Query: TNF-a,caco-2,IBD

title: Ghrelin ameliorates TNF a induced anti proliferative and pro apoptotic effects and promotes intestinal epithelial restitution
abstract: TNF a is one of the major cytokine released by the invading mucosal leukocytes during acute exacerbations of inflammatory bowel disease (IBD) It interferes with the inherent restitutive potential of mucosal epithelial cells through inhibition of proliferation, cell migration and induction of cellular apoptosis. Ghrelin is a recently discovered peptide with mitogenic and anti apoptotic properties. We hypothesized that ghrelin would modulate the TNF a induced anti proliferative, anti migratory and pro apoptotic effects to enhance mucosal healing in IBD.  Non transformed and transformed intestinal epithelial cell lines (FHs74Int, IEC 6 Caco 2) were treated with TNF a, ghrelin and ghrelin receptor antagonist in the presence or absence of specific cAMP, PI3K, EGFR, ERK1/2 inhibitors. Proliferation was determined by MTT assay, apoptosis by TUNEL staining and epithelial restitution by Podolsky's cell monolayer repair assay. Western blotting was employed to assess ghrelin's cytoprotective and prolifer ative role in the presence of TNF a in relation to EGFR, PI3K/Akt, ERK1/2 pathways and caspase 3 profiling.  Ghrelin abrogates high dose TNF alpha induced anti proliferative and pro apoptotic effects (p  Ghrelin abolishes TNF a induced anti proliferative and pro apoptotic effects and promotes intestinal epithelial restitutive behavior. Therapeutic utility of ghrelin in mucosal protection and regeneration in IBD warrants further investigation.

title: Opposite Effects of Interferon Regulatory Factor 1 and Osteopontin on the Apoptosis of Epithelial Cells Induced by TNF &agr; in Inflammatory Bowel Disease
abstract: Inflammatory bowel disease (IBD) is characterized by a damaged intestinal epithelium barrier. Interferon regulatory factor 1 (IRF1) and osteopontin (OPN) regulate cell survival and growth in a variety of circumstances but their effects on the intestinal epithelium have not been elucidated. In this study, we sought to determine the effects of OPN on intestinal epithelial cells under conditions of tumor necrosis factor (TNF) &agr; induced inflammation and whether IRF1 regulates OPN expression, the activation of downstream pathways, and inflammatory responses. The expression levels of OPN and IRF1 were assessed by immunohistochemical analyses of human IBD and experimental mouse colitis. The effects of IRF1 and OPN on inflammatory responses were investigated in vitro in NCM460 and Caco 2 cells stimulated by TNF &agr; Changes in p AKT, p P38, and p ERK levels were quantified by western blotting assays. The regulation of OPN expression by IRF1 was determined by luciferase activity and chromatin immunoprecipitation assays. IRF1 was upregulated in human IBD and in the colon epithelium of mice with dextran sulfate sodium induced colitis. Additionally, IRF1 was correlated with high sensitivity C reactive protein, erythrocyte sedimentation rate, Crohn's disease activity index, Crohn's disease endoscopic index of severity, and simple endoscopic score for Crohn's disease in Crohn's disease and with high sensitivity C reactive protein, erythrocyte sedimentation rate, Mayo score, Baron score, modified Baron score, Rachmilewitz score, ulcerative colitis endoscopic index of severity, ulcerative colitis colonoscopic index of severity, and disease duration in ulcerative colitis. The expression of OPN was significantly decreased in patients with IBD compared with controls and in dextran sulfate sodium induced experimental colitis and was also inversely correlated with clinical and endoscopic activities in both Crohn's disease and ulcerative colitis. TNF &agr; treatment upregulated IRF1 and diminished OPN in both NCM460 and Caco 2 cells. The overexpression of OPN and rhOPN ameliorated the apoptosis induced by TNF &agr; whereas the overexpression of IRF1 aggravated apoptosis, indicating opposite effects of OPN and IRF1 in inflamed epithelial cells. The luciferase and chromatin immunoprecipitation assays showed that IRF1 transcriptionally modulated the expression of OPN. TNF &agr; inhibited the OPN induced upregulation of p ERK, p P38, and p AKT. Our data suggest that during intestinal inflammation, the TNF &agr; mediated activation of IRF1 is related to the subsequent suppression of OPN expression, further reducing p AKT, p P38, and p ERK activities and resulting in aggravation of the injury to intestinal epithelial cells.

