA damage and promotes cell proliferation, thereby increasing the risk of cancer. DNA polymerase κ (Pol κ), involved in translesion DNA synthesis, counteracts mutagenesis induced by inflammation in the colon of mice. In the present study, we examined whether Pol κ suppressed inflammation-induced colon tumorigenesis by treating inactivated Polk knock-in (Polk−/−) mice with dextran sulfate sodium (DSS), an inducer of colon inflammation.ResultsMale and female Polk−/− and Polk+/+ mice were administered 2% DSS in drinking water for six consecutive days, succeeded via a recovery period of 16 days, followed by 2% DSS for another two days. DSS treatment strongly induced colitis, and the severity of colitis was higher in Polk−/− mice than in Polk+/+ mice. The mice were sacrificed after 19 weeks from the initiation of the first DSS treatment and subjected to pathological examination and mutation analysis. DSS treatment induced colonic dysplasia, and the multiplicity of dysplasia was higher in Polk−/− mice than in Polk+/+mice. Some of the dysplasias in Polk−/− mice exhibited β-catenin-stained nucleus and/or cytoplasm. Mutation frequencies in the gpt reporter gene were increased by DSS treatment in Polk−/− mice, and were higher than those in Polk+/+ mice.ConclusionsPol κ suppresses inflammation and inflammation-induced dysplasia as well as inflammation-induced mutagenesis. The possible mechanisms by which Pol κ suppresses colitis- and colitis-induced dysplasia are discussed.

Selenoprotein W Ameliorates Experimental Colitis and Promotes Intestinal Epithelial Repair.

Selenoprotein W (Selenow) is a ~9 kDa selenoprotein suggested to play a beneficial role in resolving inflammation. However, the underlying mechanisms are poorly understood. SELENOW expression in the human GI tract using ScRNAseq Gut Cell Atlas and Gene Expression Omnibus (GEO) databases revealed its expression in the small intestine and colonic epithelial, endothelial, mesenchymal, and stem cells and correlated with a protective effect in ulcerative colitis patients. Selenow KO mice treated with 4% dextran sodium sulfate (DSS) showed exacerbated acute colitis, with greater weight loss, shorter colons, and increased fecal occult blood compared to the WT counterparts. Selenow KO mice expressed higher colonic Tnfα, increased Tnfα+ macrophages in the colonic lamina propria, and exhibited loss in epithelial barrier integrity and decreased zonula occludens 1 (Zo-1) expression following DSS treatment. Expression of epithelial cellular adhesion marker (EpCam), yes-associated protein 1 (Yap1), and epidermal growth factor receptor (Egfr) were decreased along with CD24lo cycling epithelial cells in Selenow KO mice. Colonic lysates and organoids confirmed a crosstalk between Egfr and Yap1 that was regulated by Selenow. Overall, our findings suggest Selenow expression is key for efficient resolution of inflammation in experimental colitis that is mediated through the regulation of Egfr and Yap1.

Pathobionts from chemically disrupted gut microbiota induce insulin-dependent diabetes in mice

BackgroundDysbiotic gut microbiome, genetically predisposed or chemically disrupted, has been linked with insulin-dependent diabetes (IDD) including autoimmune type 1 diabetes (T1D) in both humans and animal models. However, specific IDD-inducing gut bacteria remain to be identified and their casual role in disease development demonstrated via experiments that can fulfill Koch’s postulates.ResultsHere, we show that novel gut pathobionts in the Muribaculaceae family, enriched by a low-dose dextran sulfate sodium (DSS) treatment, translocated to the pancreas and caused local inflammation, beta cell destruction and IDD in C57BL/6 mice. Antibiotic removal and transplantation of gut microbiota showed that this low DSS disrupted gut microbiota was both necessary and sufficient to induce IDD. Reduced butyrate content in the gut and decreased gene expression levels of an antimicrobial peptide in the pancreas allowed for the enrichment of selective members in the Muribaculaceae family in the gut and their translocation to the pancreas. Pure isolate of one such members induced IDD in wildtype germ-free mice on normal diet either alone or in combination with normal gut microbiome after gavaged into stomach and translocated to pancreas. Potential human relevance of this finding was shown by the induction of pancreatic inflammation, beta cell destruction and IDD development in antibiotic-treated wildtype mice via transplantation of gut microbiome from patients with IDD including autoimmune T1D.ConclusionThe pathobionts that are chemically enriched in dysbiotic gut microbiota are sufficient to induce insulin-dependent diabetes after translocation to the pancreas. This indicates that IDD can be mainly a microbiome-dependent disease, inspiring the need to search for novel pathobionts for IDD development in humans.BraSzCKLkea6-Duo39HsMTVideo

CD300b regulates intestinal inflammation and promotes repair in colitis.

