TNBS Model Research Articles

Homocysteine aggravates intestinal inflammation through promotion of 5-LOX and COX-2 in IBD

BackgroundHomocysteine (Hcy) is a pro-inflammatory molecule that has the potential to induce oxidative damage to cells and stimulate the release of inflammatory mediators. Hcy has been observed to enhance the production of inflammatory agents in vascular endothelial cells. However, the impact of Hcy on intestinal mucosal inflammation remains largely unexplored. Therefore, the objective of this study was to examine the potential of Hcy to stimulate the synthesis of inflammatory mediators and elucidate the underlying mechanisms in the intestinal mucosa.MethodsA total of 99 patients diagnosed with inflammatory bowel disease (IBD) and 10 healthy individuals were included in this study to assess the impact of homocysteine (Hcy) on the levels of leukotriene E4 (LTE4) and prostaglandin E2 (PGE2). The underlying mechanism responsible for the generation of LTE4 and PGE2 induced by Hcy was investigated using colitis rats and Caco-2 cells. 32 Sprague–Dawley rats were categorized into four groups: normal control, TNBS model, normal with Hcy injection, and TNBS model with Hcy injection. The mRNA expressions of 5-LOX, COX-2, and NF-κB were assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Caco-2 cells were subjected to treatment with varying concentrations (10, 20, 50, 100 μmol/L) of Hcy and incubated for different durations (1, 3, 6 h). The alterations in NF-κB activity, as well as the levels of Hcy, LTE4, and PGE2, were measured using enzyme-linked immunosorbent assay (ELISA).ResultsThe excretion of Hcy, LTE4, and PGE2 in urine exhibited significant increases in patients with inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC). In addition, Hcy demonstrated a significant increase in the expression of 5-LOX, COX-2, and NF-κB, as well as elevated levels of LTE4 and PGE2 in rats with colitis. Furthermore, Hcy was found to induce NF-κB activation and nuclear translocation, thereby contributing to the enhanced synthesis of LTE4 and PGE2 in Caco-2 cells.ConclusionsHcy was found to enhance the expression of 5-LOX and COX-2 by activating NF-κB, thereby augmenting the production of LTE4 and PGE2, which ultimately exacerbates colonic inflammation in individuals with inflammatory bowel disease (IBD).

Preventive role of Dietary Phytochemical Lupeol in Preclinical Ulcerative Colitis Models

Background: The primary cause of intestinal and colon inflammation is inflammatory bowel disease (IBD).Preclinical ulcerative colitis is poorly definedin this article we providea scientific validation in preclinical systemic inflammation in ulcerative colitis by the means of phytochemical lupeol as a source of triterpenoid rich food. Aim: The aim of the present study was to evaluate the bioactive phytochemical, lupeol for the management of IBD through two different experimental models. Method: The intestinal anti-inflammatory models for rat colitis were performed by using TNBS and DSS to access the use of lupeol (25μg/ml and 50μg/ml) in ulcerative colitis. Change in body weight, stool consistency, colon weight/length and histological examination of colon were performed. Results: Lupeol showed significant anti-inflammatory impact in the colon at both the doses in TNBS and DSS models. The results of lupeol showed marked evidence both by histologically and also by the tissue recovery seen in various parameters like, Change in body weight, stool consistency and colon weight/length. Conclusion: The findings of this study provide the full proof rationale for additional research using lupeol in the successful management of ulcerative colitis.

Response Variability to Drug Testing in Two Models of Chemically Induced Colitis.

The lack of knowledge regarding the pathogenesis of IBD is a challenge for the development of more effective and safer therapies. Although in vivo preclinical approaches are critical for drug testing, none of the existing models accurately reproduce human IBD. Factors that influence the intra-individual response to drugs have barely been described. With this in mind, our aim was to compare the anti-inflammatory efficacy of a new molecule (MTADV) to that of corticosteroids in TNBS and DSS-induced colitis mice of both sexes in order to clarify further the response mechanism involved and the variability between sexes. The drugs were administered preventively and therapeutically, and real-time bioluminescence was performed for the in vivo time-course colitis monitoring. Morphometric data were also collected, and colonic cytokines and acute plasma phase proteins were analyzed by qRT-PCR and ELISA, respectively-bioluminescence images correlated with inflammatory markers. In the TNBS model, dexamethasone worked better in females, while MTADV improved inflammation in males. In DSS-colitis, both therapies worked similarly. Based on the molecular profiles, interaction networks were constructed to pinpoint the drivers of therapeutic response that were highly dependent on the sex. In conclusion, our results suggest the importance of considering sex in IBD preclinical drug screening.

