Colonic Mucosal Barrier Research Articles

Low-protein diets supplemented with casein hydrolysate favor the microbiota and enhance the mucosal humoral immunity in the colon of pigs

BackgroundHigh-protein diets can increase the colonic health risks. A moderate reduction of dietary crude-protein (CP) level can improve the colonic bacterial community and mucosal immunity of pigs. However, greatly reducing the dietary CP level, even supplemented with all amino acids (AAs), detrimentally affects the colonic health, which may be due to the lack of protein-derived peptides. Therefore, this study evaluated the effects of supplementation of casein hydrolysate (peptide source) in low-protein (LP) diets, in comparison with AAs supplementation, on the colonic microbiota, microbial metabolites and mucosal immunity in pigs, aiming to determine whether a supplementation of casein hydrolysate can improve colonic health under very LP level. Twenty-one pigs (initial BW 19.90 ± 1.00 kg, 63 ± 1 days of age) were assigned to three groups and fed with control diet (16% CP), LP diets (13% CP) supplemented with free AAs (LPA) or casein hydrolysate (LPC) for 4 weeks.ResultsCompared with control diet, LPA and LPC diet decreased the relative abundance of Streptococcus and Escherichia coli, and LPC diet further decreased the relative abundance of Proteobacteria. LPC diet also increased the relative abundance of Lactobacillus reuteri. Both LP diets decreased concentrations of ammonia and cadaverine, and LPC diet also reduced concentrations of putrescine, phenol and indole. Moreover, LPC diet increased total short-chain fatty acid concentration. In comparison with control diet, both LP diets decreased protein expressions of Toll-like receptor-4, nuclear factor-κB, interleukin-1β and tumor necrosis factor-α, and LPC diet further decreased protein expressions of nucleotide-binding oligomerization domain protein-1 and interferon-γ. LPC diet also increased protein expressions of G-protein coupled receptor-43, interleukin-4, transforming growth factor-β, immunoglobulin A and mucin-4, which are indicators for mucosal defense activity.ConclusionsThe results showed that supplementing casein hydrolysate showed beneficial effects on the colonic microbiota and mucosal immunity and barrier function in comparison with supplementing free AAs in LP diets. These findings may provide new framework for future nutritional interventions for colon health in pigs.

1539 Drug-Induced Colitis as a Side Effect of Calcitonin Gene-Related Peptide (CGRP) Antagonist for Migraines

INTRODUCTION: Drug-induced colitis is a documented though relatively uncommon side effect of several drug classes, and is under-recognized and under-reported. In May 2018, an injectable calcitonin gene-related peptide (CGRP) antagonist, erenumab, was approved by the FDA for migraine prophylaxis. In the following case, this new medication is implicated as a possible cause for severe colitis resembling an inflammatory bowel disease-like picture both clinically and endoscopically. CASE DESCRIPTION/METHODS: A 33-year-old Caucasian female presented with complaints of rectal bleeding, diarrhea, and abdominal pain 2 weeks after starting a CGRP antagonist, erenumab, for chronic refractory migraine headaches. Pertinent laboratory studies showed leukocytosis with bandemia. CT Abdomen/Pelvis showed diffuse colonic wall thickening. Stool studies did not indicate any infectious etiology and an IBD panel was negative. She underwent a colonoscopy that showed severe, deep punched out ulcerations throughout the entire colon. Biopsy showed active colitis. She had a prior colonoscopy in 2009 for work up of IBS, which was endoscopically and microscopically normal. The patient's symptoms and clinical findings resolved after discontinuing the drug. A follow up colonoscopy performed one month after discharge showed evidence of ulcer healing with no new areas of inflammation. DISCUSSION: Blocking CGRP receptors of the central nervous system may reduce headaches, however blocking those of the enteric system can promote breakdown of the colonic mucosal barrier and induce a systemic inflammatory reaction. In animal models, extensive mucosal damage was noted in the subjects receiving CGRP receptor antagonists. This case implies that certain patients may be at risk for developing mucosal barrier disruption from the blockade of the calcitonin gene-related peptide receptor with erenumab or similar drugs. Further research may be needed to elucidate risk factors that may contribute to susceptibility to these deleterious effects. Once those patients that are more at risk are identified, there may be a role for endoscopic surveillance in certain patients taking these medications. While drug-induced colitis is likely to be an uncommon manifestation of blocking CGRP, clinicians should be aware of this potential side effect of targeted therapy and counsel patients receiving this therapy.