Chronic inflammation is a hallmark charataristic of various inflammatory diseases including inflammatory bowel disease. Subsequently, current therapeutic approaches target immune-mediated pathways as means for therapeutic intervention and promotion of mucosal healing and repair. Emerging data demonstrate important roles for CD300 receptor family members in settings of innate immunity as well as in allergic and autoimmune diseases. One of the main pathways mediating the activities of CD300 family members is via promotion of resolution through interactions with ligands expressed by viruses, bacteria, or dead cells (e.g., phospholipids such as PtdSer and/or ceramide). We have recently shown that the expression of CD300a, CD300b and CD300f were elevated in patients with IBD and that CD300f (but not CD300a) regulates colonic inflammation in response to dextran sodium sulphate (DSS)-induced colitis. Whether CD300b has a role in colitis or mucosal healing is largely unknown. Herein, we demonstrate a central and distinct role for CD300b in colonic inflammation and subsequent repair. We show that Cd300b-/- mice display defects in mucosal healing upon cessation of DSS treatment. Cd300b-/- mice display increased weight loss and disease activity index, which is accompanied by increased colonic histopathology, increased infiltration of inflammatory cells and expression of multiple pro-inflammatory upon cessation of DSS cytokines. Furthermore, we demonstrate that soluble CD300b (sCD300b) is increased in the colons of DSS-treated mice and establish that CD300b can bind mouse and human epithelial cells. Finally, we show that CD300b decreases epithelial EpCAM expression, promotes epithelial cell motility and wound healing. These data highlight a key role for CD300b in colonic inflammation and repair processes and suggest that CD300b may be a future therapeutic target in inflammatory GI diseases.

Long noncoding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila.

Long noncoding RNAs (lncRNAs) play important regulatory roles in stem cell self-renewal, pluripotency maintenance, and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISC regeneration in Drosophila have yet been characterized. Here, we identify a lncRNA, CR46040, that is essential for the injury-induced ISC regeneration in Drosophila. Loss of CR46040 greatly impairs ISC proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. We demonstrate that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies failed to fully activate JNK, JAK/STAT, and HIPPO signaling pathways after tissue damage, which are required for ISC proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. Our findings characterize, for the first time, a lncRNA that mediates damage-induced ISC proliferation in Drosophila and provide new insights into the functional links among the long noncoding RNAs, ISC proliferation, and tissue homeostasis.

Assessing acute colitis induced by dextran sulfate sodium in rats and its impact on gastrointestinal fluids.

Dextran sulfate sodium (DSS) is commonly used to induce colitis in rats. While the DSS-induced colitis rat model can be used to test new oral drug formulations for the treatment of inflammatory bowel disease, the effect of the DSS treatment on the gastrointestinal tract has not been thoroughly characterized. Additionally, the use of different markers to assess and confirm successful induction of colitis is somewhat inconsistent. This study aimed to investigate the DSS model to improve the preclinical evaluation of new oral drug formulations. The induction of colitis was evaluated based on the disease activity index (DAI) score, colon length, histological tissue evaluation, spleen weight, plasma C-reactive protein, and plasma lipocalin-2. Furthermore, the study investigated how the DSS-induced colitis affected the luminal pH, lipase activity, and concentrations of bile salts, polar lipids, and neutral lipids. For all evaluated parameters, healthy rats were used as a reference. The DAI score, colon length, and histological evaluation of the colon were effective disease indicators in DSS-induced colitis rats, while spleen weight, plasma C-reactive protein, and plasma lipocalin-2 were not. The luminal pH of the colon and bile salt- and neutral lipid concentrations in regions of the small intestine were lower in DSS-induced rats compared to healthy rats. Overall, the colitis model was deemed relevant for investigating ulcerative colitis-specific formulations.

Ang1 and Ang4 differentially affect colitis and carcinogenesis in an AOM-DSS mouse model.