Protective effect of Schistosoma japonicum eggs on TNBS-induced colitis is associated with regulating Treg/Th17 balance and reprogramming glycolipid metabolism in mice.

Inflammatory bowel diseases (IBDs) have been classified as modern refractory diseases. However, safe, well-tolerated, and effective treatments for IBDs are still lacking. Therefore, there is an urgent need to develop novel therapeutic targets with fewer undesirable adverse reactions. A growing body of research has shown that infection with live helminths or exposure to defined helminth-derived components can downregulate pathogenic inflammation due to their immunoregulatory ability. Here we were to explore the protective role of Schistosoma japonicum eggs on murine experimental colitis caused by trinitrobenzene sulfonic acid (TNBS) and the underlying mechanism. Frequencies of splenic Treg and Th17 cells were detected by flow cytometry. Protein and mRNA expressions of Foxp3 and RORγt were investigated by Western Blot and quantitative real-time polymerase chain reaction (qPCR), respectively. Concentrations of transforming growth factor-beta1 (TGF-β1), interleukin-10 (IL-10) and IL-17A were assessed with ELISA. Expression levels of genes related to glycolipid metabolism were measured with qPCR. The results showed that pre-exposure to S. japonicum eggs contributed to the relief of colitis in the TNBS model, evidenced by improved body weight loss, reversing spleen enlargement and colon shortening, and decreased histology scores. Compared with the TNBS group, the TNBS+Egg group had increased Treg immune response, accompanied by decreased Th17 immune response, leading to the reconstruction of Treg/Th17 balance. In addition, a ratio of Treg/Th17 was correlated negatively with the histological scores in the experiment groups. Furthermore, the regulation of Treg/Th17 balance by S. japonicum eggs was associated with inhibiting the glycolysis pathway and lipogenesis, along with promoting fatty acid oxidation in the TNBS+Egg group. These data indicate that S. japonicum eggs have a protective effect against TNBS-induced colitis, which is related to restoring Treg/Th17 balance and regulating glucose and lipid metabolism.

Immunomodulatory effects of fentanyl and morphine on DSS- and TNBS-induced colitis

Background Opioid prescription for inflammatory bowel disease (IBD)-related pain is on the rise. However, the use of strong opioids can result in severe complications, and even death, in IBD patients. This study aimed to define the role of fentanyl and morphine, two representative strong opioids, in the pathogenesis of dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced colitis. Method DSS and TNBS models were induced in C57BL/6J and Balb/c mice, respectively. Disease activity index (DAI), histopathology, enzyme-linked immunosorbent assay (ELISA), multiplex ELISA, and flow cytometry were performed to evaluate the effects of fentanyl and morphine. Result Fentanyl exacerbated DSS- and TNBS-induced colitis, while morphine exhibited no significant immunomodulatory effect. Fentanyl and morphine had no obvious effects on the serum levels of adrenocorticotropic hormone (ACTH), glucocorticoid (GC), and prostaglandin E2 (PGE-2) in DSS and TNBS models. Fentanyl elevated the proportions of Th1 cells, μ-opioid receptor (MOR) + Th1 cells, and MOR + macrophages in the colonic mucosa of DSS-treated mice, and enhanced the proportions of Th1 cells, macrophages, MOR + Th1 cells, and MOR + macrophages in the colonic mucosa of TNBS-treated mice. We found that fentanyl upregulated the levels of inflammatory cytokines/chemokines in MOR + macrophages of the colonic lamina propria mononuclear cells (LPMCs) from DSS-treated mice, whereas it had no effect on the expression of most inflammatory cytokines/chemokines in MOR + macrophages in the colonic LPMCs from TNBS-treated mice. Conclusion Our findings suggest that fentanyl exacerbates murine colitis via Th1 cell- and macrophage-mediated mechanisms, while morphine exhibits no significant immunomodulatory effect.

A Review on TNBS Model: Chemical and Procedure

A Review on TNBS Model: Chemical and Procedure

Indoleamine-2,3-dioxygenase-related anti-inflammatory effects of 3-aminobenzamide and infliximab in experimental colitis.

