Colonic Barrier Integrity Research Articles

Effect and mechanism of vitamin D on the development of colorectal cancer based on intestinal flora disorder.

To investigate the correlation between the level of circulating vitamin D and the development of colorectal cancer (CRC) and to clarify the effect and mechanism of vitamin D on the development of CRC. Serum samples from 63 patients with CRC (CRC group) and 61 healthy volunteers (normal group) were collected. Azoxymethane+dextran sodium sulfate-induced CRC mouse model and dietary models with different doses of vitamin D were established to verify whether vitamin D supplementation could reverse the occurrence and development of CRC at the overall animal level. Intestinal barrier integrity and microbial defense response were evaluated by detection of intestinal flora and expression of related genes. In the clinical serum samples, compared with the normal group, the level of 25 (OH) D3 in the CRC group was relatively low (P<0.01), which was consistent with the clinical situation in mice. Vitamin D deficiency aggravated the deterioration of enteritis and intestinal cancer in CRC mice, whereas the overall condition of CRC mice improved after vitamin D supplementation. Vitamin D has a significant regulatory effect on the homeostasis of the intestinal flora, particularly in the regulation of intestinal probiotics, Akkermansia muciniphila-mediated colon barrier integrity. Vitamin D deficiency is closely related to the high incidence of CRC, and vitamin D supplementation can inhibit the occurrence and development of CRC. Vitamin D plays a role in the reversal of CRC mainly through the regulation of intestinal flora, especially the regulation of A.muciniphila-mediated colon barrier integrity.

Cannabinoids decrease intestinal permeability and induce colonic CD103+ dendritic cells to increase T regulatory cells leading to decreased murine colitis-associated colon cancer

Abstract Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are two abundant phytocannabinoids found in Cannabis sativa. Cannabinoids bind to cannabinoid receptors 1 and 2 (CB1 and CB2), located primarily on the neurons and immune cells, respectively. Cannabis and synthetic THC and CBD are quickly gaining attention for their therapeutic potential in diseases ranging from epilepsy to multiple sclerosis, and cancer pain. Colon cancer is the third leading cause of cancer-related deaths, and treatment options are limited to surgery, chemotherapy or radiation. Many physicians are now moving towards a strategy focused on prevention. Chronic inflammation is a cause of many cancers including colon cancer. Thus, we used a murine model of colitis induced colon cancer, wherein BL6 mice were given a single administration of carcinogen (azoxymethane) followed by DSS (1%) in the drinking water daily for 1 week followed by 2 weeks without DSS. This cycle, along with bi-weekly treatment with cannabinoids (CBD, THC, THC+CBD, or vehicle; all 10mg/kg; by oral gavage) continued for 10 weeks, as the progression of cancer was monitored. Our data revealed that treatment with THC and THC+CBD resulted in decreased polyp formation in mice with colon cancer. We also showed that cannabinoids increased CD103+ dendritic cells in the colonic lamina propria that led to an increase in the CD4+Nrp1+FoxP3+ T regulatory cells (nTregs), which was dependent on CB2. Cannabinoids also increased the colonic barrier integrity as assessed by increased levels of ZO-1, through CB1, and increased mucus production. These changes collectively demonstrated cannabinoid treatment reduced the incidence of colitis-associated colon cancer by inducing an anti-inflammatory response.

Metformin prevents colonic barrier dysfunction by inhibiting mast cell activation in maternal separation-induced IBS-like rats.