Angiogenin-1 (Ang1) and angiogenin-4 (Ang4) are 14-kDa ribonucleases with potent angiogenic and antimicrobial properties. The role of Ang1 and Ang4 in chronic colitis and colitis-associated cancer has not been previously studied. Wild-type (WT) and angiogenin-1 knock-out (Ang1-KO) C57BL/6 mice were given azoxymethane, a colon carcinogen, 2 days in advance of three cycles of 3.5% dextran sodium sulfate (DSS). Disease activity index (DAI) was recorded, a colonoscopy was performed after each DSS treatment, and mice were euthanized (colitis, recovery, cancer) with tissue evaluated by histopathology. Ang1, Ang4, TNF-α, Il-1F062, IL-6, IL-10, IL-23, IL-33 mRNA levels were analyzed by RT-PCR. Ang1-KO mice exhibited more severe colitis compared to WT mice during both the acute (P<0.05) and recovery (P<0.05) phases of each DSS cycle. Consistent with these results, colonic TNF-α, IL1-β, IL-6, IL-10, and IL-33 mRNA levels were significantly upregulated in Ang1-KO mice (P<0.05). While Ang4 increased to similar levels in both WT and Ang1-KO mice during colitis and recovery phases, WT mice were distinguished by a significant upregulation of Ang1. Interestingly, despite the reduced colitis, WT mice developed significantly more tumors compared to Ang1-KO mice (P<0.05). 134 tumors formed in WT mice (4.6 tumors/mouse) while only 46 tumors formed (1.5 tumors/mice) in Ang1-KO mice, which were also characterized by a 34-fold decrease in Ang4 compared to WT mice and the complete absence of Ang1. In a mouse model of colitis-associated cancer, Ang1-KO mice develop more severe colitis, but fewer tumors compared to WT mice. Ang1 levels correlate with the severity of colitis and the development of colitis-associated cancer, while Ang4 was upregulated during both colitis and cancer. Ang1 and Ang4 play important regulatory roles in the response to chronic colitis and the development of colitis-associated cancer and may serve as novel therapeutic targets.

A30 EPITHELIAL FUNCTION OF THE CIRCADIAN CLOCK GENE, BMAL1, IS NECESSARY FOR COLONIC REGENERATION

Abstract Background The circadian clock is a self-sustained molecular oscillator which drives 24-hour physiological rhythms. It consists of the genes Bmal1 and Clock that positively regulate Cry and Per, their negative regulators, resulting in a 24-hour transcription/translation feedback loop. Shift work, which causes disruptions to 24-hour physiological rhythms, has been shown to lead to an increased incidence of inflammatory bowel disease (IBD). We have previously established that mice lacking the non-redundant circadian regulator, Bmal1, exhibit more severe colitis compared to controls. Purpose This study aims to investigate the epithelial function of Bmal1 in colonic regeneration during colitis. Method In order to assess the cell-specific role of the clock, we tested the regenerative effects of Bmal1 in intestinal epithelial tissue using Vil+/+;Bmal1flox/flox (control) and VilCre/+;Bmal1flox/flox (conditional mutant) mice. Dextran Sulfate Sodium (DSS) was applied to induce acute colitis. Disease progression was evaluated during colitis and during recovery upon removal of DSS treatment. We hypothesized that the absence of a functional circadian clock disrupts effective proliferation and regeneration of intestinal epithelial cells during colitis remission. Result(s) Vil +/+ ;Bmal1 flox/flox control and VilCre/+;Bmal1flox/flox conditional mutant mice exhibit no significant differences in disease severity or tissue histopathology during colitis. However, after the removal of DSS, VilCre/+;Bmal1flox/flox conditional mutants show increased total lesions and overall inflammation, decreased crypt density as well as a higher propensity of hyperplastic crypts in the tissue. Regenerative ability of the colon is decreased in conditional mutants: phosphorylated-histone H3 and Ki67 show persistent increases in mitosis and overall proliferation near ulcerated lesions. This suggests that, while controls complete the regenerative response, conditional mutants fail to recover from colitis and show inefficient regeneration when Bmal1 is disrupted in intestinal epithelial cells. Conclusion(s) Our results support a critical role for Bmal1 in intestinal epithelial cells during post-colitis regeneration and recovery. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; OGS, Crohn's &amp; Colitis Canada Disclosure of Interest None Declared

Raptinal ameliorates 1,2-dimethylhydrazine-induced colon cancer through p53/Bcl2/Bax/caspase-3-mediated apoptotic events in vitro and in vivo.

Colon carcinoma stands as the most familiar malignancy throughout across the globe. Raptinal induce apoptosis through the alteration of cellular events. Thus, in the present investigation, the anticancer activity of raptinal counter to 1,2-dimethylhydrazine (DMH) persuaded colon carcinoma has been evaluated through both in vivo and in vitro systems. The pharmacophore analysis demonstrated the binding efficacy of raptinal with the apoptotic proteins. The chemotherapeutic activity of raptinal was examined through HT-29 human colorectal cancer (CRC) cell line as well as DMH persuaded CRC in the rat model. The cytotoxicity analysis, flow cytometry, and DAPI analysis have been carried out on HT-29 cell line through in vitro assessment. The colon carcinoma has been induced through DMH administration and subsequently Dextran sulfate sodium treatment in male Wistar rats. After 18 weeks of raptinal treatment, the colon tissues have been investigated for aberrant crypt foci (ACF) count, antioxidant status, histology, immunohistochemical assessment, and apoptotic analysis. The raptinal therapy on HT-29 cells demonstrated a substantial % of early apoptosis followed by G0 and G1 phase arrest, which subsequently led to apoptosis. Furthermore, it inhibits ACF development with improved colonic abrasions and structural integrity of colonic mucosa with increased levels of antioxidants, proapoptotic biomarkers including p53, caspase-3, Bax and downstream effects of Bcl-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 mutation. These findings indicate the raptinal effectively reduces colon cancer by inducing apoptosis through p53/Bcl2/Bax/caspase-3 pathway and suppressing IL-6, TNF-mediated chronic inflammation in the colon cancer microenvironment.