This study aimed to investigate the presence of indoleamine-2,3-dioxygenase and bacterial translocation after the administration of 3-aminobenzamide and infliximab in the TNBS model of rat colitis. The study group was divided into five categories as follows: group 1: (control), group 2: colitis+saline, group 3: colitis+3-aminobenzamide, group 4: colitis+infliximab, and group 5: colitis+3-aminobenzamide+infliximab. Intestinal mesenteric cultures were incubated on specific agar media plates under aerobic and anaerobic conditions, bacterial translocation was evaluated and assessed as colony-forming units per gram of tissue. Colonic tissue samples were evaluated by Western blotting method to detect the presence of indoleamine-2,3-dioxygenase. The results obtained were as follows: group 1: normal gut flora; group 2: eight of nine samples had bacterial translocation, of which six of them had positive indoleamine-2,3-dioxygenase protein; group 3: five of nine samples had bacterial translocation, of which seven of them had positive indoleamine-2,3-dioxygenase; group 4: three of nine samples had bacterial translocation, of which seven of them had positive indoleamine-2,3-dioxygenase; and group 5: only one sample had exact indoleamine-2,3-dioxygenase protein. Altered expression of indoleamine-2,3-dioxygenase results in a lower bacterial translocation via infliximab compared with 3-aminobenzamide treatment. Combined treatments emphasized different approaches for the new molecules related to indoleamine-2,3-dioxygenase.

A NOVEL ORALLY ACTIVE METABOLITE REVERSES CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS

Abstract BACKGROUND Intestinal fibrosis is a debilitating complication of Crohn’s disease (CD) patients. Our groups and others discovered that intestinal metabolites are associated with intestinal fibrosis. We selected the most relevant metabolites and determined their anti-fibrogenic efficacies in three mouse models of intestinal fibrosis. METHODS We compared the intestinal metabolite profiles between non-IBD, stricturing CD, and non-stricturing CD patients in inflammatory bowel disease (IBD) multi-omics database to identify CD stricture-related metabolites. We compared the fecal metabolite profiles between trinitrobenzene sulfonic acid (TNBS)-treated mice with and without anti-fibrogenic drug CSA13 treatment to identify mouse intestinal fibrosis-related metabolites. RESULTS Compared to non-stricturing CD patients and non-IBD patients, stricturing CD patients had reduced fecal levels of hydrocinnamate (H), inosine (I), taurine (T), tauroursodeoxycholate (TUDCA), and sphingosine (S). Oral CSA13 increased fecal levels of inosine (I) and sphingosine (S) in the TNBS-treated mice. Overall disease activity (ODA) is a comprehensive assessment of histology and fibrosis scores and intestinal mRNA expression of fibrosis and inflammation markers. Sphingosine (10mg/kg/day for 7-14 days via oral gavages) showed the best anti-fibrogenic effects against spontaneous ileal fibrosis in 42-week-old SAMP1/YitFc mice (11% ODA), cecal fibrosis in Salmonella-infected mice (10% ODA), and colonic fibrosis in TNBS-treated mice (9% ODA). The other four metabolites were ineffective in reversing intestinal fibrosis. Sphingosine (10 microM), but not other metabolites, directly inhibited collagen expression in human colonic CCD-18Co fibroblasts and primary human intestinal fibroblasts from CD patients (CD-HIF). RNA-seq and proteomics identified that low MAPK3 and high ZEB1 mRNA expression in intestines are associated with intestinal fibrosis in CD patients. Sphingosine activated ERK1/2 phosphorylation and inhibited TGF-b1-induced ZEB1 expression in CCD-18Co fibroblasts. The sphingosine-mediated inhibition of collagen synthesis was reversed by ERK1/2 inhibitor PD98059 and ZEB1 overexpression. The anti-fibrogenic effect of sphingosine in the TNBS model was reversed by Zeb1 overexpression. Stricturing CD patients have higher circulating granulocyte-colony stimulating factor (G-CSF) levels than non-stricturing CD patients. Csf3 promoted collagen expression in CD-HIF, while Csf3 inhibition diminished cecal fibrosis in Salmonella-infected mice. Sphingosine diminished G-CSF secretion in primary human peripheral blood mononuclear cells (PBMCs). Sphingosine also inhibited cecal Csf3 mRNA expression and reduced circulating G-CSF levels in Salmonella-infected mice. CONCLUSIONS Sphingosine exerts the most potent anti-fibrogenic effects by modulating ERK1/2, ZEB1, and G-CSF expression.