311 Colorectal Neoplasm in Patients With Cryptogenic Pyogenic Liver Abscess, Do They Need Routine Screening Colonoscopy: A Systematic Review and Meta-Analysis

INTRODUCTION: Patients with cryptogenic pyogenic liver abscess (PLA) do not have a definite source of infection and it could be a manifestation of compromised colonic mucosal barrier due to colorectal cancer (CRC). Association of PLA and CRC is unclear. Evidence is weak and limited to small sized studies. As a result, the need for screening colonoscopy in PLA patients is subject to debate and currently not established. METHODS: We conducted a comprehensive search of multiple electronic databases and conference proceedings (from inception through January 2019) to identify studies that reported on the prevalence of CRC in PLA patients. Our goal was to evaluate the pooled rate of prevalence of CRC in patients with cryptogenic PLA. Heterogeneity was measured by I2 statistics and funnel-plot was used to assess publication bias. RESULTS: 12 studies were included in the analysis. Majority of them were from East Asian countries. 18,607 patients were diagnosed with PLA in study group and 60,130 patients were in control group. 63% were males in the age range of 56 to 94 years. 90.5% of the lesions were left sided and 93.1% were positive for Klebsiella pneumoniae. A total of 18,607 PLA patients were reported, of which 17,906 patients underwent screening colonoscopy. A total of 713 colorectal lesions were found, of which 648 were diagnosed with CRC. 60,130 patients were used as control, of which 509 patients were diagnosed with CRC. The pooled rate of prevalence of CRC was 7.9% (95% CI 5-12.1, I2 = 92.4, relative risk = 6.6) in patients with PLA, as compared to 1.2% (95% CI 0.3-5.7, I2 = 93.4) in control, with statistical significance (P = 0.001). Relative risk = 6.6. Publication bias was present. CONCLUSION: In conclusion, our meta-analysis demonstrates that patients with cryptogenic PLA have a 7-fold risk of having CRC, when compared to people without PLA. Although limited by heterogeneity, our results support the need for a screening colonoscopy in patients diagnosed with cryptogenic PLA, especially if positive for Klebsiella pneumoniae.

Effect of arabinoxylan on colonic bacterial metabolites and mucosal barrier in high-fat diet-induced rats.

The aim of this study was to demonstrate the effect of arabinoxylan on colonic mucosal barrier and metabolomic profiles in high‐fat diet‐induced rats. A total of 20 six‐week‐old male rats were arranged randomly to two groups (n = 10/group), including a high‐fat diet (HFD) group and a high‐fat supplemented with arabinoxylan diet (AXD) group. Results showed that feeding AXD reduced serum lipopolysaccharide in rats after 5 weeks. In colonic digesta, Escherichia coli population was reduced, while Lactobacillus, Bifidobacterium, and Bacteroidetes populations were increased in AXD group. Metabolomics assay found that the different abundances of 84 metabolites were observed, involving lipid, carbohydrate, and nitrogenous metabolism in colonic digesta. In colonic mucosa, AXD up‐regulated gene level of tight‐junction‐related proteins. Meanwhile, lower TNF‐α and IL‐1β levels were related to TLR4/NF‐κB/MyD88 pathway in AXD group. In conclusion, arabinoxylan could change colonic microbial metabolism and improve the colonic mucosal barrier via modulating intestinal microflora and tight junction proteins.

Musca domestica Cecropin (Mdc) Alleviates Salmonella typhimurium-Induced Colonic Mucosal Barrier Impairment: Associating With Inflammatory and Oxidative Stress Response, Tight Junction as Well as Intestinal Flora.