title: Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway.
abstract: Intestinal epithelial barrier dysfunction, which involves myosin light chain kinase (MLCK) activation, contributes to the occurrence and progression of inflammation in inflammatory bowel disease (IBD) Wogonoside helps maintain intestinal homeostasis in mice with dextran sulfate sodium (DSS) induced colitis, but it is unclear whether it modulates intestinal barrier function.  Here, we demonstrate that wogonoside protects against intestinal barrier dysfunction in colitis via the MLCK/pMLC2 pathway both in vivo and in vitro.  Caco 2 cell monolayers treated with the proinflammatory cytokine TNF a showed barrier dysfunction and were assessed in the absence and presence of wogonoside for various physiological, morphological, and biochemical parameters. Colitis was induced by 3% DSS in mice, which were used as an animal model to explore the pharmacodynamics of wogonoside. We detected MLCK/pMLC2 pathway proteins via western blot analysis, assessed the cytokines IL 13 and IFN g via ELISA, tested bacterial translocation via fluorescence in situ hybridization (FISH) and a proper sampling of secondary lymphoid organs for bacterial culture. In addition, the docking affinity of wogonoside and MLCK was observed with DS2.5 software.  Wogonoside alleviated the disruption of transepithelial electrical resistance (TER) in TNF a exposured Caco 2 cell; FITC dextran hyperpermeability; loss of the tight junction (TJ) proteins occludin, ZO 1 and claudin 1 in Caco 2 cell monolayers; and bacterial translocation in colitic mice. Moreover, wogonoside reduced the levels of the proinflammatory cytokines IL 13 and IFN g to maintain intestinal immune homeostasis. Transmission electron microscopy (TEM) confirmed that wogonoside ameliorated the destruction of intestinal epithelial TJs. Wogonoside not only inhibited the cytoskeletal F actin rearrangement induced by TNF a, stabilized the cytoskeletal structure, suppressed MLCK protein expression, and reduced MLC2 phosphorylation. In addition, the results of molecular docking analysis showed that wogonoside had a high affinity for MLCK and formed hydrogen bonds with the amino acid residue LYS261 and p bonds with LYS229.  Collectively, our study indicates that wogonoside alleviates colitis by protecting against intestinal barrier dysfunction, and the potential mechanism may involve regulation of TJs via the MLCK/pMLC2 signaling pathway. Meanwhile, our study also explains the success of S. baicalensis in the treatment of ulcerative colitis (UC)

title: Role of microRNA 449a in the progress of inflammatory bowel disease in children
abstract: This study aimed to investigate the expression and role of microRNA 449a (miR ) in inflammatory bowel disease (IBD) in children, and further to explore the underlying mechanism. To study the role of miR 449a in IBD development, an in vitro inflammatory model of IBD was conducted by stimulating the normal intestinal epithelial cells Caco 2 with 2% dextran sulfate sodium (DSS) The results showed that the miR 449a level was significantly higher in children with IBD than that in children without IBD. Notch1 was a direct target gene of miR 449a. The increased levels of TNF a, IL 1b and IL 6, reduced cell viability and enhanced cell apoptosis in Caco 2 cells caused by DSS administration were significantly inhibited by miR 449a inhibition. Besides, DSS significantly decreased the level of Notch1, Hes 1, Hey 1 and Bcl 2, and increased Bax expression, and these changes were prevented by miR 449a inhibitor. Importantly, all the effects of miR 449a inhibitor on Caco 2 cells were eliminated by Notch1 gene silencing. In conclusion, the data of our study indicated that miR 449a was up regulated in IBD in children, and miR 449a inhibitor could inhibit inflammatory response and cell apoptosis by targeting Notch1 in IBD in vitro. The miR 449a/Notch1 axis might be a novel target for IBD treatment.

title: The protective effects of resveratrol on ulcerative colitis via changing the profile of Nrf2 and IL 1b protein.
abstract: Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) with increasing incidence and prevalence in developed countries. The presence of inflammatory cytokines is considered the main detrimental factor in severe types of IBD. The Nrf2 transcription factor plays an important role in reducing the expression of inflammatory agents such as interleukin (IL) 1b and increasing reparative factors such as IL 11. Resveratrol, a plant derived phenolic compound, reduces the damage in chronic experimentally induced colitis. Twenty patients with UC and also 20 healthy controls were recruited in this study. The proteins expression of Nrf2 and IL 1b was assessed in colonic biopsies by Western blotting. Caco 2 cells were challenged with TNF a (in vitro simulation of UC) in the presence or not of 190 nM (24 h) and 75 nM (48 h) Resveratrol. Then, Nrf2 and IL 1b in gene and protein expression were measured by real time PCR and Western blotting in different treatments. Finally, IL 11 proteins expression was measured in culture supernatant by ELISA. A significant increase of IL 1b protein was detected in inflamed colonic tissues from UC patients compared with the control individuals. In Caco 2 cells challenged with TNF a, protein expression of IL 1b and p Nrf2 showed an increase, while gene expression of Nrf2 did not show a significant difference. After treatment with Resveratrol, both IL 1b mRNA and protein levels were reduced, while IL 11 protein levels showed any increase. The p Nrf2 is a dominant form which is prevalent in inflamed tissues from UC patients. Resveratrol can reverse the inflammatory effects of TNF a by reducing IL 1b and increasing IL 11 production.