Protective Effect of Saffron in Mouse Colitis Models Through Immune Modulation.

People with inflammatory bowel disease (IBD) including ulcerative colitis are at risk for colorectal cancer. Despite available effective drugs used to treat IBD, many patients fail or lose response over time with some displaying drug-induced adverse events. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD has not been explored extensively. To establish whether saffron treatment alleviates inflammation in experimental colitis. Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron doses (7.5, 15, 20, 25mg/kg body weight) or vehicle through daily gavage. On day 11, mice were euthanized and analyzed for gross and microscopic inflammation. Distal colon segments were collected for mRNA and protein expression of HO-1 protein and GPX2, (the downstream targets of NRF-2). Nrf-2 translocation from cytosol to nucleus was confirmed by immunofluorescence, and further Nrf-2 protein expression in nuclear and cytosolic fraction of colon was analyzed by immunoblot. Immune cells were isolated from the lamina propria of mouse colon for flow cytometry-based immunophenotyping. Colitis was also induced in C57BL/6 Ahr knockout and wild type mice to explore the involvement of Ahr-dependent pathways in saffron's protective effect(s). The therapeutic effect of saffron was further validated in another TNBS model of colitis. Saffron 20mg/kg body weightshowed improved colon gross and histology features and led to better body weight, colon length, histology score, and reduced disease activity index (DAI). Saffron significantly decreased pro-inflammatory macrophages (M1), while increasing anti-inflammatory macrophages (M2) and IL10 + dendritic cells. Saffron treatment also enhanced CD3 + T and CD3 + CD8 + T cells followed by increase in different CD3 + CD4 + T cells subsets like CD25 + T cells, FoxP3 + CD25 + regulatory T cells, and CD4 + FOXP3 + CD25-regulatory T cells. Immunoblot analysis showed a significant increase in HO-1/GPX2 protein expression. With saffron treatment, Nrf-2 translocation into nucleus from cytosol also supports the involvement of Nrf-2 and its downstream targets in the protective effect of saffron. Further, we demonstrated that saffron in part exert anti-inflammatory effect through activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways. These data demonstrate saffron's therapeutic potential and its protective role in part via Ahr/Nrf-2 pathways and regulatory innate and adaptive immune cells.

Leptin-resistant Zucker rats with trinitrobenzene sulfonic acid colitis present a reduced inflammatory response but enhanced epithelial damage.

The role of leptin in the development of intestinal inflammation remains controversial, since proinflammatory and anti-inflammatory effects have been described. This study describes the effect of the absence of leptin signaling in intestinal inflammation. Experimental colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to lean and obese Zucker rats (n = 10). Effects on inflammation and mucosal barrier were studied. Bacterial translocation and LPS concentration were evaluated together with colonic permeability to 4-kDa FITC-dextran. Obese Zucker rats showed a lower intestinal myeloperoxidase and alkaline phosphatase activity, reduced alkaline phosphatase sensitivity to levamisole, and diminished colonic expression of Nos2, Tnf, and Il6, indicating attenuated intestinal inflammation, associated with attenuated STAT3, AKT, and ERK signaling in the colonic tissue. S100a8 and Cxcl1 mRNA levels were maintained, suggesting that in the absence of leptin signaling neutrophil activation rather than infiltration is hampered. Despite the lower inflammatory response, leptin resistance enhanced intestinal permeability, reflecting an increased epithelial damage. This was shown by augmented LPS presence in the portal vein of colitic obese Zucker rats, associated with induction of tissue nonspecific alkaline phosphatase, LPS-binding protein, and CD14 hepatic expression (involved in LPS handling). This was linked to decreased ZO-1 immunoreactivity in tight junctions and lower occludin expression. Our results indicate that obese Zucker rats present an attenuated inflammatory response to TNBS, but increased intestinal epithelial damage allowing the passage of bacterial antigens.NEW & NOTEWORTHY Obese Zucker rats, which are resistant to leptin, exhibit a diminished inflammatory response in the trinitrobenzenesulfonic acid (TNBS) model of colitis, suggesting leptin role is proinflammatory. At the same time, obese Zucker rats present a debilitated intestinal barrier function, with increased translocation of LPS. Zucker rats present a dual response in the TNBS model of rat colitis.

