Calcium-rich dairy matrix protects better than mineral calcium against colonic luminal haem-induced alterations in male rats.

The haemoglobin content in meat is consistently associated with an increased risk of colorectal cancer, whereas calcium may play a role as a chemopreventive agent. Using rodent models, calcium salts have been shown to prevent the promotion of haem-induced and red meat-induced colorectal carcinogenesis by limiting the bioavailability of the gut luminal haem iron. Therefore, this study aimed to compare impacts of dietary calcium provided as calcium salts or dairy matrix on gut homoeostasis perturbations by high haeminic or non-haeminic iron intakes. A 3-week intervention study was conducted using Fischer 344 rats. Compared to the ferric citrate-enriched diet, the haemoglobin-enriched diet led to increased faecal, mucosal, and urinary lipoperoxidation-related biomarkers, resulting from higher gut luminal haem iron bioavailability. This redox imbalance was associated to a dysbiosis of faecal microbiota. The addition of calcium to haemoglobin-enriched diets limited haem iron bioavailability and counteracted redox imbalance, with improved preventive efficacy when calcium was provided in dairy matrix. Data integration revealed correlations between haem-induced lipoperoxidation products and bacterial communities belonging to Peptococcaceae, Eubacterium coprostanoligenes group, and Bifidobacteriaceae. This integrated approach provides evidence of the benefits of dairy matrix as a dietary calcium vehicle to counteract the deleterious side-effects of meat consumption.

Effects of sodium nitrite reduction, removal or replacement on cured and cooked meat for microbiological growth, food safety, colon ecosystem, and colorectal carcinogenesis in Fischer 344 rats.

Epidemiological and experimental evidence indicated that processed meat consumption is associated with colorectal cancer risks. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Compared to the reference level (120 mg/kg of ham), sodium nitrite removal and reduction (90 mg/kg) similarly decreased preneoplastic lesions in F344 rats, but only reduction had an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level and an effective lipid peroxidation control. Among the three nitrite salt alternatives tested, none of them led to a significant gain when compared to the reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions in rats despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions in rats but the concomitant presence of nitrosyl iron in feces. Except the vegetable stock, other alternatives were less efficient than sodium nitrite in reducing L. monocytogenes growth.

Revisiting definition and assessment of intestinal trans-epithelial passage.

This study aims to remind that Intestinal Passage (IP) measurement is a complex task that cannot be achieved by a unique measure of an orally given exogenous marker in blood or urine. This will be illustrated in the case of NOD mice. Indeed, various methods have been proposed to measure IP. Among them ex vivo measurement in Ussing chambers of luminal to serosal fluxes of exogenous markers and in vivo measurement of exogenous markers in blood or urine after oral gavage are the more commonly used. Even though they are commonly used indifferently, they do not give the same information and can provide contradictory results. Published data showed that diabetic status in female Non Obese Diabetic (NOD) mice increased FD4 concentration in blood after gavage but did not modify FD4 fluxes in Ussing chamber. We observed the same results in our experimental conditions and tracked FD4 concentrations in blood over a kinetic study (Area Under the Curve-AUC). In vivo measurements are a dynamic process and address not only absorption (IP and intestinal surface) but also distribution, metabolism and excretion (ADME). Diabetic status in NOD mice was associated with an increase of intestinal length (absorptive surface), itself positively correlated with AUC of FD4 in blood. We concluded that increased intestinal length induced by diabetic status will extend the absorptive surface and increase FD4 concentration in plasma (in vivo measurement) despite no modification on IP of FD4 (ex vivo measurement). In addition, this study characterized intestinal function in diabetic NOD mice. Diabetic status in NOD female mice increases intestinal length and decreases paracellular IP (FSS) without affecting transcellular IP (HRP, FD4). Histological studies of small and large intestine did not show any modification of intestinal circumference nor villi and crypt size. Finally, diabetic status was not associated with intestinal inflammation (ELISA).