Intestinal barrier dysfunction is a key etiologic factor of irritable bowel syndrome (IBS). Metformin improves intestinal barrier function, although the underlying mechanism has yet to be fully explained. This study evaluates the protective effect of metformin on colonic barrier integrity and explores the underlying cellular mechanisms. IBS-like rats were induced by maternal separation. Metformin was administered daily by gavage at 08:30, and rat pups were then separated from their mother. Visceral hyperalgesia and depression-like behaviors were evaluated by colorectal distension, sucrose preference tests, and forced swimming tests. Intestinal integrity was analyzed using sugar probes and transmission electron microscopy. Inflammatory factors and the levels of corticotropin-releasing factor were assessed by PCR and ELISA. The number of mast cells was evaluated by toluidine blue staining. Protein expression and localization were determined using Western blot and immunochemistry. Metformin pretreatment (a) reduced visceral hypersensitivity to colorectal distension, immobility time and enhanced sucrose consumption; (b) decreased urine lactulose/mannitol ratio and sucralose output; (c) inhibited the dilation of tight junction and prevented claudin-4 translocation; (d) inhibited mast cell activation and downregulated the expression of IL-6, IL-18, tryptase, PAR-2, and ERK activation; (e) inhibited claudin-4 phosphorylation at serine sites and interactions between clau-4 and ZO-1. Metformin may block mast cell activation to reduce PAR-2 expression and subsequently inhibit ERK activation and clau-4 phosphorylation at serine sites to normalize the interaction of clau-4 and ZO-1 and clau-4 distribution. Metformin may be clinically beneficial for patients with IBS or IBS-like symptoms.

Effect of С60 fullerenes on the intensity of colon damage and hematological signs of ulcerative colitis in rats

The application of pristine С60 fullerene aqueous colloid solution (C60FAS; 0.5 mg/kg body weight) for rats experienced acute colitis, either intraperitoneally or intrarectally (1) restores the colonic mucosa healing and epithelial barrier integrity, evidenced by autopsies and histological findings and the normalization of phenolsufonphthalein dye daily excretion; (2) attenuates the consequences of hemorrhages, such as signs of anemia and increased platelet count; (3) improves the liver redox status suppressing the lipid peroxidation (malonic dialdehyde and protein carbonyl group levels tended down) and stimulating the antioxidant defense system (glutathione peroxidase activity grew up). In addition, C60FAS intrarectally increases the monocyte count and decreases content of neutrophil granulocytes, i.e. diminishes the rate of the inflammatory process and activates the processes of colon tissues restoring with monocytes involvement. Therefore, intrarectal administration of C60FAS may serve as an efficient tool of ulcerative colitis therapy.

Translational safety biomarkers of colonic barrier integrity in the rat.

The intestinal barrier controls intestinal permeability, and its disruption has been associated with multiple diseases. Therefore, preclinical safety biomarkers monitoring barrier integrity are essential during the development of drugs targeting the intestines, particularly if starting treatment early after onset of disease. Classical toxicology endpoints are not sensitive enough and therefore our objective was to identify non-invasive markers enabling early in vivo detection of colonic barrier perturbation. Male Sprague-Dawley rats were dosed intracolonically via the rectum, using sodium caprate or ibuprofen as tool compounds to alter barrier integrity. Several potentially translational biomarkers and probe molecules related to permeability, inflammation or tissue damage were evaluated, using various analytical platforms, including immunoassays, targeted metabolomics and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry. Several markers were identified that allow early in vivo detection of colonic barrier integrity changes, before histopathological evidence of tissue damage. The most promising permeability markers identified were plasma fluorescein isothiocyanate-dextran 4000 and a lactulose/mannitol/sucralose mixture in urine. These markers showed maximum increases over 100-fold or approximately 10-50-fold, respectively. Intracolonic administration of the above probe molecules outperformed oral administration and inflammatory or other biomarkers, such as α2 -macroglobulin, calprotectin, cytokines, prostaglandins and a panel of metabolic molecules to identify early and subtle changes in barrier integrity. However, optimal timing of probe administration and sample collection is important for all markers evaluated. Inclusion of these probe molecules in preclinical toxicity studies might aid in risk assessment and the design of a clinical biomarker plan, as several of these markers have translational potential.