Neutrophil Extracellular Traps Impair Intestinal Barrier Function during Experimental Colitis.

Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis.

The Prophylactic Use of Bovine Colostrum in a Murine Model of TNBS-Induced Colitis

Simple SummaryColostrum is the first milk secreted by the mammary glands, and it is very rich in bioactive components. Recently, the importance of bovine colostrum (BC) as a nutraceutical product has been emerging with regards to gastrointestinal diseases. One of the most widespread gastrointestinal disorders is the inflammatory bowel disease (IBD), a multifactorial chronic condition that has a powerful impact on the social life of millions of people. Because current therapy protocols neither ensure complete recovery from IBD nor are free of secondary side effects, the present study assessed the impact of a short-term prophylactic oral administration of BC in a murine model of TNBS-induced colitis. BC administration was both well tolerated and did not induce any pathological symptoms. It considerably modulated the response to inflammation through modifications of the TLR4 and cytokines gene expression profiles as well as that of the intestinal microbiota. Although further studies are needed to develop a precise therapeutic protocol of BC administration, it seems to have the potential to be used as a natural supplement in the treatment of IBD.This study investigated the effects of a short-term administration of bovine colostrum (BC) in a TNBS model of induced colitis. Colitis was induced by TNBS treatment after seven days of BC (BC group, n = 12) or saline (control group, n = 12) administration in mice. Clinical signs, histopathological characteristics, expression levels of Toll-like receptor 4 (TLR4), pro- and anti-inflammatory cytokines, and microbial composition were assessed. BC was well tolerated and did not induce any histological damage or clinical symptoms. After TNBS treatment, the BC group showed a reduction in body weight (BW) loss compared to Control (p < 0.05). Moreover, expression levels of TLR4 (p < 0.01), Interleukin-1β (IL-1β; p < 0.001), Interleukin-8 (IL-8; p < 0.001), and Interleukin-10 (IL-10; p < 0.001) were lower in mice administered with BC. Finally, Escherichia coli were higher (p < 0.05), while Enterococci (p < 0.001), Lactobacillus spp. (p < 0.001), and Bifidobacterium spp. (p < 0.05) were lower in Control than BC group. This study confirms that pre-treatment with BC modulates the expression of genes and the count of microbes involved in the etiopathogenesis of colitis.

Ephrin-B2 signaling in the spinal cord as a player in post-inflammatory and stress-induced visceral hypersensitivity.

Ephrin-B2/EphB receptor signaling contributes to persistent pain states such as postinflammatory and neuropathic pain. Visceral hypersensitivity (VHS) is a major mechanism underlying abdominal pain in patients with irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) in remission, but the underlying pathophysiology remains unclear. Here, we evaluated the spinal ephrin-B2/EphB pathway in VHS in 2 murine models of VHS, that is, postinflammatory TNBS colitis and maternal separation (MS). Wild-type (WT) mice and mice lacking ephrin-B2 in Nav 1.8 nociceptive neurons (cKO) were studied. VHS was induced by: 1. intracolonic instillation of TNBS or 2. water avoidance stress (WAS) in mice that underwent maternal separation (MS). VHS was assessed by quantifying the visceromotor response (VMRs) during colorectal distention. Colonic tissue and spinal cord were collected for histology, gene, and protein expression evaluation. In WT mice, but not cKO mice, TNBS induced VHS at day 14 after instillation, which returned to baseline perception from day 28 onwards. In MS WT mice, WAS induced VHS for up to 4weeks. In cKO however, visceral pain perception returned to basal level by week 4. The development of VHS in WT mice was associated with significant upregulation of spinal ephrin-B2 and EphB1 mRNA expression or protein levels in the TNBS model and upregulation of spinal ephrin-B2 protein in the MS model. No changes were observed in cKO mice. VHS was not associated with persistent intestinal inflammation. Overall, our data indicate that the ephrin-B2/EphB1 spinal signaling pathway is involved in VHS and may represent a novel therapeutic target.