Anxiety is a potential effect modifier of the association between red and processed meat consumption and cancer risk: findings from the NutriNet-Santé cohort.

PURPOSE: Red and processed meats are recognized by the International Agency for Research on Cancer as probably carcinogenic and carcinogenic to humans, respectively. Heme iron has been proposed as a central factor responsible for this effect. Furthermore, anxiety affects the intestinal barrier function by increasing intestinal permeability. The objective of this work was to assess how anxiety modifies the association between red and processed meat consumption and cancer risk in the NutriNet-Santé prospective cohort (2009-2019). METHODS: Using multi-adjusted Cox models in a sample of 101,269 subjects, we studied the associations between the consumption of red and processed meat, the amount of heme iron coming from these meats and overall, colorectal, prostate, and breast cancer risks, overall and separately among participants with and without anxiety. RESULTS: An increase in red and processed meat consumption was associated with an increased risk of developing colorectal cancer in the total population (HR for an increase of 50 g/day = 1.18 (1.01-1.37), p = 0.03). After stratification on anxiety, the HR 50 g/day was 1.42 (1.03-1.94, p = 0.03) in anxious participants and 1.12 (0.94-1.33, p = 0.20) in other participants. Similar trends were observed for overall cancer risk. Analyses conducted with heme iron also provided similar results. CONCLUSIONS: Our results strengthen the existing body of evidence supporting that red and processed meat consumption and heme iron intake are associated with an increased risk of overall and more specifically colorectal cancer, and suggest that anxiety modifies these associations, with an increased risk in anxious participants.

Heme-Iron-Induced Production of 4-Hydroxynonenal in Intestinal Lumen May Have Extra-Intestinal Consequences through Protein-Adduct Formation.

Some epidemiological studies show that heme iron consumption, in red meat, is associated to the development of several chronic diseases, including cancers and cardio-metabolic diseases. As heme iron intestinal absorption is finely regulated, we hypothesized that heme iron may act indirectly, through the peroxidation of dietary lipids, in food or in the intestinal lumen during digestion. This heme-iron-induced lipid peroxidation provokes the generation of toxic lipid oxidation products that could be absorbed, such as 4-hydroxynonenal (HNE). In a first experiment, heme iron given to rats by oral gavage together with the linoleic-acid-rich safflower oil induced the formation of HNE in the intestinal lumen. The HNE major urinary metabolite was elevated in the urine of the treated rats, indicating that this compound has been absorbed. In a second experiment, we showed that stable isotope-labeled HNE given orally to rats was able to reach non-intestinal tissues as a bioactive form and to make protein-adducts in heart, liver and skeletal muscle tissues. The presence of HNE-protein adducts in those tissues suggests a putative biological role of diet-originating HNE in extra-intestinal organs. This finding could have major consequences on the onset/development of chronic diseases associated with red meat over-consumption, and more largely to peroxidation-prone food consumption.

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice.

BACKGROUND: Food-grade TiO2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice. RESULTS: The absorption of TiO2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer's patches (PP). Lower absorption occurred in the colon, while TiO2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected. CONCLUSIONS: After a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GC-associated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be major absorption routes for transepithelial uptake of dietary TiO2.

Haem iron reshapes colonic luminal environment: impact on mucosal homeostasis and microbiome through aldehyde formation.

BACKGROUND: The World Health Organization classified processed and red meat consumption as "carcinogenic" and "probably carcinogenic", respectively, to humans. Haem iron from meat plays a role in the promotion of colorectal cancer in rodent models, in association with enhanced luminal lipoperoxidation and subsequent formation of aldehydes. Here, we investigated the short-term effects of this haem-induced lipoperoxidation on mucosal and luminal gut homeostasis including microbiome in F344 male rats fed with a haem-enriched diet (1.5 µmol/g) 14-21 days. RESULTS: Changes in permeability, inflammation, and genotoxicity observed in the mucosal colonic barrier correlated with luminal haem and lipoperoxidation markers. Trapping of luminal haem-induced aldehydes normalised cellular genotoxicity, permeability, and ROS formation on a colon epithelial cell line. Addition of calcium carbonate (2%) to the haem-enriched diet allowed the luminal haem to be trapped in vivo and counteracted these haem-induced physiological traits. Similar covariations of faecal metabolites and bacterial taxa according to haem-induced lipoperoxidation were identified. CONCLUSIONS: This integrated approach provides an overview of haem-induced modulations of the main actors in the colonic barrier. All alterations were closely linked to haem-induced lipoperoxidation, which is associated with red meat-induced colorectal cancer risk.