Salmonella typhimurium, a Gram-negative food-borne pathogen, induces impairment in intestinal mucosal barrier function frequently. The injury is related to many factors such as inflammation, oxidative stress, tight junctions and flora changes in the host intestine. Musca domestica cecropin (Mdc), a novel antimicrobial peptide containing 40 amino acids, has potential antibacterial, anti-inflammatory, and immunological functions. It remains unclear exactly whether and how Mdc reduces colonic mucosal barrier damage caused by S. typhimurium. Twenty four 6-week-old male mice were divided into four groups: normal group, control group (S. typhimurium-challenged), Mdc group, and ceftriaxone sodium group (Cs group). HE staining and transmission electron microscopy (TEM) were performed to observe the morphology of the colon tissues. Bacterial load of S. typhimurium in colon, liver and spleen were determined by bacterial plate counting. Inflammatory factors were detected by enzyme linked immunosorbent assay (ELISA). Oxidative stress levels in the colon tissues were also analyzed. Immunofluorescence analysis, RT-PCR, and Western blot were carried out to examine the levels of tight junction and inflammatory proteins. The intestinal microbiota composition was assessed via 16s rDNA sequencing. We successfully built and evaluated an S. typhimurium-infection model in mice. Morphology and microcosmic change of the colon tissues confirmed the protective qualities of Mdc. Mdc could inhibit colonic inflammation and oxidative stress. Tight junctions were improved significantly after Mdc administration. Interestingly, Mdc ameliorated intestinal flora imbalance, which may be related to the improvement of tight junction. Our results shed a new light on protective effects and mechanism of the antimicrobial peptide Mdc on colonic mucosal barrier damage caused by S. typhimurium infection. Mdc is expected to be an important candidate for S. typhimurium infection treatment.

Dopamine promotes colonic mucus secretion through dopamine D5 receptor in rats.

Dopamine regulates gastrointestinal mucosal barrier. Mucus plays important roles in the protection of intestinal mucosa. Here, the regulatory effect of dopamine on rat colonic mucus secretion was investigated. RT-PCR, immunofluorescence, Periodic Acid-Schiff reagent assay, Alcian blue-Periodic Acid-Schiff staining, and enzyme-linked immunosorbent assay were used to observe the expression of dopamine receptor and the direct effect of dopamine on the colonic mucus. Mice injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) destroying enteric dopamine (DA) neurons, rats microinjected with 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra damaging central dopaminergic neurons, and dopamine D5 receptor-downregulated transgenic mice were used to detect the effect of endogenous enteric dopamine or dopamine receptors on distal colonic mucus. Our results indicated that D5 immunoreactivity was widely distributed on the colonic goblet cells. Dopamine dose-dependently increased rat distal colonic mucus secretion in vitro. D1-like receptor antagonist SCH23390 inhibited dopamine (1 μΜ)-induced distal colonic mucus secretion. D1-like receptor agonist SKF38393 promoted mucin 2 (MUC2) secretion and increased the intracellular cAMP level of colonic mucosa. D5 receptor-downregulated transgenic mice showed a decreased colonic MUC2 content. MPTP-treated mice exhibited lower colonic dopamine content and decreased colonic mucus content. 6-OHDA rats had an increase in the dopamine content in colonic mucosa but decreases in the protein levels of D1 and D5 receptors and MUC2 content in the colonic mucosa. These findings reveal that dopamine is able to promote distal colonic mucus secretion through the D5 receptor, which provides important evidence to better understand the possible role of dopamine in the colonic mucosal barrier.

Changes of intestinal mucosal barrier in mice with chronic ulcerative colitis

Objective: To study the damage and mechanism of intestinal mucosal barrier function in mice with ulcerative colitis induced by Dextran sulphate sodium (DSS). Methods: Mice models of chronic ulcerative colitis induced by DSS were established. The mice were completely randomized into normal control group and DSS group, 25 mice in each group. The body weight and colon length of the mice were monitored. The pathological examination of colon tissue was confirmed the success of the model and assessed the integrity of the colonic mucosal barrier; Evan's Blue's intestinal permeability analysis assessed the function of colon mucosal barrier; immunofluorescence staining and Western blot were used to evaluate the expression of intestinal mucosal barrier integrity-related proteins. Results: Compared with the normal control group, the DSS group had lower body weight [(25.6±0.7)g vs (23.5±0.7)g, t=2.14, P<0.05], and the colon length was shorter [(7.3±0.4)cm vs (5.6±0.2)cm, t=3.975, P<0.001]; colonic pathological results showed that the intestinal mucosa became thinner and part of the intestinal mucosa was defective; Evan's Blue instilled into the intestinal lumen was more abundant into the intestinal mucosa, and the optical density at 620 nm (OD(620))/colon tissue weight (g) was higher [(0.11±0.01) vs (0.15±0.01), t=4.174, P<0.05]; immunofluorescence and Western blot results showed lower expression of ZO-1, Claudin-1, and F-actin in colonic mucosa. Conclusion: The structure and function of intestinal mucosal barrier in DSS-induced chronic ulcerative colitis mice is impaired.

Chemopreventive Effects of Silibinin on Colitis-Associated Tumorigenesis by Inhibiting IL-6/STAT3 Signaling Pathway.

Inflammatory bowel disease (IBD), characterized by sustained inflammation, is a latent risk factor of colon tumorigenesis. Silibinin has been reported to be anti-inflammatory and antineoplastic, but its efficacy on colitis-associated cancer (CAC) has not been reported. Interlukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) is the key signaling pathway involved in CAC. We evaluated the chemopreventive effect of silibinin on a CAC mouse model and determined its impact on IL-6/STAT3 signaling. Intestinal tumor cells (IMCE and HCT-116 cell lines) were also treated by graded concentration of silibinin, and cellular viability was determined. Silibinin (750 mg/kg/day) was administered to an azoxymethane/dextran sulfate sodium (AOM/DSS) C57BL/6 mouse model for 10 weeks by gavage. Body weight, colon length, and the amount and diameter of colon tumors were documented, respectively. Specimens were subjected to H&E staining for colitis and tumor scoring, immunohistochemical staining and terminal deoxynucleotidyl transferase dUTP nick end labeling for proliferation assessment, and immunofluorescent staining for intestinal mucosa barrier assessment. Production of inflammatory cytokines was determined by real-time PCR. IL-6/STAT3 pathway activation was evaluated through immunohistochemical staining and western blot. In the current study, silibinin significantly inhibited the viability of intestinal tumor cells. The production of inflammatory cytokines and the phosphorylation of STAT3 were both inhibited in intestinal tumor cells. Meanwhile, silibinin decreased the amount and size of tumors in AOM/DSS mice. Colitis and tumor scores were decreased accompanying with inhibition of colonic tumor cell proliferation and promotion of cellular apoptosis. Additionally, silibinin could reduce the production of inflammatory cytokines and attenuate the impairment of colonic mucosal barrier. Furthermore, STAT3 phosphorylation was significantly suppressed by silibinin. In conclusion, silibinin could protect against colitis-associated tumorigenesis in mice via inhibiting IL-6/STAT3, which showed promising chemopreventive potential of CAC.

P098 Aryl hydrocarbon receptor ameliorates intestinal barrier dysfunction in colitis by regulating the myosin light-chain kinase signalling pathway

Accumulating evidence suggests that the aryl hydrocarbon receptor (AhR) plays an important role in regulating the function of the intestinal barrier, especially the immune response, in patients with inflammatory bowel disease and in mouse models. However, whether AhR aids to protect the intestinal mucosal mechanical barrier remains unknown. In this study, we hypothesised that AhR ameliorates intestinal mucosal mechanical barrier in colitis. Experimental colitis was induced in male C57BL/6 wild-type mice and AhR knockout (AhR KO) mice using 2,4,6-trinitrobenzenesulfonic acid (TNBS). Correspondingly, the AhR agonist 6-formylindolo [3,2-b] carbazole (FICZ) and the AhR antagonist CH223191 were used to treat Caco-2 monolayers in the presence of tumour necrosis factor α (TNF-α). Colonic mucosal barrier permeability and myosin light-chain kinase (MLCK) signalling pathway were investigated both in vivo and vitro. In vivo experiments, it was found that TNBS-induced colitis was more severe in AhR KO mice than in C57BL/6 wild-type mice. The intestinal permeability was significantly higher in AhR KO mice than in wild-type mice induced by TNBS. The expression and fluorescence of tight junction protein in AhR KO mice were lower than that in wild-type mice induced by TNBS. The increased expression of MLCK and phosphorylated MLC (pMLC) indicated that this pathway was open. In cell culture experiments, treatment with FICZ caused retention of the tight junction protein, alleviated the increase of intestinal permeability, and mitigated epithelial injury. However, treatment with CH223191 facilitated the unblocking of the MLCK-pMLC signalling pathway and repressed the protein expression of tight junction in vitro. AhR plays an important role in ameliorating epithelial barrier dysfunction in colitis through regulating the MLCK signalling pathway, suggesting that AhR may be a suitable entrance of addressing this condition.