Butyrate ameliorated-NLRC3 protects the intestinal barrier in a GPR43-dependent manner

BackgroundIntestinal barrier dysfunctions are related to dysbacteriosis and chronic gut inflammation in type 2 diabetes. Although there is emerging evidence that the chronic gut inflammatory response is stimulated by nucleotide-binding oligomerization domain-like receptors (NLRs), the relationship and precise mechanism between NLRC3 and the colonic epithelial barrier remains largely elusive. MethodsWe investigated the function and mechanism of NLRC3 in the colonic tissues of diabetic mice and colonic epithelial cell lines. The regulatory mechanism between NLRC3, butyrate and tight junctions was elucidated via a transepithelial electrical resistance measurement, transmission electron microscopy, RNA interference and western blotting. ResultsIn this study, we found that NLRC3 expression was decreased in the colonic tissues of diabetic mice. NLRC3 over-expression ameliorated colonic epithelial barrier integrity and up-regulated tight junction proteins in colonic epithelial cells. Knockdown of TRAF6 diminished NLRC3-induced ZO-1/occludin expression. In addition, we demonstrated that butyrate could stimulate NLRC3 expression in both diabetic mice and colonic epithelial cells. GPR43 on colonic epithelial cells is involved in the activation of NLRC3 induced by butyrate. ConclusionOur findings demonstrated that NLCR3 could ameliorate colonic epithelial barrier integrity in diabetes mellitus in a TRAF6-dependent manner, and NLCR3 was stimulated by butyrate via binding GPR43 on colonic epithelial cells.

Dietary Fermentable Fibers Attenuate Chronic Kidney Disease in Mice by Protecting the Intestinal Barrier

BackgroundChronic kidney disease (CKD) is a worldwide health problem. Although the pathogenesis of CKD is still unclear, recent studies suggest that systemic inflammation caused by a dysregulated microflora and an impaired intestinal barrier is involved in CKD development. ObjectiveWe investigated the effects of the fermentable dietary fibers (DFs), unmodified guar gum (GG), and partially hydrolyzed GG (PHGG) (i.e., substances with distinct viscosity characteristics) on CKD development, with a particular focus on colonic tight junction (TJ) barriers in mice. MethodsMale 7-wk-old ICR mice were fed an AIN-93G diet that contained 0.25% adenine for 2 wk to induce CKD. Mice fed adenine were then divided into 3 groups and fed the unsupplemented diet (CKD) or a diet containing 10% PHGG (CKD+PHGG) or GG (CKD+GG) for 3 wk. Control (CON) mice were fed an AIN-93G diet without adenine throughout the 5-wk experiment. Plasma urea concentration; the colonic TJ proteins zonula occludens (ZO) 1, ZO2, occludin, junctional adhesion molecule A (JAMA), and claudin isoforms; renal inflammatory cytokines tumor necrosis factor α (Tnfa), interleukin (Il ) 1β (Il1b), and Il6; and cecal short-chain fatty acids (SCFAs) and microflora were analyzed. ResultsCompared with the CON, CKD+PHGG, and CKD+GG groups, the CKD group had a 2.2- to 4.4-fold higher plasma urea concentration and greater expression of inflammatory cytokine genes in the kidney, including Tnfa (4.4- to 48-fold), Il1b (4.6- to 56-fold), and Il6 (8.8- to 115-fold). The CON, CKD+PHGG, and CKD+GG groups had greater expression of colonic TJ proteins including ZO1 (2.9- to 3.7-fold), ZO2 (3.4- to 4.3-fold), occludin (3.0- to 3.3-fold), JAMA (4.4- to 5.4-fold), and claudin 7 (2.1- to 2.6-fold) and higher cecal SCFA (1.8- to 3.5-fold) and Lactobacillus (2.7- to 4.0-fold) concentrations than the CKD group. ConclusionSupplemental feeding with fermentable DFs, such as GG and PHGG, might be effective for the prevention or management of CKD by restoring colonic barrier integrity and microflora composition, as shown in mice.

Colon dysregulation in methamphetamine self-administering HIV-1 transgenic rats.