Metabolic Effects of a Chronic Dietary Exposure to a Low-Dose Pesticide Cocktail in Mice: Sexual Dimorphism and Role of the Constitutive Androstane Receptor.

BACKGROUND: Epidemiological evidence suggests a link between pesticide exposure and the development of metabolic diseases. However, most experimental studies have evaluated the metabolic effects of pesticides using individual molecules, often at nonrelevant doses or in combination with other risk factors such as high-fat diets. OBJECTIVES: We aimed to evaluate, in mice, the metabolic consequences of chronic dietary exposure to a pesticide mixture at nontoxic doses, relevant to consumers' risk assessment. METHODS: A mixture of six pesticides commonly used in France, i.e., boscalid, captan, chlorpyrifos, thiofanate, thiacloprid, and ziram, was incorporated in a standard chow at doses exposing mice to the tolerable daily intake (TDI) of each pesticide. Wild-type (WT) and constitutive androstane receptor-deficient (CAR-/-) male and female mice were exposed for 52 wk. We assessed metabolic parameters [body weight (BW), food and water consumption, glucose tolerance, urinary metabolome] throughout the experiment. At the end of the experiment, we evaluated liver metabolism (histology, transcriptomics, metabolomics, lipidomics) and pesticide detoxification using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Compared to those fed control chow, WT male mice fed pesticide chow had greater BW gain and more adiposity. Moreover, these WT males fed pesticide chow exhibited characteristics of hepatic steatosis and glucose intolerance, which were not observed in those fed control chow. WT exposed female mice exhibited fasting hyperglycemia, higher reduced glutathione (GSH):oxidized glutathione (GSSG) liver ratio and perturbations of gut microbiota-related urinary metabolites compared to WT mice fed control chow. When we performed these experiments on CAR-/- mice, pesticide-exposed CAR-/- males did not exhibit BW gain or changes in glucose metabolism compared to the CAR-/- males fed control chow. Moreover, CAR-/- females fed pesticide chow exhibited pesticide toxicity with higher BWs and mortality rate compared to the CAR-/- females fed control chow. CONCLUSIONS: To our knowledge, we are the first to demonstrate a sexually dimorphic obesogenic and diabetogenic effect of chronic dietary exposure to a common mixture of pesticides at TDI levels, and to provide evidence for a partial role for CAR in an in vivo mouse model. This raises questions about the relevance of TDI for individual pesticides when present in a mixture. https://doi.org/10.1289/EHP2877.

Oral treatment with plecanatide or dolcanatide attenuates visceral hypersensitivity via activation of guanylate cyclase-C in rat models.

AIM: To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS: Transport of fluorescein isothiocyanate (FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1 (ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid (TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress (PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests. RESULTS: Treatment of T84 and Caco-2 monolayers with lipopolysaccharide (LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION: Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent anti-nociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.

The role of mucoprotectants in the management of gastrointestinal disorders.