P071 MODULATION OF THE MUCUS LAYER BY BIFIDOBACTERIUM DENTIUM PROVIDES PROTECTION IN A MODEL OF COLITIS

Abstract Background The intestinal mucus layer serves as a critical interface between the environment and the host. Patients with inflammatory bowel disease (IBD), particularly ulcerative colitis, exhibit reduced synthesis and secretion of the mucus protein MUC2 and decreased mucus thickness. This in turn promotes immune activation and inflammation. The clinical relevance of the mucus layer emphasizes the need to address strategies to modulate this barrier. Although bifidobacteria represent only 3–6% of the healthy adult fecal microbiota, their presence has been associated with numerous health benefits, including bolstering mucus production. However, the molecular mechanisms that underlie these positive effects appear to be strain-specific and are not well defined. We hypothesized that the human-derived Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion via specific metabolites. We also speculated that modulation of goblet cells would be beneficial during colitis. Methods &amp; Results In silico genome analysis revealed that B. dentium lacked the enzymatic repertoire required for degradation of mucin glycans. Consistent with these findings, we found that B. dentium could not use mucin glycans as a primary carbon source in vitro. To examine mucus modulation in vivo, germ-free mice were mono-associated with live or heat-killed B. dentium. Live B. dentium mono-associated mice exhibited increased colonic expression of goblet cell markers Krüppel-Like Factor 4 (Klf4), Relmβ, trefoil factor 3 (Tff3), Muc2, and several mucin glycosyltransferases compared to both heat-killed B. dentium and germ-free counterparts. Likewise, live B. dentium mono-associated colon had increased acidic mucin-filled goblet cells as denoted by MUC2 and PAS-AB staining. In vitro, B. dentium secreted products, including acetate, were able to increase MUC2 levels in T84 cells, mouse colonoids and human colonoids. We also identified that B. dentium secreted products, such as GABA, stimulated autophagy-mediated calcium signaling and MUC2 release. To identify whether B. dentium could enhance MUC2 production in mice harboring a complete microbiota, specific pathogen free mice were treated with live B. dentium by oral gavage. Administration of B. dentium increased the inner mucus layer compared to controls. Moreover, in a TNBS model of colitis, B. dentium treated mice had increased goblet cell numbers and MUC2 mRNA. Mirroring these findings, B. dentium treated mice lost less weight, had improved histology and had decreased levels of TNF, KC (IL-8), and IL-6. Conclusions This work illustrates that B. dentium enhances the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways with a net increase in mucin production. Ultimately, these pathways may be targeted for the development of novel therapeutics.

4-Methylesculetin, a natural coumarin with intestinal anti-inflammatory activity, elicits a glutathione antioxidant response by different mechanisms

4-methylesculetin (4 ME) is a natural antioxidant coumarin with protective effects on the intestinal inflammation, in which oxidative stress plays a key role in its aetiology and pathophysiology. Based on this, we examined the antioxidant molecular mechanisms involved in the intestinal anti-inflammatory activity of the 4 ME. For this purpose, we investigated the effects of the 4 ME on the modulation of gene expression and antioxidant-related enzyme activities in TNBS model of intestinal inflammation as well as the molecular interaction between 4 ME and glutathione reductase. Our results showed that 4 ME modulated glutathione-related enzymes, mainly increasing glutathione reductase activity. These effects were related to upregulation of glutathione reductase and Nrf2 gene expression. Fluorescence and nuclear magnetic resonance data showed that interaction between 4 ME and glutathione reductase is collisional, hydrophobic and spontaneous, in which C4 methyl group is the second epitope most buried into glutathione reductase. Molecular modelling calculation showed Lys70-B, Arg81-A, Glu381-B, Asp443-A, Ser444-A, Glu447-B and Ser475-A participated in electrostatic interaction, Lys70-B, Glu381-B and Arg81-A acted in the hydrophobic interactions and Trp73, Phe377 and Ala446 are responsible for the hydrogen bonds. Based on this, our results showed 4 ME acted by different mechanisms to control oxidative stress induced by intestinal damage, controlling the imbalance between myeloperoxidase activity and glutathione production, upregulating the glutathione S-transferase and glutathione reductase activities, preventing the Nrf2 and glutathione gene expression downregulation with consequent glutathione maintenance. Finally, 4 ME interacted at molecular level with glutathione reductase, stabilizing its enzymatic activity and reducing oxidative stress to take place in intestinal inflammatory process.

Renin-angiotensin system promotes colonic inflammation by inducing TH17 activation via JAK2/STAT pathway.

Previous studies suggest that the renin-angiotensin system (RAS) is a pathogenic factor for colitis. The goal of this study was to elucidate the molecular mechanism whereby angiotensin II (ANG II) promotes colonic inflammation. We found that renin was highly induced in colonic biopsies from patients with ulcerative colitis or Crohn's disease, and colonic renin and ANG II levels were markedly increased in a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model, indicating that the colonic RAS is activated in colitis. Renin transgenic (RenTg) mice exhibited increased phosphorylation in Janus kinase-2 (JAK2) and signal transducer and activator of transcription1/3 (STAT1/3) within colonic mucosa at baseline and following TNBS induction, suggesting that ANG II promotes colonic inflammation via the JAK2/STAT1/3 pathway. Treatment with pan-JAK inhibitor tofacitinib blocked JAK2 and STAT1/3 phosphorylation, attenuated T helper (TH)1 and TH17 responses, alleviated colitis, and prevented death of RenTg mice in TNBS model. ANG II stimulated JAK2/STAT1/3 phosphorylation in both Jurkat T lymphocytes and HCT116 epithelial cells. In vitro polarization assays demonstrated that ANG II directly promoted TH17 polarization, but not TH1 polarization, via JAK2/STAT1/3. ANG II stimulation of transforming growth factor-β1 (TGFβ1), IL-6, myosin light chain kinase, and p53 upregulated modulator of apoptosis in HCT116 cells was also mediated by JAK2/STAT1/3. These observations suggest that ANG II promotes TH17 polarization directly as well as indirectly by inducing production of TH17-polarizing cytokines (e.g., TGFβ1 and IL-6) from colonic epithelial cells, both via the JAK2/STAT pathway. Therefore, colonic RAS promotes colonic inflammation, at least in part, by stimulating TH17 activation. NEW & NOTEWORTHY This study demonstrates that the local renin-angiotensin system in the colon is activated in colitis development, which promotes mucosal T helper cell activation through the JAK2/STAT pathway. These observations provide molecular evidence that the renin-angiotensin system is a pathogenic factor for the development of inflammatory bowel diseases.

MiR-146a regulates the crosstalk between intestinal epithelial cells, microbial components and inflammatory stimuli

Regulation of miR-146a abundance and its role in intestinal inflammation and particularly in intestinal epithelial cells (IECs) has been poorly studied. Here we study the relationship between bacterial antigens and inflammatory stimuli, and miR-146a expression using IEC lines and models of colitis (trinitrobenzenesulfonic acid (TNBS), dextran sulfate sodium (DSS) and the CD4 + CD62L + T cell transfer model). Specific bacterial antigens and cytokines (LPS, flagelin and IL-1β/TNF) stimulate miR-146a expression, while peptidoglycan, muramyldipeptide and CpG DNA have no effect. Overexpression of miR-146a by LPS depends on the activation of the TLR4/MyD88/NF-kB and Akt pathways. Accordingly, the induction of miR-146a is lower in TLR4, but not in TLR2 knock out mice in both basal and colitic conditions. miR-146a overexpression in IECs induces immune tolerance, inhibiting cytokine production (MCP-1 and GROα/IL-8) in response to LPS (IEC18) or IL-1β (Caco-2). Intestinal inflammation induced by chemical damage to the epithelium (DSS and TNBS models) induces miR-146a, but no effect is observed in the lymphocyte transfer model. Finally, we found that miR-146a expression is upregulated in purified IECs from villi vs. crypts. Our results indicate that miR-146a is a key molecule in the interaction among IECs, inflammatory stimuli and the microbiota.

Pim-1 inhibitor attenuates trinitrobenzene sulphonic acid induced colitis in the mice

Pim-1 kinase has been implicated in inflammatory bowel disease (IBD). This study aimed to evaluate the application of Pim-1 inhibitor (PIM-Inh) for the treatment of IBD. Mouse model of IBD was established by the treatment with trinitrobenzene sulphonic acid (TNBS). The results showed that disease activity index score was significantly decreased, colon length was significantly increased while Wallace score and pathological score were significantly decreased after PIM-Inh treatment compared to TNBS model group. In addition, GATA3 and ROR-γt mRNA and protein levels significantly increased but Foxp3 mRNA and protein levels significantly decreased in mice with TNBS treatment compared to mice without TNBS treatment. Administration of PIM-Inh caused significant decreases in GATA3, T-bet and ROR-γt mRNA and protein levels as well as significant increases in FOXP3 mRNA and protein levels. In conclusion, our data suggest that Pim-1 kinase inhibitor could attenuate IBD by promoting T-cell differentiation into Foxp3+ regulatory T-cells and is a promising agent for IBD therapy.

Gp96 Peptide Antagonist gp96-II Confers Therapeutic Effects in Murine Intestinal Inflammation.

The expression of heat shock protein gp96 is strongly correlated with the degree of tissue inflammation in ulcerative colitis and Crohn's disease, thereby leading us to the hypothesis that inhibition of expression via gp96-II peptide prevents intestinal inflammation. We employed daily injections of gp96-II peptide in two murine models of intestinal inflammation, the first resulting from five daily injections of IL-12/IL-18, the second via a single intrarectal application of TNBS (2,4,6-trinitrobenzenesulfonic acid). We also assessed the effectiveness of gp96-II peptide in murine and human primary cell culture. In the IL-12/IL-18 model, all gp96-II peptide-treated animals survived until day 5, whereas 80% of placebo-injected animals died. gp96-II peptide reduced IL-12/IL-18-induced plasma IFNγ by 89%, IL-1β by 63%, IL-6 by 43% and tumor necrosis factor (TNF) by 70% compared to controls. The clinical assessment Disease Activity Index of intestinal inflammation severity was found to be significantly lower in the gp96-II-treated animals when compared to vehicle-injected mice. gp96-II peptide treatment in the TNBS model limited weight loss to 5% on day 7 compared with prednisolone treatment, whereas placebo-treated animals suffered a 20% weight loss. Histological disease severity was reduced equally by prednisolone (by 40%) and gp96-II peptide (35%). Mice treated with either gp96-II peptide or prednisolone exhibited improved endoscopic scores compared with vehicle-treated control mice: vascularity, fibrin, granularity, and translucency scores were reduced by up to 49% by prednisolone and by up to 30% by gp96-II peptide. In vitro, gp96-II peptide reduced TLR2-, TLR4- and IL-12/IL-18-induced cytokine expression in murine splenocytes, with declines in constitutive IL-6 (54%), lipopolysaccharide-induced TNF (48%), IL-6 (81%) and in Staphylococcus epidermidis-induced TNF (67%) and IL-6 (81%), as well as IL-12/IL-18-induced IFNγ (75%). gp96-II peptide reduced IL-1β, IL-6, TNF and GM-CSF in human peripheral blood mononuclear cells to a similar degree without affecting cell viability, whereas RANTES, IL-25 and MIF were twofold to threefold increased. gp96-II peptide protects against murine intestinal inflammation by regulating inflammation in vivo and in vitro, pointing to its promise as a novel treatment for inflammatory bowel disease.

Of mice and men: a novel dietary supplement for the treatment of ulcerative colitis.

Background:Curcumin, green tea polyphenols and selenium possess anti-inflammatory and anti-oxidant properties. Individually they have demonstrated some efficacy in animal models and human subjects with inflammatory bowel disease (IBD). To evaluate the efficacy and safety of Coltect [Curcumin (500 mg), green tea (250 mg) and selenium (100 µg)] in vivo and in patients with ulcerative colitis (UC).Methods:Each component was compared to placebo in a DSS mice colitis model. The efficacy was validated in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) rat colitis model. Twenty patients with mild-to-moderate UC received two Coltect tablets twice daily for 8 weeks. Enrollees underwent sigmoidoscopy at study entrance and closure, and physical and laboratory evaluation at baseline, 4 and 8 weeks.Results:Coltect showed a synergistic therapeutic effect in the DSS and TNBS models. Disease activity was significantly higher in the placebo versus the treated group (p < 0.05). Selenium was the more active component. The contribution of green tea was minor. In the TNBS model, the Wallace scores for macroscopic lesions were 4.8 ± 1.5 (treatment) and 8.2 ± 0.5 (placebo) (p = 0.01). In humans, Coltect was well tolerated and effective. Fourteen subjects (70%) improved: nine (45%) went into complete remission, four (20%) experienced marked improvement and one (5%) experienced moderate improvement at the end of the trial. Clinical activity index decreased significantly at 4 and 8 weeks (p < 0.001). Two patients had no change in their symptoms, and one withdrew after 4 weeks. Flare-up in four subjects caused three to withdraw from the study after less than 4 weeks. Endoscopic improvement was observed in 11 (69%) patients, and four patients (25%) achieved complete remission.Conclusions:Coltect may serve as a first-line or add-on therapy in patients with mild-to-moderate UC.