The integrity and function of the gut is impaired in HIV-infected individuals, and gut pathogenesis may play a role in several HIV-associated disorders. Methamphetamine is a popular illicit drug abused by HIV-infected individuals. However, the effect of methamphetamine on the gut and its potential to exacerbate HIV-associated gut pathology is not known. To shed light on this scenario, we evaluated colon barrier pathology in a rat model of the human comorbid condition. Intestinal barrier integrity and permeability were assessed in drug-naïve Fischer 344 HIV-1 transgenic (Tg) and non-Tg rats, and in Tg and non-Tg rats instrumented with jugular cannulae trained to self-administer methamphetamine or serving as saline-yoked controls. Intestinal permeability was determined by measuring the urine content of orally gavaged sugars. Intestinal barrier integrity was evaluated by immunoblotting or immunofluorescence of colon claudin-1 and zonula occludens-1 (ZO-1), two major tight junction proteins that regulate gut epithelial paracellular permeability. Both non-Tg and Tg rats self-administered moderate amounts of methamphetamine. These amounts were sufficient to increase colon permeability, reduce protein level of claudin-1, and reduce claudin-1 and ZO-1 immunofluorescence in Tg rats relative to non-Tg rats. Methamphetamine decreased tight junction immunofluorescence in non-Tg rats, with a similar, but non-significant trend observed in Tg rats. However, the effect of methamphetamine on tight junction proteins was subthreshold to gut leakiness. These findings reveal that both HIV-1 proteins and methamphetamine alter colon barrier integrity, and indicate that the gut may be a pathogenic site for these insults.

Navy and black bean supplementation primes the colonic mucosal microenvironment to improve gut health

Common beans (Phaseolus vulgaris L.) are enriched in non-digestible fermentable carbohydrates and phenolic compounds that can modulate the colonic microenvironment (microbiota and host epithelial barrier) to improve gut health. In a comprehensive assessment of the impact of two commonly consumed bean varieties (differing in levels and types of phenolic compounds) within the colonic microenvironment, C57Bl/6 mice were fed diets supplemented with 20% cooked navy bean (NB) or black bean (BB) flours or an isocaloric basal diet control (BD) for 3 weeks. NB and BB similarly altered the fecal microbiota community structure (16S rRNA sequencing) notably by increasing the abundance of carbohydrate fermenting bacteria such as Prevotella, S24–7 and Ruminococcus flavefaciens, which coincided with enhanced short chain fatty acid (SCFA) production (microbial-derived carbohydrate fermentation products) and colonic expression of the SCFA receptors GPR-41/-43/-109a. Both NB and BB enhanced multiple aspects of mucus and epithelial barrier integrity vs. BD including: (i) goblet cell number, crypt mucus content and mucin mRNA expression, (ii) anti-microbial defenses (Reg3γ), (iii) crypt length and epithelial cell proliferation, (iv) apical junctional complex components (occludin, JAM-A, ZO-1 and E-cadherin) mRNA expression and (v) reduced serum endotoxin concentrations. Interestingly, biomarkers of colon barrier integrity (crypt height, mucus content, cell proliferation and goblet cell number) were enhanced in BB vs. NB-fed mice, suggesting added benefits attributable to unique BB components (e.g., phenolics). Overall, NB and BB improved baseline colonic microenvironment function by altering the microbial community structure and activity and promoting colon barrier integrity and function; effects which may prove beneficial in attenuating gut-associated diseases.

Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity.

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2+/+ and CerS2-/- mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2-/- mice developed more severe disease than CerS2+/+ mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2-/- mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2-/- mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2-/- mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4+ and Th17+ T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.

Fecal Supernatants from Active Ulcerative Colitis Patients Impair Colonic Epithelial Barrier Integrity and Activate Pelvic Mucosal Extrinsic Afferent Nerves