INTRODUCTION: The intestinal barrier controls the absorption of nutrients and water whilst helping to prevent the entry of toxins and pathogenic micro-organisms from the lumen into the tissues. Deficiencies in the barrier are associated with various gastrointestinal and extra digestive disorders. Areas covered: This review provides an overview of the relationship between increased intestinal permeability and disease, and considers the role of mucosal protectants (mucoprotectants) in restoring normal intestinal barrier function, with a particular focus on diarrheal disorders. Expert commentary: Impairment of the intestinal barrier characterizes a variety of diseases, and there is ongoing interest in the development of pharmacological approaches targeting the reduction of intestinal permeability. These include corticosteroids, aminosalicylates and anti-tumor necrosis factor-α (TNF-α), which act by reducing inflammation; probiotics, which modulate the production of mucin and epithelial tight junction proteins; and mucoprotectants, which form a protective film over the epithelium. Recently, preclinical and clinical data highlight, the ability of new mucoprotectants, such as gelatin tannate and xyloglucan, to protect the intestinal mucosa and to exert anti-diarrheal effects. In the future the ability of these substances to enhance the intestinal barrier may extend their use in the management of a variety of gastro-intestinal diseases associated with 'leaky gut'.

A pilot study on faecal MMP-9: a new noninvasive diagnostic marker of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading malignancies worldwide, therefore cheap noninvasive screening methods are of great importance. Matrix-metalloproteinase-9 (MMP-9) has a role in the progression of CRC, and its level is elevated in tumour biopsies. Faecal MMP-9 levels are increased in active ulcerative colitis patients, but in CRC patients, they have never been measured. We aimed to assess the faecal MMP-9 levels in patients undergoing total colonoscopy according to endoscopic and histological diagnosis. METHODS: One hundred and nine patients provided faecal samples for MMP-9 analysis. A total colonoscopy was performed; suspicious lesions were evaluated by histology. Faecal MMP-9 levels were measured by ELISA. RESULTS: The number of patients allocated to different groups were: negative/diverticulosis: 34 (referred to as controls); hyperplastic polyps: 15; adenomas: 32 (22 at high risk); and CRC: 28. Faecal MMP-9 was significantly increased in CRC compared with all other groups (P<0.001). Faecal MMP-9 was suitable to distinguish CRC patients from controls (sensitivity: 89.3%; specificity: 91.2%). By means of a lower cutoff level, faecal MMP-9 identified high-risk adenomas besides CRC (sensitivity: 76%; specificity: 85.3%). This lower cutoff level screened 59% of high-risk adenomas. CONCLUSIONS: Faecal MMP-9 may be a promising new noninvasive marker in CRC.

Protective effects of ß-casofensin, a bioactive peptide from bovine ß-casein, against indomethacin-induced intestinal lesions in rats.

SCOPE: ß-casofensin, also known as peptide ß-CN(94-123), is a milk bioactive peptide that modulates the intestinal barrier through its action on goblet cells. Here, we evaluated whether oral administration of ß-casofensin can prevent indomethacin-induced injury of the jejunum in rats. METHODS AND RESULTS: Rats received ß-casofensin (0.01-100 µM) or tap water by daily gavage (4 µL/g) for eight days, then two subcutaneous injections of indomethacin (10 mg/kg, days 9 and 10) and were euthanized on day 12. In vitro, we investigated the effects of ß-casofensin on the restitution of a wounded monolayer. Preventive administration of ß-casofensin (100 µM) reduced intestinal macroscopic and microscopic damage induced by indomethacin. ß-casofensin also prevented the depletion of goblet cells and increased myeloperoxidase activity, as well as tumor necrosis factor-ɑ (TNF-ɑ) expression and immunostaining of active caspase-3 in the jejunum of rats treated with indomethacin. In wound healing experiments, ß-casofensin promoted epithelial restitution with no effect on cell proliferation. This effect was inhibited by pre-incubation with an anti-CC chemokine receptor 6 (CCR6) neutralizing antibody. CONCLUSIONS: ß-casofensin exerts protective effects in indomethacin-induced enteritis through preservation of goblet cells and improvement in wound healing. ß-casofensin could therefore become vital in nutritional programs for the prevention of intestinal diseases.

Persistent visceral allodynia in rats exposed to colorectal irradiation is reversed by mesenchymal stromal cell treatment.