title: Anti Inflammatory Effects of Analogues of N Acyl Homoserine Lactones on Eukaryotic Cells
abstract: Since acyl homoserine lactone (AHL) profiling has been described in the gut of healthy subjects and patients with inflammatory bowel disease (IBD) the potential effects of these molecules on host cells have raised interest in the medical community. In particular, natural AHLs such as the 3 oxo C12 HSL exhibit anti inflammatory properties. Our study aimed at finding stable 3 oxo C12 HSL derived analogues with improved anti inflammatory effects on epithelial and immune cells.  We first studied the stability and biological properties of the natural 3 oxo C12 HSL on eukaryotic cells and a bacterial reporter strain. We then constructed and screened a library of 22 AHL derived molecules. Anti inflammatory effects were assessed by cytokine release in an epithelial cell model, Caco 2, and a murine macrophage cell line, RAW264.7, (respectively, IL 8 and IL 6) upon exposure to the molecule and after appropriate stimulation (respectively, TNF a 50 ng/mL and IFN g 50 ng/mL, and LPS 10 ng/mL and IFN g 20 U/mL)  We found two molecules of interest with amplified anti inflammatory effects on mammalian cells without bacterial activating properties in the reporter strain. The molecules furthermore showed improved stability in biological medium compared to the native 3 oxo C12 HSL.  We provide new bio inspired AHL analogues with strong anti inflammatory properties that will need further study from a therapeutic perspective.

title: Oleoylethanolamide Ameliorates Dextran Sulfate Sodium Induced Colitis in Rats
abstract: Oleoylethanolamide (OEA) is an endogenous fatty acid ethanolamide known for its anti inflammatory effects and its influence on gut microbiota composition; however, the effects of OEA in inflammatory bowel disease (IBD) remain unknown. During in vitro experiments, OEA downregulated the expression of tumor necrosis factor (TNF) a and reduced phosphorylation of inhibitor of kappa (Ik) Ba induced by lipopolysaccharide in human embryonic kidney cells. Moreover, OEA downregulated the expression of interleukin (IL) 8 and IL 1b and inhibited the phosphorylation of IkBa and p65 induced by TNF a in human enterocytes (Caco 2) The effect of OEA in reducing the expression of IL 8 was blocked by the peroxisome proliferator activated receptor (PPAR) a antagonist. During in vivo experiments on rats, colitis was induced by the oral administration of 8% dextran sulfate sodium from day 0 through day 5, and OEA (20 mg/kg) was intraperitoneally injected once a day from day 0 for 6 days. OEA administration significantly ameliorated the reduction in body weight, the increase in disease activity index score, and the shortening of colon length. In rectums, OEA administration reduced the infiltration of macrophages and neutrophils and tended to reduce the histological score and the expression of inflammatory cytokines. Administration of OEA produced significant improvement in a colitis model, possibly by inhibiting the nuclear factor kappa B signaling pathway through PPAR a receptors. OEA could be a potential new treatment for IBD.

title: Aryl Hydrocarbon Receptor Activation Modulates Intestinal Epithelial Barrier Function by Maintaining Tight Junction Integrity
abstract: Activation of Aryl hydrocarbon receptor (AhR) is involved in the control of intestinal mucosal homeostasis. Intestinal barrier dysfunction contributes to the development of many intestinal diseases, such as inflammatory bowel disease (IBD) In this study, we investigated the mechanisms of AhR activation in the maintenance of intestinal barrier function. Adult C57BL/6 mice were treated with dextran sulphate sodium (DSS) for 7 days, with or without 6 Formylindolo(3,2 b)carbazole (FICZ) a ligand of AhR. We found that AhR activation by FICZ attenuated the decreased TJ protein expression in the colonic mucosa of the DSS induced mice. Further, the increase of both MLC phosphorylation and MLCK expression in the mice with DSS induced colitis was also significantly inhibited by FICZ induced AhR activation. For in vitro experiments, Caco 2 cells were treated with tumour necrosis factor alpha (TNF a)/interferon gamma (IFN g) for 48 h, with or without FICZ. AhR activation prevented TNF a/IFN g induced decrease in TER and morphological disruption of the TJs in Caco 2 monolayers. It also inhibited TNF a/IFN g induced increase in MLCK expression and MLC phosphorylation by suppression of NF kB p65 signaling pathway. Thus, AhR activating factors might have potential as therapeutic agents for the treatment of patients with IBD.