IntroductionUlcerative Colitis is characterised by colonic mucosal inflammation. Microbial dysbiosis is well documented in active UC, but little is understood regarding the physiological implications this has on epithelial barrier integrity or colonic extrinsic afferent signalling. We aimed to determine the effects of fecal supernatants from active UC on epithelial barrier and colo‐rectal mucosal extrinsic pelvic afferent nerves.MethodsExcreted fecal samples from 10 patients with active UC (endoscopic MAYO subscore ≥2) were mixed with Ringers (Ussing) or Krebs solution (electrophysiology) at 1.5gm / 3 ml, centrifuged, and the supernatant filtered and pooled. Pooled fecal supernatants (FSN) were run over a 30kDa size exclusion column. Neat, high and low molecular weight FSN were applied to CACO‐2 monolayers or healthy mouse colon tissue in Ussing chamber experiments over a 2 hour period. Extracellular electrophysiological responses from pelvic colonic extrinsic afferent nerves were recorded from healthy mice. Mucosal afferents were characterised as responding to fine mucosal stroking with 10 mg Von Frey Hair. Neat FSN was applied to mucosal afferents for 5 min and mechanical sensitivity to graded mucosal stroking with Von Frey Hair re‐assessed.ResultsNeat UC FSN potently decreased the resistance of CACO‐2 monolayers and mouse colon over a 2 hour period compared to vehicle. Low molecular weight FSN also decreased the resistance of mouse colon, yet increased the resistance of CACO‐2 monolayers. High molecular weight FSN had no effect on either mouse tissue or CACO‐2 monolayers resistance. Neat UC FSN directly activated mucosal colonic afferent endings immediately following addition, but had no effect on mechanical sensitivity to mucosal stroking.DiscussionMediators present in faecal samples impair the resistance of the epithelial barrier and directly activate mucosal afferent nerve endings. The &lt;30kDa fraction had the most potent activity on epithelial barrier. Further characterisation of the active mediator(s) and mechanisms has the potential to lead to new targets for the treatment of barrier dysfunction and nerve activation in active UC.Support or Funding InformationSupported by NHMRC Australia

Epithelial response to a high-protein diet in rat colon

BackgroundHigh-protein diets (HPD) alter the large intestine microbiota composition in association with a metabolic shift towards protein degradation. Some amino acid-derived metabolites produced by the colon bacteria are beneficial for the mucosa while others are deleterious at high concentrations. The aim of the present work was to define the colonic epithelial response to an HPD. Transcriptome profiling was performed on colonocytes of rats fed an HPD or an isocaloric normal-protein diet (NPD) for 2 weeks.ResultsThe HPD downregulated the expression of genes notably implicated in pathways related to cellular metabolism, NF-κB signaling, DNA repair, glutathione metabolism and cellular adhesion in colonocytes. In contrast, the HPD upregulated the expression of genes related to cell proliferation and chemical barrier function. These changes at the mRNA level in colonocytes were not associated with detrimental effects of the HPD on DNA integrity (comet assay), epithelium renewal (quantification of proliferation and apoptosis markers by immunohistochemistry and western blot) and colonic barrier integrity (Ussing chamber experiments).ConclusionThe modifications of the luminal environment after an HPD were associated with maintenance of the colonic homeostasis that might be the result of adaptive processes in the epithelium related to the observed transcriptional regulations.

Hydrogen sulfide improves colonic barrier integrity in DSS-induced inflammation in Caco-2 cells and mice

Intestinal barrier involves in the pathogeny of inflammatory bowel disease (IBD) and hydrogen sulfide (H2S) has been reported to improve intestinal barrier integrity. Thus, this study investigated the effects of GYY4137, a slow-release H2S donor, on DSS-induced inflammation and intestinal dysfunction. In vitro model, cellular permeability was significantly increased and expression of tight junctions (ZO-1, Cauldin4, and Occludin) was downregulated in Caco-2 cells. GYY4137 treatment markedly attenuated DSS-induced inflammation and barrier dysfunction. Cystathionine β-synthase (CBS)-siRNA transfection further demonstrated that endogenous H2S system involves in DSS-induced inflammation and mediates barrier function. In vivo model, DSS exposure caused colonic inflammation and injury in mice and GYY4137 injection alleviated inflammatory response and improved intestinal barrier via reducing intestinal permeability and upregulating of tight junctions. In conclusion, endogenous H2S system involves in DSS-induced inflammation and H2S addition alleviated inflammation and intestinal dysfunction in vitro and in vivo.

Vasoactive intestinal polypeptide promotes intestinal barrier homeostasis and protection against colitis in mice.

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis.

Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

Dietary polyphenols modulate intestinal barrier defects and inflammation in a murine model of colitis

Some polyphenols have been shown to promote the tight junction (TJ) barrier in intestinal cells. The present study investigated the ameliorative effects of polyphenols, curcumin, quercetin, naringenin or hesperetin, on experimental colitis with a particular focus on the TJ barrier in mice. Administration of dextran sulphate sodium (DSS) caused severe colon damage, indicated by body weight loss, clinical scores, and colon shortening, and TJ barrier impairment, indicated by FITC-dextran permeability. Supplemental feeding of polyphenols (0.3% (w/w)), curcumin, quercetin, naringenin or hesperetin, partially restored these symptoms although the levels of amelioration differed among the polyphenols tested. Feeding naringenin led to more effective restoration. The feeding of each polyphenol restored the expression of TJ proteins, such as zonula occludens-1, occludin, junctional adhesion molecule-A, and claudin-3, impaired by DSS administration. The colon barrier integrity correlated closely with the level of inflammation. Collectively, supplemental feeding of these polyphenols restores DSS-induced colitis, at least in part, through regulation of the colonic TJ barrier.

Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occludin and reduces fecal calprotectin: a secondary analysis of healthy Wistar Furth rats

Animal studies have demonstrated the potential of grape seed extract (GSE) to prevent metabolic syndrome, obesity, and type 2 diabetes. Recently, metabolic endotoxemia induced by bacterial endotoxins produced in the colon has emerged as a possible factor in the etiology of metabolic syndrome. Improving colonic barrier function may control endotoxemia by reducing endotoxin uptake. However, the impact of GSE on colonic barrier integrity and endotoxin uptake has not been evaluated. We performed a secondary analysis of samples collected from a chronic GSE feeding study with pharmacokinetic end points to examine potential modulation of biomarkers of colonic integrity and endotoxin uptake. We hypothesized that a secondary analysis would indicate that chronic GSE administration increases colonic expression of intestinal tight junction proteins and reduces circulating endotoxin levels, even in the absence of an obesity-promoting stimulus. Wistar Furth rats were administered drinking water containing 0.1% GSE for 21 days. Grape seed extract significantly increased the expression of gut junction protein occludin in the proximal colon and reduced fecal levels of the neutrophil protein calprotectin, compared with control. Grape seed extract did not significantly reduce serum or fecal endotoxin levels compared with control, although the variability in serum levels was widely increased by GSE. These data suggest that the improvement of gut barrier integrity and potential modulation of endotoxemia warrant investigation as a possible mechanism by which GSE prevents metabolic syndrome and associated diseases. Further investigation of this mechanism in high-fat feeding metabolic syndrome and obesity models is therefore justified.

Role of EP4 receptor and prostaglandin transporter in prostaglandin E2-induced alteration in colonic epithelial barrier integrity

Prostaglandin E(2) (PGE(2)) is a proinflammatory lipid mediator produced in excess in inflammatory bowel disease (IBD). PGE(2) couples to and signals via four different E-prostanoid (EP) receptors, namely EP1, EP2, EP3, and EP4. In this study, we determined a role for PGE(2) and EP4 receptors in altering colonic epithelial barrier integrity. In healthy colonic mucosa, EP4 receptors were localized on apical plasma membrane of epithelial cells at the tip of mucosal folds, whereas, in patients with IBD and in rats with dextran sodium sulfate (DSS)-induced colitis, they were diffusely overexpressed throughout the mucosa. Similarly, expression of EP4 receptor was polarized in T84 colonic epithelial monolayer and mimics the normal epithelium. Apical exposure of T84 monolayer with high levels of PGE(2) decreased barrier integrity, which was abrogated by an EP4 receptor antagonist. To reveal the mechanism of vectorial transport of basally produced PGE(2) toward apical EP4 receptors, we identified prostaglandin transporters (PGT) in human colonic epithelia. PGT were least expressed on epithelial cells at the colonic mucosal folds of control subjects but overexpressed in epithelial cells of patients with IBD or animals with DSS-induced colitis. T84 monolayer also expressed PGT, which increased twofold following stimulation with TNF-α. Importantly, in T84 monolayer stimulated with TNF-α, there was a corresponding increase in the uptake and vectorial transport of (3)H-PGE(2) to the apical surface. Knockdown or pharmacological inhibition of PGT significantly decreased vectorial transport of (3)H-PGE(2). These studies unravel a mechanism whereby EP4 receptor and PGT play a role in PGE(2)-induced alteration of epithelial barrier integrity in colitis.

W1695 Influence of Gastrointestinal Food Allergy On Colonic Immune Activation and Barrier Integrity in the Irritable Bowel Syndrome

Nematode infection results in a polarized Th2 cytokine response and development of alternatively activated macrophages (AAMΦ).IL-13 upregulation of 5-HT2A and PAR-2 is essential for infection-induced changes in smooth muscle hypercontractile response to 5-HT and the PAR-2 agonist, SLIGRL, respectively. Little is known, however, about the interaction between macrophages and these mediators.Aim:To determine the interaction between IL-13 and 5HT or SLIGRL on macrophage development.Methods: Macrophages were collected from uninfected C57BL/6 mice (naive) by peritoneal lavage 4 days after intraperitoneal injections of thioglycate. Macrophages were plated and exposed to vehicle, PAR-2 agonist, SLIGRL(200μM), or 5-HT(50μM)in the presence or absence of IL-13(10ng/ml)for 24 hours. Macrophages were harvested and real-time PCR was used to measure mRNA expression of specific markers. To determine the persistence of these effects we studied macrophages harvested from mice 20 weeks post H. polygyrus(Hp)infection (primed).Results:In naive macrophages, IL-13 upregulated expression of the AAMΦ marker, arginase-1 (1.0±0.1 vs 4.6±1.3 fold). Exposure of naive macrophages to SLIGRL (1.4±0.9 fold) or 5-HT (0.5±0.01) alone did not affect expression of arginase-1; however, SLIGRL markedly potentiated the effects of IL-13 on arginase-1 (4.6±1.3 vs 23.8±0.9). In macrophages harvested from previously Hp infected mice (primed), expression of AAMΦmakers was not different from control. Exposure to SLIGRL or 5-HT alone significantly upregulated by arginase1 and CD206 (table). The effect of SLIGRL was associated with increased expression of PAR-2 (1±0.9 vs 4.9±1.7 fold) as well as IL-4Rα (1±0.1 vs 2.3±0.5 fold) and IL-13Rα1 (1±0.2 vs 3.1±0.3), the receptor components for the type-2 IL-4 receptor that binds IL-13.Conclusions:During infection, exposure to PAR-2 agonists promotes the development of AAMΦ critical for the infection induced changes in smooth muscle contractility. Long lasting effects of infection include the ability of PAR-2 agonists or 5-HT to induce the AAMΦ phenotype by increasing the sensitivity of macrophages to IL-13. This may be important in the development of a Th2 memory response and the persistent effects of infection on gut function.

Unbalanced Fatty Acid Nutrition High in n‐3 Fatty Acids During Early Development Programs Intestinal Permeability and Later Susceptibility to Experimentally‐Induced Colitis in Rats

Intrauterine and early postnatal life are periods of physiological plasticity, when adverse environmental events may have long‐lasting effects. The n‐6 and n‐3 fatty acids are important membrane components, precursors for eicosanoids, and ligands for transcription factors. We investigated whether the n‐6 and n‐3 fatty acid supply in gestation and milk‐feeding had enduring effects on colonic barrier integrity and mucosal responsiveness to a chemical insult in rats. Rats were fed diets identical in all nutrients except fatty acids, with 20 % energy from safflower oil to provide high linoleic acid (LA, 18:2n‐6), canola oil with balanced LA and á‐linolenic acid (ALA, 18:3n‐3), or safflower plus fish oil to provide eicosapentaenoic acid (EPA, 20:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3) throughout gestation and lactation. Male offspring were weaned to chow, and colitis was induced with dinitrobenzene sulfonic acid at 3‐mth age. At 15 days age, colonic crypt depth was highest in the safflower, but paracellular permeability was highest in the fish oil group. Reduced colonic crypt depth and impaired paracellular permeability persisted in fish oil group at 3 mth, with more severe inflammatory response. We show a maternal diet high in n‐3, but low in n‐6 fatty acids has lasting deleterious effects on colonic barrier integrity, predisposing to an exaggerated inflammatory response in later life. Funded by NSERC.