The inhibitory effect of oral administration of lentinan on DSS-induced inflammation is exerted by the migration of T cells activated in the ileum to the colon

Oral administration of lentinan ameliorated dextran sulfate sodium (DSS)-induced colitis through Dectin-1 receptor on intestinal epithelial cells. However, it is unclear where lentinan affects in the intestine to prevent the inflammation. We found that the administration of lentinan has induced migration of CD4+ cells from the ileum to the colon by using Kikume Green-Red (KikGR) mice in this study. This result suggests that the oral lentinan treatment could accelerate the migration of Th cells in lymphocyte from ileum into the colon during lentinan intake. Then, C57BL/6 mice were administered 2% DSS to induce colitis. The mice were administered lentinan daily via oral or rectal route before DSS administration. Its rectal administration also suppressed DSS-induced colitis, but its suppressive effects were lower compared to when orally administered, indicating that the biological responses to lentinan in the small intestine contributed to the anti-inflammatory effects. In normal mice (without DSS treatment), the expression of Il12b was significantly increased in the ileum by the oral administration of lentinan, but not by rectal one. On the other hand, no change was observed in the colon by either administration method. In addition, Tbx21 was significantly increased in the ileum. These suggested that IL-12 was increased in the ileum and Th1 cells differentiated in dependence on it. Therefore, Th1 predominant condition in the ileum could influence immunity in the colon and improve the colitis.

Musculin does not modulate the disease course of Experimental Autoimmune Encephalomyelitis and DSS colitis

Previous evidences show that Musculin (Msc), a repressor member of basic helix-loop-helix transcription factors, is responsible in vitro for the low responsiveness of human Th17 cells to the growth factor IL-2, providing an explanation for Th17 cells rarity in inflammatory tissue. However, how and to what extent Musculin gene can regulate the immune response in vivo in an inflammatory context is still unknown. Here, exploiting two animal models of inflammatory diseases, the Experimental Autoimmune Encephalomyelitis (EAE) and the dextran sodium sulfate (DSS)-induced colitis, we evaluated the effect of Musculin gene knock-out on clinical course, performing also a deep immune phenotypical analysis on T cells compartment and an extended microbiota analysis in colitis-sick mice. We found that, at least during the early phase, Musculin gene has a very marginal role in modulating both the diseases. Indeed, the clinical course and the histological analysis showed no differences between wild type and Msc knock-out mice, whereas immune system appeared to give rise to a regulatory milieu in lymph nodes of EAE mice and in the spleen of DSS colitis-sick mice. Moreover, in the microbiota analysis, we found irrelevant differences between wild type and Musculin knock-out colitis-sick mice, with a similar bacterial strains’ frequency and diversity after the DSS treatment. This work strengthened the idea of a negligible Msc gene involvement in these models.

DSS-induced acute colitis causes dysregulated tryptophan metabolism in brain: an involvement of gut microbiota

Inflammatory bowel disease can cause pathological changes of certain organs, including the gut and brain. As the major degradation route of tryptophan (Trp), Kynurenine (Kyn) pathway are involved in multiple pathologies of brain. This study sought to explore the effects of Dextran sulphate sodium (DSS)-induced colitis on serum and brain Trp metabolism (especially the Kyn pathway) and its mechanisms. We induced acute colitis and sub-chronic colitis with 3% DSS and 1% DSS respectively and found more severe intestinal symptoms in acute colitis than sub-chronic colitis. Both of the colitis groups altered Trp-Kyn-Kynurenic acid (Kyna) pathway in serum by regulating the expression of rate-limiting enzyme (IDO-1, KAT2). Interestingly, only 3% DSS group activated Trp-Kyn pathway under the action of metabolic enzymes (IDO-1, TDO-2 and KAT2) in brain. Furthermore, intestinal flora 16S rRNA sequencing showed significantly changes in both DSS-induced colitis groups, including microbial diversity, indicator species, and the abundance of intestinal microflora related to Trp metabolism. The functional pathways of microbiomes involved in inflammation and Trp biosynthesis were elevated after DSS treatment. Moreover, correlation analysis showed a significant association between intestinal flora and Trp metabolism (both in serum and brain). In conclusion, our study suggests that DSS-induced acute colitis causes dysregulation of Trp-Kyn-Kyna pathways of Trp metabolism in serum and brain by affecting rate-limiting enzymes and intestinal flora.