Each year, millions of people worldwide are treated for primary or recurrent pelvic malignancies, involving radiotherapy in almost 50% of cases. Delayed development of visceral complications after radiotherapy is recognized in cancer survivors. Therapeutic doses of radiation may lead to the damage of healthy tissue around the tumor and abdominal pain. Because of the lack of experimental models, the underlying mechanisms of radiation-induced long-lasting visceral pain are still unknown. This makes managing radiation-induced pain difficult, and the therapeutic strategies proposed are mostly inefficient. The aim of our study was to develop an animal model of radiation-induced visceral hypersensitivity to (1) analyze some cellular and molecular mechanisms involved and (2) to test a therapeutic strategy using mesenchymal stromal cells (MSCs). Using a single 27-Grays colorectal irradiation in rats, we showed that such exposure induces a persistent visceral allodynia that is associated with an increased spinal sensitization (enhanced p-ERK neurons), colonic neuroplasticity (as increased density of substance P nerve fibers), and colonic mast cell hyperplasia and hypertrophy. Mast cell stabilization by ketotifen provided evidence of their functional involvement in radiation-induced allodynia. Finally, intravenous injection of 1.5 million MSCs, 4 weeks after irradiation, induced a time-dependent reversion of the visceral allodynia and a reduction of the number of anatomical interactions between mast cells and PGP9.5+ nerve fibers. Moreover, unlike ketotifen, MSC treatment has the key advantage to limit radiation-induced colonic ulceration. This work provides new insights into the potential use of MSCs as cellular therapy in the treatment of pelvic radiation disease.

Oral tolerance failure upon neonatal gut colonization with Escherichia coli producing the genotoxin colibactin.

The intestinal barrier controls the balance between tolerance and immunity to luminal antigens. When this finely tuned equilibrium is deregulated, inflammatory disorders can occur. There is a concomitant increase, in urban populations of developed countries, of immune-mediated diseases along with a shift in Escherichia coli population from the declining phylogenetic group A to the newly dominant group B2, including commensal strains producing a genotoxin called colibactin that massively colonized the gut of neonates. Here, we showed that mother-to-offspring early gut colonization by colibactin-producing E. coli impairs intestinal permeability and enhances the transepithelial passage of luminal antigen, leading to an increased immune activation. Functionally, this was accompanied by a dramatic increase in local and systemic immune responses against a fed antigen, decreased regulatory T cell population, tolerogenic dendritic cells, and enhanced mucosal delayed-type hypersensitivity response. Conversely, the abolition of colibactin expression by mutagenesis abrogates the alteration of oral tolerance induced by neonatal colonization by E. coli. In conclusion, the vertical colonization by E. coli producing the genotoxin colibactin enhances intestinal translocation and subsequently alters oral tolerance. Thus, early colonization by E. coli from the newly dominant phylogenetic group B2, which produces colibactin, may represent a risk factor for the development of immune-mediated diseases.

Maternally acquired genotoxic Escherichia coli alters offspring's intestinal homeostasis.

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

Multispecies probiotic protects gut barrier function in experimental models.