The Effect of Low Protein on Colonic Mucosal Barrier in Pigs

The Effect of Low Protein on Colonic Mucosal Barrier in Pigs

Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

Bile acids and epithelial-derived human β-defensins (HβDs) are known to be important factors in the regulation of colonic mucosal barrier function and inflammation. We hypothesized that bile acids regulate colonic HβD expression and aimed to test this by investigating the effects of deoxycholic acid (DCA) and ursodeoxycholic acid on the expression and release of HβD1 and HβD2 from colonic epithelial cells and mucosal tissues. DCA (10-150 µM) stimulated the release of both HβD1 and HβD2 from epithelial cell monolayers and human colonic mucosal tissue in vitro In contrast, ursodeoxycholic acid (50-200 µM) inhibited both basal and DCA-induced defensin release. Effects of DCA were mimicked by the Takeda GPCR 5 agonist, INT-777 (50 μM), but not by the farnesoid X receptor agonist, GW4064 (10 μM). INT-777 also stimulated colonic HβD1 and HβD2 release from wild-type, but not Takeda GPCR 5-/-, mice. DCA stimulated phosphorylation of the p65 subunit of NF-κB, an effect that was attenuated by ursodeoxycholic acid, whereas an NF-κB inhibitor, BMS-345541 (25 μM), inhibited DCA-induced HβD2, but not HβD1, release. We conclude that bile acids can differentially regulate colonic epithelial HβD expression and secretion and discuss the implications of our findings for intestinal health and disease.-Lajczak, N. K., Saint-Criq, V., O'Dwyer, A. M., Perino, A., Adorini, L., Schoonjans, K., Keely, S. J. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation.

Changes of adherens junctions between the epithelial cells of intestinal mucosal barrier in irritable bowel syndrome

Objective: To investigate whether there are changes in adherens junctions(AJ), including E-cadherin (E-cad) and β-catenin (β-cat), in the colonic mucosa of irritable bowel syndrome (IBS). Methods: Colorectal dilatation combined with restraint stress was used to establish IBS SD rat model. There were 8 rats in the model group and 8 rats in normal control group. From each rat, the colon tissue 5 centimeters away from ileocecum (I) and the junction of the rectum and sigmoid colon (R) were taken, in which E-cad and β-cat mRNA, protein level and structural changes were detected using real-time quantitative-PCR, Western blot, and immunofluorescence. According to the Rome Ⅲ criteria, IBS with diarrhea (IBS-D) patients receiving colonoscopy (n=17) and healthy volunteers (n=17) were enrolled in Center of Endoscopy of the First Affiliated Hospital of Zhejiang Chinese Medicine University. The colon tissues of I and R locations were collected from them. Colonic epithelial cell AJ protein expression and AJ structural changes in the tissues were detected using Western blot, immunofluorescence and transmission electron microscope (TEM). Results: (1)Both in I and R sites, the mRNA and protein expression of E-cad in IBS rats were both significantly lower than in normal SD rats (all P<0.05). The mRNA expression of β-cat showed no significant change in IBS SD rats compared with the normal SD rats; the protein expression of β-cat was decreased in I(P<0.05), no significantly different in R. (2)In I and R of IBS-D patients, the expression of E-cad protein were significantly decreased(I: 0.85±0.30 vs 1.24±0.34, P=0.00; R: 0.86±0.17 vs 1.14±0.48, P=0.05); the protein expression of β-cat also showed significant reduction both in I(0.85±0.39 vs 1.22±0.51, P=0.04)and R (0.92±0.22 vs 1.16±0.31, P=0.02) of IBS-D patients, compared with the healthy controls. Immunofluorescence results showed that E-cad and β-cat in colonic epithelial cells of I and R in the healthy controls were distributed along the cell membrane, demonstrating honeycomb linear fluorescence; however, in IBS-D patients, the structure of E-cad and β-cat were fuzzy, with disrupted distribution in the cytoplasm. Under TEM, the gaps of AJ in I and R of IBS-D patients appeared wider than in the healthy volunteers. Conclusions: In IBS-D patients, there are wider gaps of AJ, and decreased expression with structural disorder of E-cad and β-cat in the colon. The change of AJ plays an important role in the damage of colonic mucosal barrier and the increase of intestinal epithelial permeability in IBS-D patients, but the mechanism is yet to be further explored.

The immune-regulating effect of Xiao\u2019er Qixingcha in constipated mice induced by high-heat and high-protein diet

BackgroundXiao’er Qixingcha (EXQ) has been extensively applied to relieve dyspepsia and constipation in children for hundreds of years in China. However, the therapeutic mechanism underlying its efficacy remained to be defined. The present study aimed to clarify the possible laxative and immune-regulating effects of EXQ on two models of experimental constipation in mice, which mimicked the pediatric constipation caused by high-heat and high-protein diet (HHPD).MethodsThe two models of constipated mice were induced by HHPD or HHPD + atropine respectively. To investigate the laxative and immune-regulating activities of EXQ, animals were treated with three doses of EXQ (0.75, 1.5 and 3 g/kg) for 7 consecutive days. The fecal output parameters (number and weight), weight of intestinal content and, the thymus and spleen indexes were measured. The levels of sIgA, IL-10, TNF-α and LPS in colon and serum were determined by ELISA. Furthermore, the pathological changes of colon tissue were examined after routine H&E staining.ResultsBoth HHPD and HHPD + atropine treatments obviously inhibited the fecal output and reduced the colonic sIgA, prominently increased the levels of IL-10 and TNF-α in colonic tissue and elevated the contents of LPS in serum and colonic tissues. In contrast, oral administration of EXQ significantly improved the feces characters and dose-dependently decreased the intestinal changes in both models. In HHPD model test, EXQ efficaciously boosted the sIgA level in a dose-dependent manner, significantly elicited decreases in TNF-α and IL-10 levels, and evidently decreased the spleen and thymus indexes. In HHPD + atropine model test, EXQ treatment reversed the pathological changes by not only dramatically decreasing the spleen index and the levels of LPS and IL-10, but also markedly elevating the thymus index. Furthermore, microscopic observation revealed that EXQ treatment maintained the integrity of colonic mucosa, and protected the colonic tissues from inflammation in the both models.ConclusionsEXQ exhibited prominent laxative activity and effectively protected the colonic mucosal barrier in two models of constipated mice, of which the mechanism might be closely associated with its propulsive and immune-regulating properties. The current results not only validated the rationale for the clinical application of EXQ in pediatric constipation related symptoms, but also threw new light on the immune-inflammatory responses accompanied with chronic constipation pathology.

Constipation-Predominant Irritable Bowel Syndrome Females Have Normal Colonic Barrier and Secretory Function.

The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function. A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g 13C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression. There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of 13C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm2 vs. healthy: 29.8 (1.9) Ω cm2) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm2 vs. healthy: 17.6 (1.7) Ω cm2); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) μA cm-2) vs. healthy (56.9 (4.9) μA cm-2), P=0.05. Ach-evoked ΔIsc was similar. Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.

Histomorphological analysis of the colonic barrier in a mouse model of obesity

Background and Aim. Obesity is a metabolic disorder with an increasing incidence in Western countries and childhood. It is characterized by low-grade systemic inflammation and several comorbidities, including alterations of gastrointestinal (GI) functions, which impact negatively on patients’ quality of life. There is currently limited information on the morpho-functional features of the GI tract in obese subjects. Of note, the intestinal barrier function has been found to be altered in obese subjects, even before the occurrence of body weight increase [1]. In this light, the present study was carried out to assess, in a mouse model of diet-induced obesity, whether high fat diet (HFD) is associated with morphological alterations of the colonic mucosal barrier. Methods. C57BL/6 mice (n=5/group) were fed with standard diet (SD, 18% calories from fat) or HFD (60% calories from fat). After 8 weeks, body weight, and levels of blood cholesterol, triglycerides and glucose were evaluated. Malondialdehyde (MDA, colorimetric assay), IL-1β and IL-6 levels (ELISA assays) were examined in colonic tissues. Morphological features of colonic mucosal structures (lining epithelial cells, goblet cells, inflammatory infiltrates and enteric glia) were examined by histochemistry and immunohistochemistry. Results. HFD mice displayed significant differences at both molecular and histomorphological level, as compared with SD animals: increased body weight and blood metabolic indexes; increased MDA, IL-1β and IL-6 levels in colonic tissues; altered pattern of claudin-1 expression along with upregulation of transmembrane 16A protein and induced nitric oxide synthase in the enteric epithelium facing the lumen; increased proliferation rate of crypts; altered composition of goblet cell mucous; mucosal gliosis and infiltrates with mixed inflammatory cells. Conclusions. After 8 weeks, HFD intake led to significant alterations of systemic metabolic indexes, colonic tissue inflammation, and colonic mucosal barrier in obese mice, as compared with controls. Morphological studies can be useful to allow the characterization of histopathological patterns of colonic wall remodelling and inflammation underlying bowel motor dysfunctions associated with obesity.

Restoring Mucosal Barrier Function and Modifying Macrophage Phenotype with an Extracellular Matrix Hydrogel: Potential Therapy for Ulcerative Colitis.

Despite advances in therapeutic options, more than half of all patients with ulcerative colitis [UC] do not achieve long-term remission, many require colectomy, and the disease still has a marked negative impact on quality of life. Extracellular matrix [ECM] bioscaffolds facilitate the functional repair of many soft tissues by mechanisms that include mitigation of pro-inflammatory macrophage phenotype and mobilization of endogenous stem/progenitor cells. The aim of the present study was to determine if an ECM hydrogel therapy could influence outcomes in an inducible rodent model of UC. The dextran sodium sulphate [DSS]-colitis model was used in male Sprague Dawley rats. Animals were treated via enema with an ECM hydrogel and the severity of colitis was determined by clinical and histological criteria. Lamina propria cells were isolated and the production of inflammatory mediators was quantified. Mucosal permeability was assessed in vivo by administering TRITC-dextran and in vitro using transepithelial electrical resistance [TEER]. ECM hydrogel therapy accelerated healing and improved outcome. The hydrogel was adhesive to colonic tissue, which allowed for targeted delivery of the therapy, and resulted in a reduction in clinical and histological signs of disease. ECM hydrogel facilitated functional improvement of colonic epithelial barrier function and the resolution of the pro-inflammatory state of tissue macrophages. The present study shows that a non-surgical and non-pharmacological ECM-based therapy can abate DSS-colitis not by immunosuppression but by promoting phenotypic change in local macrophage phenotype and rapid replacement of the colonic mucosal barrier.

The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21(Cip1/Waf1), and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21(Cip1/Waf1). Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.

Rapid disruption of intestinal epithelial tight junction and barrier dysfunction by ionizing radiation in mouse colon in vivo: protection by N-acetyl-l-cysteine.

The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and β-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.

Moxibustion regulates inflammatory mediators and colonic mucosal barrier in ulcerative colitis rats.

To observe the efficacy and mechanism of grain-sized moxibustion at different acupoints in a rat model of ulcerative colitis (UC). Sprague-Dawley rats were randomly divided into control, UC model, grain-sized moxibustion at a single acupoint (CV 12), grain-sized moxibustion at two acupoints (CV 12 and CV 4), grain-sized moxibustion at three acupoints (CV 12, CV 4, and ST 36), and medication groups (n = 8/group). The UC model was established by enema of trinitrobenzene sulfonic acid. Direct moxibustion was used once a day for 7 d. Disease activity index (DAI) was evaluated before and after the treatment. Morphologic changes of intestinal tissue were observed under an optical microscope. The expression of tumor necrosis factor (TNF)-α and p38 mitogen-activated protein kinase (p38MAPK) in colonic tissue was detected using Western blot, and the levels of occludin and zonula occludens-1 (ZO-1) mRNAs were detected using reverse transcription PCR. Compared with the control group, the intestinal mucosae were incomplete in the model group, glandular structures were irregular, and submucosae were edematous, hyperemic, and infiltrated with inflammatory cells. The DAI scores and expression of TNF-α and p38MAPK were increased significantly in the model group compared to controls (Ps < 0.01), while the mRNA levels of occludin and ZO-1 were reduced significantly (Ps < 0.01). Compared with the model group, colonic mucosa and the arrangement of glands were complete and regular in the treatment groups. DAI scores and the expression of TNF-α and p38MAPK were reduced significantly in moxibustion groups compared to controls (Ps < 0.01), while the mRNA levels of occludin and ZO-1 were increased significantly (Ps < 0.01). The improvements in the above indices in the three acupoints group and the medication group were superior to those in the single and two acupoints groups (all P < 0.05). Reduction of TNF-α and p38MAPK and increased expression of occludin and ZO-1 in colonic tissue represent a potential mechanism for improved intestinal mucosal tissue repair with grain-sized moxibustion.