title: Effects and Mechanism of Constitutive TL1A Expression on Intestinal Mucosal Barrier in DSS Induced Colitis
abstract: role of TL1A in the intestinal mucosa barrier in inflammatory bowel disease (IBD) is still unclear. This study was aimed to investigate the expression levels of tight junction protein (TJ) myosin light chain kinase (MLCK) MyD88 and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and how TL1A influences the intestinal barrier in IBD.MethodsThe mouse models of IBD were built using FMS TL1A GFP transgenic mice and wild type mice. The morphological and histopathological changes, bacterial translocation, permeability of colonic mucosa, and LPS level were assessed. Caco 2 cells were used to further investigate the association between TL1A and TNF a and LPS. The protein level and mRNA changes of TJ proteins including ZO 1, occluding, JAMA, claudin 1, claudin 2, and claudin 3 were investigated using Western blot and real time PCR. Protein changes of MLCK, MyD88 and TNF receptor associated factor 6 (TRAF6) and TNF a mRNA in the mouse colon were further assessed.ResultsThe IBD models were successfully built. Cooper HS score and histopathological score of the colon were higher in DSS/WT group than in control/WT group (P 0.05) higher in DSS/Tg group than in control/Tg group (P 0.05) and higher in DSS/Tg group than in DSS/WT group. PAS, colonic permeability of the colon, and FITC D examination showed the similar results and trends. Compared with control/WT group, the levels of TL1A and claudin 2 were higher and the levels of ZO 1, occludin, JAMA, claudin 1, and claudin 3 were lower in DSS/WT group (P 0.05) Compared with control/Tg group, the levels of TL1A and claudin 2 were higher and the levels of ZO 1, occludin, JAMA, claudin 1, and claudin 3 were lower in DSS/Tg group. Compared with Caco 2 TNF a group, the expression level of occludin and claudin 1 in Caco 2 LV TNFSF15 TNF a group was significantly lower (P 0.05) p MLC level was significantly higher. Compared with Caco 2 LPS group, the expression level of occludin and claudin 1 significantly decreased in Caco 2 LV TNFSF15 LPS group; MyD88 and TRAF6 expression level significantly increased.ConclusionThe results suggested that TL1A could impair intestinal epithelial barrier in the mouse model of IBD and might regulate TJ expression via MLCK/p MLC pathway and LPS mediated MyD88/TRAF6 pathway.

title: Implication of TNF related apoptosis inducing ligand in inflammatory intestinal epithelial lesions.
abstract: Few data exist on the molecular events causing intestinal epithelial destruction during inflammatory processes, such as inflammatory bowel disease (IBD) In this work, we analyzed the potential implication of tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) in these inflammatory lesions.  TRAIL and TRAIL receptor expression were analyzed in normal, inflammatory ileum/colon and human intestinal epithelial cell (IEC) lines (HIEC) Caco 2, and HT 29 using RNase protection assay, real time and reverse transcription polymerase chain reaction (RT PCR) immunohistochemistry, and Western blot analysis. TRAIL induced activation of NF kappaB was determined by electrophoretic mobility shift assay. Caspase recruitment domain (CARD)15 expression and interleukin (IL)8 production were studied by RT PCR and enzyme linked immunosorbent assay. Apoptosis was monitored using Annexin V/caspase 3 assays.  Normal mature IEC expressed low TRAIL levels, whereas, in inflammatory lesions, TRAIL messenger RNA and protein were markedly up regulated in IEC and lamina propria lymphocytes at levels comparable with trinitrobenzene sulfonic acid induced colitis. Interferon gamma and TNF alpha potently induced TRAIL in IEC. In vitro analyses revealed a dual biologic effect of TRAIL on HIEC: Under noninflammatory conditions, TRAIL up regulated via nuclear factor kappaB CARD15 and IL 8, whereas, under inflammatory conditions, TRAIL became a potent inducer of apoptosis in HIEC, which was confirmed ex vivo using ileal organ cultures. TNF alpha markedly increased the expression of the proapoptotic receptor TRAIL R2. TRAIL induced IEC apoptosis required a functional caspase cascade.  TRAIL is a new inflammatory mediator implicated in the homeostasis of intestinal epithelial barrier functions. TRAIL is highly up regulated in IEC in inflammatory ileum and colon. It may augment in an auto /paracrine fashion the elimination of IEC via apoptosis.