AIM: To investigate the effect of the probiotic combination Lactibiane Tolerance(®) (LT) on epithelial barrier function in vitro and in vivo. METHODS: The effect of the multispecies probiotic LT was assessed on several models of epithelial barrier function both in vitro (in basal and inflammatory conditions) and in vivo [visceral hypersensitivity induced by chronic stress or by colonic perfusion of a fecal supernatant (FSN) from patients with irritable bowel syndrome (IBS)]. In vitro, we measured the permeability of confluent T84 cell monolayers incubated with or without LT by evaluating the paracellular flux of macromolecules, in basal conditions and after stimulation with lipopolysaccharide (LPS) or with conditioned medium of colonic biopsies from IBS patients (IBS-CM). In vivo, male C57/Bl6 mice received orally NaCl or LT for 15 d and were submitted to water avoidance stress (WAS) before evaluating visceral sensitivity by measuring the myoelectrical activity of the abdominal muscle and the paracellular permeability with (51)Cr-EDTA. Permeability and sensitivity were also measured after colonic instillation of FSN. Tight-junctions were assessed by immunoblotting and TLR-4 expression was evaluated by immunohistochemistry RESULTS: Incubation of T84 cell monolayers with LT in basal conditions had no significant effect on permeability (P > 0.05 vs culture medium). By contrast, addition of LT bacterial bodies (LT) completely prevented the LPS-induced increase in paracellular permeability (P < 0.01 vs LPS 10 ng/mL (LPS 10); P < 0.01 vs LPS 100 ng/mL (LPS 100), P > 0.05 vs culture medium). The effect was dose dependent as addition of 10(9) LT bacterial bodies induced a stronger decrease in absorbance than 10(6) LT (10(9) LT + LPS 10: -20.1% ± 13.4, P < 0.01 vs LPS 10; 10(6) LT + LPS 10: -11.6% ± 6.2, P < 0.01 vs LPS 10; 10(9) LT + LPS 100: -14.4% ± 5.5, P < 0.01 vs LPS 100; 10(6) LT + LPS 100: -11.6% ± 7.3, P < 0.05 vs LPS 100). Moreover, the increase in paracellular permeability induced by culturing T84 cells with conditioned medium of colonic biopsies from IBS patients (IBS-CM) was completely inhibited in the presence of 10(9) LT (P < 0.01 vs IBS-CM). LT also significantly prevented the epithelial disruption induced by intracolonic infusion of fecal supernatant from IBS patients (P < 0.01 vs IBS FSN) or water avoidance stress P < 0.01 vs WAS) in C57/Bl6 mice and increased the expression of occludin in vitro and in vivo, as assessed by immnunoblotting. The WAS-induced effect on visceral sensitivity was prevented by LT treatment since values obtained for all steps of colorectal distension were significantly (P < 0.01) different from the WAS group. Finally, LT down-regulated the response mediated through TLR-4 in vitro (decrease in tumor necrosis factor α secretion in response to LPS: -65.8% for 10(9) LT and -52.5% for 10(6) LT, P < 0.01 vs LPS) and in vivo (inhibition of WAS induced an increase in TLR-4 expression in the LT treated mice colon, P < 0.01 vs WAS). CONCLUSION: The probiotic LT mix prevented the disruption to the epithelial barrier induced by LPS, stress or colonic soluble factors from IBS patients and prevented visceral hypersensitivity.

Luminal cysteine-proteases degrade colonic tight junction structure and are responsible for abdominal pain in constipation-predominant IBS.

OBJECTIVES: Luminal serine-proteases lead to increased colonic paracellular permeability and visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Other proteases, namely cysteine-proteases (CPs), increase airway permeability by digesting epithelial tight junction proteins. In this study, we focused on constipation-predominant IBS (IBS-C) and we aimed to (i) evaluate CP levels in two cohorts of IBS patients, (ii) test if IBS-C fecal supernatant (FSN) affects permeability, and visceral sensitivity after repeated administrations in mice, and (iii) evaluate occludin expression in IBS-C colonic biopsies. METHODS: Fecal CP activity was determined using selective substrate and inhibitor (E64). The effect of papain, as positive control, and IBS-C FSN administrations were evaluated on colonic paracellular permeability and mucosal occludin levels in mice and T84 monolayers. Occludin protein levels were evaluated in IBS-C colonic biopsies. Sensitivity to colorectal distension (CRD) was measured after repeated administrations of IBS-C FSN. RESULTS: We found in a subset of IBS-C patients an enhanced fecal CP activity, in comparison with healthy controls and IBS-D patients. CP activity levels positively correlated with disease severity and abdominal pain scoring. This association was confirmed by receiver operating characteristic curve analysis. In mice, repeated application of IBS-C FSN into colon triggered increased permeability, linked to the enzymatic degradation of occludin, and was associated with enhanced visceral sensitivity to CRD. Finally, occludin levels were found decreased in colonic biopsies from IBS-C patients, and IBS-C FSNs were able to degrade recombinant human occludin in vitro. All these effects were abolished by preincubation of IBS-C FSN with a CP inhibitor, E64. CONCLUSIONS: These data suggest that luminal CPs may represent a new factor contributing to the genesis of symptoms in IBS.

A new soy germ fermented ingredient displays estrogenic and protease inhibitor activities able to prevent irritable bowel syndrome-like symptoms in stressed female rats.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) often associated with psychological distress, is characterized by increased gut permeability and visceral sensitivity. In animals, stress increases intestinal paracellular permeability (IPP), visceral sensitivity and colonic proteolytic activity. Estradiol reduces IPP and affects visceral sensitivity in non-stressed ovariectomized rats, but whether estrogens affect stress-induced hyperpermeability and hypersensitivity in cyclic females remains unclear. We aimed to evaluate (i) the effects of a phytoestrogen-rich soy germ fermented ingredient (SG) on visceral hypersensitivity, hyperpermeability and other symptoms in stressed intact female rats, (ii) the mechanisms of action involved on the basis of both estrogenic and protease inhibitor activities of SG. METHODS: Female rats received orally for 15-d either SG, 17ß-estradiol benzoate (EB), or vehicles, with or without the estrogen receptor (ER) antagonist ICI182.780 before stress. Visceral sensitivity, IPP, faecal proteolytic activity, plasma corticosterone, rat mast cell protease II immunostaining, and occludin expression were assessed. RESULTS: Stress increased IPP (concomitantly to a drop in occludin expression), visceral sensitivity, faecal proteolytic activity and plasma corticosterone. Similarly to EB, SG prevented the stress-induced hyperpermeability, and hypersensitivity, without changes in plasma corticosterone. SG inhibited the increase in faecal proteolytic activity, enhanced occludin expression, and reduced the colonic mast cell density. All SG effects, except decrease on faecal proteolytic activity, were blocked by ICI182.780. CONCLUSION: A 2-wk oral treatment with SG prevented the stress-induced hyperpermeability and visceral hypersensitivity in cyclic rats through ER activation, and blocked the increase in colonic proteolytic activity, suggesting that SG can be promising in IBS management.

Fecal MMP-9: a new noninvasive differential diagnostic and activity marker in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is characterized by frequent relapses, with the presence of colorectal inflammation and mucosal lesions. Matrix-metalloprotease 9 (MMP-9) is elevated in colonic biopsies, urine, and blood plasma of UC patients. MMP-9 has been suggested as a predictor of UC in the urine of children; however, 20% of the controls tested positive. So far, fecal MMP-9 levels have never been measured. Our aims were: 1) to compare fecal MMP-9 levels in UC patients to control subjects and a functional gastrointestinal disorder characterized by diarrhea (IBS-D); 2) to test the correlation between UC disease activity and fecal levels of MMP-9; and 3) to correlate fecal MMP-9 levels with a known fecal marker of UC activity, calprotectin. METHODS: UC (n = 47), IBS-D (n = 23) patients, and control subjects (n = 24) provided fecal samples for MMP-9 analysis. In UC patients, disease severity was evaluated by the Mayo score. Fecal MMP-9 and calprotectin levels were measured by enzyme-linked immunosorbent assay and lateral flow assay, respectively. RESULTS: MMP-9 was undetectable or ≤0.22 ng/mL in the feces of all controls and IBS-D patients. In UC patients, fecal MMP-9 levels significantly correlated with the overall Mayo score (P < 0.001), the endoscopic score (P < 0.001), and the serum C-reactive protein levels (P = 0.002). Additionally, in UC patients fecal MMP-9 levels showed a significant correlation with a known disease activity marker, fecal calprotectin (P = 0.014). CONCLUSIONS: These results highlight fecal MMP-9 as a useful tool in the differential diagnosis of diarrheic disorders and in the noninvasive evaluation of disease activity and mucosal healing in UC.

A low dose of fermented soy germ alleviates gut barrier injury, hyperalgesia and faecal protease activity in a rat model of inflammatory bowel disease.

Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1ß and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD.