Partial Restraint Stress Research Articles

Translational evaluation of Gelsectan® effects on gut barrier dysfunction and visceral pain in animal models and irritable bowel syndrome with diarrhoea.

Gelsectan® is a formulation of xyloglucan (XG), pea protein, grape seed extract (PPGS) and xylo-oligosaccharides (XOS). Our aim was to examine the effect of Gelsectan® on rectal sensitivity in an animal model, abdominal pain in irritable bowel syndrome with diarrhoea (IBS-D) subjects and intestinal permeability in both conditions. Animals: Wistar rats received gavage with XOS, XG+PPGS or XG+PPGS+XOS, as a single dose or for 7days before a partial restraint stress (PRS). Visceromotor response to rectal distension and total gut paracellular permeability to 51Cr-EDTA were assessed. Humans: IBS-D and control patients were involved. After initial colonoscopy with biopsy sampling Gelsectan® was administered to IBS-D patients for 12weeks. Stool count and pain scores were documented. After treatment, colonoscopy was repeated. The permeability of biopsy samples was measured in Ussing-chambers. Adherent mucus layer, Muc-2 expression as well as TNFα, Interferon IFNγ were evaluated by histology/immunohistochemistry and ELISA assays, respectively. Animal studies: In control rats, PRS significantly increased visceromotor response as well as gut paracellular permeability. Single dose administration of XG+PPGS+XOS failed to reverse PRS, but 7days of oral treatment reversed PRS-induced rectal hypersensitivity and gut hyperpermeability. Human studies: Gelsectan® treatment significantly reduced and abdominal pain. Intestinal permeability in IBS-D patients was elevated compared with controls, Gelsectan® restored permeability in the ascendent colon. Periodic acid-Schiff-stained mucus layer was significantly thinner in IBS-D patients compared with controls, In both segments, mucus thickness and the proportion of Muc-2 positive cells were not affected by Gelsectan® treatment. IFNγ tissue level in the sigmoid colon shows modest mucosal inflammation in IBS-D. Gelsectan® prevented rectal hypersensitivity in rats, abdominal pain in human and intestinal hyperpermeability in rat and human studies respectively. These effects involve restoration of gut permeability. Based on this translational study, Gelsectan® can be considered as an effective therapy for IBS-D symptoms.

NHE3 inhibitor tenapanor maintains intestinal barrier function, decreases visceral hypersensitivity, and attenuates TRPV1 signaling in colonic sensory neurons.

The pathogenesis of irritable bowel syndrome (IBS) is multifactorial, characterized in part by increased intestinal permeability, and visceral hypersensitivity. Increased permeability is associated with IBS severity and abdominal pain. Tenapanor is FDA-approved for the treatment of IBS with constipation (IBS-C) and has demonstrated improvements in bowel motility and a reduction in IBS-related pain; however, the mechanism by which tenapanor mediates these functions remains unclear. Here, the effects of tenapanor on colonic pain signaling and intestinal permeability were assessed through behavioral, electrophysiological, and cell culture experiments. Intestinal motility studies in rats and humans demonstrated that tenapanor increased luminal sodium and water retention and gastrointestinal transit versus placebo. A significantly reduced visceral motor reflex (VMR) to colonic distension was observed with tenapanor treatment versus vehicle in two rat models of visceral hypersensitivity (neonatal acetic acid sensitization and partial restraint stress; both P < 0.05), returning VMR responses to that of nonsensitized controls. Whole cell voltage patch-clamp recordings of retrogradely labeled colonic dorsal root ganglia (DRG) neurons from sensitized rats found that tenapanor significantly reduced DRG neuron hyperexcitability to capsaicin versus vehicle (P < 0.05), an effect not mediated by epithelial cell secretions. Tenapanor also attenuated increases in intestinal permeability in human colon monolayer cultures caused by incubation with proinflammatory cytokines (P < 0.001) or fecal supernatants from patients with IBS-C (P < 0.005). These results support a model in which tenapanor reduces IBS-related pain by strengthening the intestinal barrier, thereby decreasing permeability to macromolecules and antigens and reducing DRG-mediated pain signaling.NEW & NOTEWORTHY A series of nonclinical experiments support the theory that tenapanor inhibits IBS-C-related pain by strengthening the intestinal barrier. Tenapanor treatment reduced visceral motor responses to nonsensitized levels in two rat models of hypersensitivity and reduced responses to capsaicin in sensitized colonic nociceptive dorsal root ganglia neurons. Intestinal permeability experiments in human colon monolayer cultures found that tenapanor attenuates increases in permeability induced by either inflammatory cytokines or fecal supernatants from patients with IBS-C.

Inhibition of Canonical Transient Receptor Potential Channels 4/5 with Highly Selective and Potent Small-Molecule HC-070 Alleviates Mechanical Hypersensitivity in Rat Models of Visceral and Neuropathic Pain.

Transient receptor potential channels C4/C5 are widely expressed in the pain pathway. Here, we studied the putative analgesic efficacy of the highly selective and potent TRPC4/C5 antagonist HC-070 in rats. Inhibitory potency on human TRPC4 was assessed by using the whole-cell manual patch-clamp technique. Visceral pain sensitivity was assessed by the colonic distension test after intra-colonic trinitrobenzene sulfonic acid injection and partial restraint stress. Mechanical pain sensitivity was assessed by the paw pressure test in the chronic constriction injury (CCI) neuropathic pain model. We confirm that HC-070 is a low nanomolar antagonist. Following single oral doses (3-30 mg/kg in male or female rats), colonic hypersensitivity was significantly and dose-dependently attenuated, even fully reversed to baseline. HC-070 also had a significant anti-hypersensitivity effect in the established phase of the CCI model. HC-070 did not have an effect on the mechanical withdrawal threshold of the non-injured paw, whereas the reference compound morphine significantly increased it. Analgesic effects are observed at unbound brain concentrations near the 50% inhibitory concentration (IC50) recorded in vitro. This suggests that analgesic effects reported here are brought about by TRPC4/C5 blocking in vivo. The results strengthen the idea that TRPC4/C5 antagonism is a novel, safe non-opioid treatment for chronic pain.

Efficacy of a Product Containing Xyloglucan and Pea Protein on Intestinal Barrier Function in a Partial Restraint Stress Animal Model.

Functional abdominal bloating and distension (FABD) are common and frequent symptoms in patients with pre-existing gastrointestinal (GI) disorders. FABD is characterized by recurrent abdominal fullness and bloating. The pathophysiology of FABD is still unclear. However, the plausible mechanisms involved are small intestinal bacterial overgrowth (SIBO), imbalance of gut microbiota, visceral hypersensitivity, intestinal permeability alteration, and disruption of intestinal barrier function. Thus, the creation of a barrier on the wall of the intestine could represent an alternative therapeutic strategy to prevent FABD. This study aimed to investigate the effect of two natural substances, Xyloglucan (XG) and Pea-protein (PP), known for their mucosal-protective properties, in an in vivo model of Partial restraint-stress (PRS). Our results showed that the pre-treatment with a product containing XG and PP in stressed-rats was able to reduce the number of abdominal contractions and visceral hypersensitivity. Moreover, XG and PP were able to reduce intestinal permeability alteration, restoring tight-junctions (TJs) expression and decreased the lactulose–mannitol ratio, a quantitative marker used to measure intestinal permeability, compared to PRS-group. In conclusion, the data obtained revealed that the product containing XG and PP was able to restore the normal intestinal-barrier function; therefore, it could be considered a therapeutic strategy to manage FABD.

Delta-Opioid Receptors Play a Role in the Control of Selected Parameters Related to Stress and Brain Plasticity Under Non-stress and/or Stress Conditions.

There is some evidence that delta-opioid receptors may be involved in the brain processes related to neuroprotection. The aim of the present studies was to test the hypothesis that endogenous opioid peptides acting via delta-opioid receptors can protect against stress-induced changes in factors related to brain plasticity and stress hormone release. Forty male adult Wistar rats were used. Half of the animals were exposed to sustained partial restraint stress (hypokinesis) lasting48h. Rats were treated with vehicle (isotonic saline) or the delta-opioid receptor antagonist naltrindole (3mg/kg/ml, s.c.) six times a day. The stressfulness of the model was confirmed by increased plasma concentrations of corticosterone and prolactin, the increase in anxiety behavior in the open field test, as well as the reduction of BrdU incorporation into newly formed DNA in the hippocampus. Treatment with naltrindole potentiated the stress-induced rise in aldosterone concentrations. The blockade of delta-opioid receptors resulted in a decrease in hippocampal BDNF gene expression independently of control or stress conditions. Treatment with naltrindole enhanced plasma concentrations of copeptin, a stable precursor of vasopressin. In conclusion, these results suggest that endogenous opioid peptides might play an inhibitory role in aldosterone release under stress conditions and in the control of vasopressin release independently of stress exposure. Endogenous opioids might stimulate hippocampal gene expression of the important neurotrophic factor BDNF via delta-opioid receptors.

The multicomponent medication Spascupreel attenuates stress-induced gut dysfunction in rats.

BackgroundIrritable bowel syndrome (IBS) is a common disorder worldwide. It is characterized by abdominal pain/discomfort and changes in bowel habits. Due to the multifactorial pathophysiology and the heterogeneity of IBS patients, appropriate treatment of IBS is still a challenge. Spascupreel (SP‐11), as a multicomponent medication, has the potential to modulate multiple pathophysiological pathways simultaneously. Therefore, the objective of the current study was to investigate the effects of oral SP‐11 treatment on stress‐induced changes of peripheral and central functions in a rat model mimicking human IBS.MethodsNaïve Wistar rats were treated with SP‐11 (0.9 tab/kg) or NaCl 0.9% by oral gavage for 4 days before 2‐hour partial restraint stress (PRS) procedure. Twenty minutes after PRS, central and peripheral stress‐induced changes affecting IBS were assessed. These include the hypothalamic‐pituitary‐adrenal (HPA) axis response through plasma ACTH and corticosterone measurements, visceral pain in response to colorectal distension, gut permeability, colonic mast cell number, and sensitization as well as gut transit time.ResultsTreatment with SP‐11 reduced the HPA axis activation in response to PRS. At the gut level, a reduction in colonic hypersensitivity to colorectal distension, a normalization of gut transit time acceleration, a reduced mast cell sensitization, and a trend toward reduced gut hyperpermeability were observed.ConclusionsThese data suggest that stress‐induced IBS signs can be reduced using SP‐11 in rats. The observed effects and the good tolerability of the drug make SP‐11 an innovative candidate in the management of IBS.

GPER‐mediated, oestrogen‐dependent visceral hypersensitivity in stressed rats is associated with mast cell tryptase and histamine expression

Visceral hypersensitivity (VH) is common in irritable bowel syndrome (IBS), and female patients are more likely to seek healthcare services for IBS-related abdominal pain. Oestrogen has been reported to mediate pain modulation via its receptor, and mast cells are known to participate in the development of visceral hypersensitivity. Our previous studies showed that the G-protein-coupled oestrogen receptor (GPER, also known as GPR30) was expressed by mast cells in human colonic tissues and was associated with IBS type and severity of visceral pain. However, whether GPER is involved in oestrogen-dependent visceral hypersensitivity via mast cell degranulation is still unknown. Rats were subjected to wrap partial restraint stress to induce visceral hypersensitivity and were ovariectomized (OVX) to eliminate the effects of oestrogen on visceral hypersensitivity. OVX rats were treated with oestrogen, an oestrogen receptor α and β antagonist (ICI 182.780), a GPER antagonist (G15) or a GPER agonist (G1), to evaluate the effects of oestrogen via its receptor. The colorectal distention test was performed to assess visceral sensitivity. Immunofluorescence studies were performed to evaluate GPER and mast cell tryptase co-expression. Mast cell number with degranulation was detected by specific staining. Mast cell tryptase expression in rat colon was also investigated by Western blot and immunohistochemistry. Substance P and histamine expression were examined by ELISA. GPER was expressed by the majority of tryptase-positive mast cells in the colonic mucosa. Stressed rats showed increased visceral sensitivity, increased mast cell degranulation, mast cell tryptase expression, and increased colon histamine levels. Ovariectomy reduced stress-induced VH in female rats and decreased mast cell degranulation, mast cell tryptase expression, and histamine levels, whereas oestrogen replacement reversed these effects. In OVX rats, the GPER antagonist G15 counteracted the enhancing effects of oestrogen on stress-induced VH, mast cell degranulation, mast cell tryptase, and histamine expression, whereas VH was preserved after treatment with ICI 182.780. On the other hand, pretreatment with the selective GPER agonist G1 at doses between 1 and 20μg/kg significantly increased VH, mast cell tryptase, and histamine expression in OVX-stressed rats, mimicking the effects of oestrogen. GPER plays a pivotal role in the regulation of mast cell degranulation, mast cell tryptase expression, and histamine levels and contributes to the development of colonic hypersensitivity in a female rat model of IBS.

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

AIMTo investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity.METHODSTransport 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.RESULTSTreatment 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.CONCLUSIONOur 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.

Oral probiotic treatment ofLactobacillus rhamnosusLcr35®prevents visceral hypersensitivity to a colonic inflammation and an acute psychological stress

This study evaluated the efficacy of a repeated oral treatment with two active pharmaceutical ingredients (Lcr Lenio® and Lcr Restituo® ) derivated from the probiotic bacterial strain Lactobacillus rhamnosus Lcr35® in two animal models mimicking different features of irritable bowel syndrome (IBS). IBS is characterized by visceral pain associated with alteration of bowel transit. IBS patients present visceral hypersensitivity with peripheral and central origins. The injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the proximal colon as well as an acute partial restraint stress (PRS) produces colonic hypersensitivity measured in conscious rats by a decrease in pain threshold in response to distal colonic distension. Visceral hypersensitivity was produced by injection of TNBS 7days before colonic distension or by acute PRS on testing day. Treatments were performed once a day during eight consecutive days. This study indicates that an 8-day probiotic treatment (Lcr Lenio and Lcr Restituo) produces an antihypersensitivity activity in both TNBS and PRS visceral pain models. As this probiotic strain attenuates peripherally and centrally induced visceral hypersensitivity in rats, it may be active in treatment of IBS symptoms. An immunomodulatory effect of the probiotics was highlighted in the TNBS model on the IL-23 secretion, suggesting a mechanism of action involving a regulation of the local IL-23/Th17 immune activation. Two formulas of Lcr35® probiotic strain show very encouraging results for the treatment of IBS patients. Further studies are needed to better understand the role and mechanisms of probiotics on the pathogenesis of IBS.

Changes of excitatory and inhibitory neurotransmitters in the colon of rats underwent to the wrap partial restraint stress.

Animal models proposed to reproduce some of the human irritable bowel syndrome (IBS) symptoms are based on the hypothesis that psychosocial stressors play a pivotal role in the IBS etio-pathology. We investigated the wrap restraint stress (WRS) model with the aim to analyze the morphological changes of the entire colonic wall of these animals that showed some of the human IBS symptoms such as visceral hypersensitivity. Male Wistar rats were used and WRS was maintained for 2 h. Abdominal contractions (AC) were recorded in the colon-rectum by balloon distension. Fecal pellets were quantitated. Colonic specimens were examined by routine histology, immunohistochemistry and western blot. WRS animals were characterized by: (i) increase in AC number and fecal pellets mean weight; (ii) clusters of mononucleated cells, increase in eosinophilic granulocytes and mast cells in the mucosa; (iii) increase in CGRP-immunoreactive (IR) nerve fibers in the lamina propria; (iv) decrease in myenteric NK1r-IR and nNOS-IR neurons and in submucous nNOS-IR neurons; (v) decrease in SP-IR nerve fibers in the muscle wall; (vi) reduction in S100β-IR glia in the entire colonic wall; (vii) increase in CRF1r-IR myenteric neurons; (viii) no change in ChAT-IR neurons, smooth muscle cells and interstitial cells of Cajal. The present results support the consistency of the WRS as a potential model where part of the human IBS signs and symptoms are reproduced. The changes in glial cells and in excitatory and inhibitory neurotransmitters might represent the substrate for the dysmotility and hypersensitivity.

Anti-nociceptive effect of Faecalibacterium prausnitzii in non-inflammatory IBS-like models.

Visceral pain and intestinal dysbiosis are associated with Irritable Bowel Syndrome (IBS), a common functional gastrointestinal disorder without available efficient therapies. In this study, a decrease of Faecalibacterium prausnitzii presence has been observed in an IBS-like rodent model induced by a neonatal maternal separation (NMS) stress. Moreover, it was investigated whether F. prausnitzii may have an impact on colonic sensitivity. The A2-165 reference strain, but not its supernatant, significantly decreased colonic hypersensitivity induced by either NMS in mice or partial restraint stress in rats. This effect was associated with a reinforcement of intestinal epithelial barrier. Thus, F. prausnitzii exhibits anti-nociceptive properties, indicating its potential to treat abdominal pain in IBS patients.

Endogenous CRF in rat large intestine mediates motor and secretory responses to stress.

Corticotropin-releasing factor (CRF) mediates our body's overall responses to stress. The role of central CRF in stress-stimulated colonic motility is well characterized. We hypothesized that transient perturbation in expression of enteric CRF is sufficient to change stress-induced colonic motor and secretory responses. Sprague-Dawley rats (adult, male) were subjected to 1-h partial restraint stress (PRS) and euthanized at 0, 4, 8, and 24h. CRF mRNA and peptide levels in the colon were quantified by real-time RT-PCR, enzyme immuno-assay and immunohistochemistry. Double-stranded RNA (dsRNA) designed to target CRF (dsCRF) was injected into the colonic wall to attain RNA interference-mediated inhibition of CRF mRNA expression. DsRNA for β-globin was used as a control (dsControl). Four days after dsRNA injection, rats were subjected to 1-h PRS. Fecal output was measured. Ussing chamber techniques were used to assess colonic mucosal ion secretion and transepithelial tissue conductance. Exposure to PRS elevated CRF expression and increased CRF release in the rat colon. Injection of dsCRF inhibited basal CRF expression and prevented the PRS-induced increase in CRF expression, whereas CRF expression in dsControl-injected colons remained high after PRS. In rats treated with dsControl, PRS caused a significant increase in fecal pellet output, colonic baseline ion secretion, and transepithelial tissue conductance. Inhibition of CRF expression in the colon prevented PRS-induced increase in fecal output, baseline ion secretion, and transepithelial tissue conductance. These results provide direct evidence that transient perturbation in peripherally expressed CRF prevents colonic responses to stress.

Role of insular cortex in visceral hypersensitivity model in rats subjected to chronic stress

Abnormal processing of visceral sensation at the level of the central nervous system has been proven to be important in the pathophysiologic mechanisms of stress related functional gastrointestinal disorders. However, the specific mechanism is still not clear. The insular cortex (IC) was considered as one important visceral sensory area. Moreover, the IC has been shown to be involved in various neuropsychiatric diseases such as panic disorders and post-traumatic stress disorder. However, whether the IC is important in psychological stress related visceral hypersensitivity has not been studied yet. In our study, through destruction of the bilateral IC, we explored whether the IC played a critical role in the formation of visceral hypersensitivity induced by chronic stress on rats. Chronic partial restraint stress was used to establish viscerally hypersensitive rat model. Bilateral IC lesions were generated by N-methyl-D-day (door) aspartate. After a recovery period of 7 days, 14-day consecutive restraint stress was performed. The visceromotor response to colorectal distension was monitored by recording electromyogram to measure rats׳ visceral sensitivity. We found that bilateral insular cortex lesion could markedly inhibit the formation of visceral hypersensitivity induced by chronic stress. The insular cortex plays a critical role in the pathophysiology of stress-related visceral hypersensitivity.

Tu1969 Probiotic (Probio'Stick®) Attenuates Hypothalamic-Pituitary-Adrenal (HPA) Axis Response to Chronic Psychological Stress Through an Improvement of Cellular Proliferation and Changes Gene Expression in the Brain Mice

BACKGROUND: Irritable Bowel Syndrome (IBS) is a common disorder characterized by visceral hypersensitivity and increased intestinal permeability. Abnormalities of gut flora as well as gut immune cells subtype variation have also been observed in IBS. Stress events can affect the onset and course of IBS. Clinical trials showed a beneficial effect of fermented milk (FM) containing Bifidobacterium lactis CNCM-I2494 in constipation predominant IBS (IBS-C) subjects. Recently, we reported that this FM product, dose dependently suppresses visceral hypersensitivity by normalizing intestinal epithelial barrier dysfunctions induced by acute stress in rats. Beside it has been shown that the relief of IBS symptoms by Bifidobacterium. infantis 35624 is associated with normalization in the ratio of proinflammatory/antiinflammatory cytokines IL-12/IL10. Therefore, the aim of this study was to investigate the modulatory effect of FM product containing Bifidobacterium lactis CNCM-I2494 on gut immune barrier impairment promoted by an acute stress in rats. MATERIAL ANDMETHODS: Wistar rats (200-250g) were orally given FM product containing Bifidobacterium lactis CNCM-I2494 for 15 days (1 mL/rat-corresponding to 108 colony forming units of Bifidobacterium lactis CNCM I-2494) or saline. At day 15, paracellular permeability to dextran-FITC (4kD), and transmucosal flux of the protein antigen, horseradish peroxidase 40kD (HRP), were measured in Ussing chamber at different gut levels (ileal Peyer's patches, ileal and colonic segments) in both basal and after 2h of partial restraint stress (PRS) session. Moreover, IL10 and IL12 levels were measured by biochemistry assay on Peyer's patches. RESULTS: In saline treated animals, compared to basal condition, acute stress significantly increased dextran-FITC flux in Peyer's patches and in colonic segment and also substantially increased HRP flux in all gut segments tested as well as in Peyer's patches. FM consumption did not affect the in vitro measured intestinal permeability in basal condition but reduced the increase of both dextran-FITC and HRP flux induced by acute stress. Moreover, in comparison with basal condition, acute stress decreased the ratio of IL10/IL12 in ileal Peyer's patches, illustrating a pro-inflammatory effect. All these stress immune changes were significantly counteracted by 15 days of FM consumption. CONCLUSIONS: These data show in rats, that the consumption of a FM containing Bifidobacterium lactis CNCM-I2494 i) not only reduces the increased gut permeability function but also normalize local immune platform permeability (ileal Payer' patches) altered by acute stress; ii) protects the intestinal immunity barrier by modulating cytokines levels towards an anti-inflammatory profile.

Tu1967 Microbiome Alterations of Lactose Intolerant Individuals in Response to Dietary Intervention With Galacto-Oligosaccharides May Help Negate Symptoms of Lactose Intolerance

BACKGROUND: Irritable Bowel Syndrome (IBS) is a common disorder characterized by visceral hypersensitivity and increased intestinal permeability. Abnormalities of gut flora as well as gut immune cells subtype variation have also been observed in IBS. Stress events can affect the onset and course of IBS. Clinical trials showed a beneficial effect of fermented milk (FM) containing Bifidobacterium lactis CNCM-I2494 in constipation predominant IBS (IBS-C) subjects. Recently, we reported that this FM product, dose dependently suppresses visceral hypersensitivity by normalizing intestinal epithelial barrier dysfunctions induced by acute stress in rats. Beside it has been shown that the relief of IBS symptoms by Bifidobacterium. infantis 35624 is associated with normalization in the ratio of proinflammatory/antiinflammatory cytokines IL-12/IL10. Therefore, the aim of this study was to investigate the modulatory effect of FM product containing Bifidobacterium lactis CNCM-I2494 on gut immune barrier impairment promoted by an acute stress in rats. MATERIAL ANDMETHODS: Wistar rats (200-250g) were orally given FM product containing Bifidobacterium lactis CNCM-I2494 for 15 days (1 mL/rat-corresponding to 108 colony forming units of Bifidobacterium lactis CNCM I-2494) or saline. At day 15, paracellular permeability to dextran-FITC (4kD), and transmucosal flux of the protein antigen, horseradish peroxidase 40kD (HRP), were measured in Ussing chamber at different gut levels (ileal Peyer's patches, ileal and colonic segments) in both basal and after 2h of partial restraint stress (PRS) session. Moreover, IL10 and IL12 levels were measured by biochemistry assay on Peyer's patches. RESULTS: In saline treated animals, compared to basal condition, acute stress significantly increased dextran-FITC flux in Peyer's patches and in colonic segment and also substantially increased HRP flux in all gut segments tested as well as in Peyer's patches. FM consumption did not affect the in vitro measured intestinal permeability in basal condition but reduced the increase of both dextran-FITC and HRP flux induced by acute stress. Moreover, in comparison with basal condition, acute stress decreased the ratio of IL10/IL12 in ileal Peyer's patches, illustrating a pro-inflammatory effect. All these stress immune changes were significantly counteracted by 15 days of FM consumption. CONCLUSIONS: These data show in rats, that the consumption of a FM containing Bifidobacterium lactis CNCM-I2494 i) not only reduces the increased gut permeability function but also normalize local immune platform permeability (ileal Payer' patches) altered by acute stress; ii) protects the intestinal immunity barrier by modulating cytokines levels towards an anti-inflammatory profile.

Tu1966 Protective Effect of a Fermented Milk Dairy Product Containing Bifidobactrium Lactis CNCM I-2494 on Gut Immune Barrier Impairment Promoted by Acute Stress in Rats

BACKGROUND: Irritable Bowel Syndrome (IBS) is a common disorder characterized by visceral hypersensitivity and increased intestinal permeability. Abnormalities of gut flora as well as gut immune cells subtype variation have also been observed in IBS. Stress events can affect the onset and course of IBS. Clinical trials showed a beneficial effect of fermented milk (FM) containing Bifidobacterium lactis CNCM-I2494 in constipation predominant IBS (IBS-C) subjects. Recently, we reported that this FM product, dose dependently suppresses visceral hypersensitivity by normalizing intestinal epithelial barrier dysfunctions induced by acute stress in rats. Beside it has been shown that the relief of IBS symptoms by Bifidobacterium. infantis 35624 is associated with normalization in the ratio of proinflammatory/antiinflammatory cytokines IL-12/IL10. Therefore, the aim of this study was to investigate the modulatory effect of FM product containing Bifidobacterium lactis CNCM-I2494 on gut immune barrier impairment promoted by an acute stress in rats. MATERIAL ANDMETHODS: Wistar rats (200-250g) were orally given FM product containing Bifidobacterium lactis CNCM-I2494 for 15 days (1 mL/rat-corresponding to 108 colony forming units of Bifidobacterium lactis CNCM I-2494) or saline. At day 15, paracellular permeability to dextran-FITC (4kD), and transmucosal flux of the protein antigen, horseradish peroxidase 40kD (HRP), were measured in Ussing chamber at different gut levels (ileal Peyer's patches, ileal and colonic segments) in both basal and after 2h of partial restraint stress (PRS) session. Moreover, IL10 and IL12 levels were measured by biochemistry assay on Peyer's patches. RESULTS: In saline treated animals, compared to basal condition, acute stress significantly increased dextran-FITC flux in Peyer's patches and in colonic segment and also substantially increased HRP flux in all gut segments tested as well as in Peyer's patches. FM consumption did not affect the in vitro measured intestinal permeability in basal condition but reduced the increase of both dextran-FITC and HRP flux induced by acute stress. Moreover, in comparison with basal condition, acute stress decreased the ratio of IL10/IL12 in ileal Peyer's patches, illustrating a pro-inflammatory effect. All these stress immune changes were significantly counteracted by 15 days of FM consumption. CONCLUSIONS: These data show in rats, that the consumption of a FM containing Bifidobacterium lactis CNCM-I2494 i) not only reduces the increased gut permeability function but also normalize local immune platform permeability (ileal Payer' patches) altered by acute stress; ii) protects the intestinal immunity barrier by modulating cytokines levels towards an anti-inflammatory profile.

Influence of simethicone and alverine on stress-induced alterations of colonic permeability and sensitivity in rats: beneficial effect of their association

Alverine, an antispasmodic agent for the treatment of irritable bowel syndrome (IBS), may be combined with simethicone, a protective agent of the mucosa. Stress is a major factor triggering abdominal pain in IBS and causing hypersensitivity to colonic distension in animals through an increased colonic permeability. The antinociceptive effects of alverine and simethicone, separately or in association, were evaluated on stress-induced colonic hypersensitivity to distension in rats. The influence of simethicone on altered permeability was also tested. Groups of 8-10 female adult Wistar rats (200-250 g) housed individually were used. Gut paracellular permeability was evaluated after 2 h of partial restraint stress using oral gavage with ⁵¹Cr-EDTA and 24 h of urine collection. The number of abdominal cramps during colonic distension was evaluated in animals equipped with electrodes on their abdominal striated muscles. At 200 mg/kg p.o. twice a day, but not at lower doses, simethicone reduced stress-induced increase of colonic permeability and hypersensitivity to distension. Administered alone at 10 mg/kg p.o., alverine also reduced stress-induced hypersensitivity to distension; lower doses were inactive. However, alverine administered at an inactive dose with simethicone suppressed stress-induced hypersensitivity to distension. We conclude that both simethicone and alverine have visceral antinociceptive effects by two different mechanisms and that simethicone exerts a potentiating effect on the antinociceptive action of alverine.

Prevention of gut leakiness by a probiotic treatment leads to attenuated HPA response to an acute psychological stress in rats

Intestinal barrier impairment is incriminated in the pathophysiology of intestinal gut disorders associated with psychiatric comorbidity. Increased intestinal permeability associated with upload of lipopolysaccharides (LPS) translocation induces depressive symptoms. Gut microbiota and probiotics alter behavior and brain neurochemistry. Since Lactobacillus farciminis suppresses stress-induced hyperpermeability, we examined whether (i) L. farciminis affects the HPA axis stress response, (ii) stress induces changes in LPS translocation and central cytokine expression which may be reversed by L. farciminis, (iii) the prevention of "leaky" gut and LPS upload are involved in these effects. At the end of the following treatments female rats were submitted to a partial restraint stress (PRS) or sham-PRS: (i) oral administration of L. farciminis during 2 weeks, (ii) intraperitoneal administration of ML-7 (a specific myosin light chain kinase inhibitor), (iii) antibiotic administration in drinking water during 12 days. After PRS or sham-PRS session, we evaluated LPS levels in portal blood, plasma corticosterone and adrenocorticotropic hormone (ACTH) levels, hypothalamic corticotropin releasing factor (CRF) and pro-inflammatory cytokine mRNA expression, and colonic paracellular permeability (CPP). PRS increased plasma ACTH and corticosterone; hypothalamic CRF and pro-inflammatory cytokine expression; CPP and portal blood concentration of LPS. L. farciminis and ML-7 suppressed stress-induced hyperpermeability, endotoxemia and prevented HPA axis stress response and neuroinflammation. Antibiotic reduction of luminal LPS concentration prevented HPA axis stress response and increased hypothalamic expression of pro-inflammatory cytokines. The attenuation of the HPA axis response to stress by L. farciminis depends upon the prevention of intestinal barrier impairment and decrease of circulating LPS levels.

A marketed fermented dairy product containing Bifidobacterium lactis CNCM I‐2494 suppresses gut hypersensitivity and colonic barrier disruption induced by acute stress in rats

Fermented milk (FM) containing Bifidobacterium lactis CNCM I-2494 and yogurt strains improves irritable bowel syndrome (IBS) symptoms in constipated IBS patients. In rats, stressful events exacerbate IBS symptoms and result in the alteration of gut sensitivity and permeability via epithelial cell cytoskeleton contraction. In a stress model, we aimed at evaluating the effect of B. lactis CNCM I-2494 as a pure strain or contained in an FM product on visceral sensitivity and the impact of this FM on intestinal barrier integrity. Visceral sensitivity was analyzed in rats subjected to partial restraint stress (PRS). Rats received during 15 days the B. lactis as a pure strain (10(6) to 10(10) CFU mL(-1)), B. lactis in an FM product (10(8) CFU g(-1), diluted or not), or a control product. Gut paracellular permeability, colonic occluding and Jam-A proteins, and blood endotoxin levels were determined in rats receiving B. lactis in an FM product submitted or not to a PRS. The FM product showed a dose-dependent inhibitory effect on stress-induced visceral hypersensitivity. A similar antihyperalgesic effect was observed at 10(10) CFU mL(-1) of pure B. lactis administration. The FM product prevented the increase in intestinal permeability induced by PRS and restored occludin and JAM-A expressions to control levels. The FM product abolished the increase concentration of blood endotoxin induced by PRS. This study illustrates that a probiotic food containing B. lactis CNCM I-2494 strain reduces visceral hypersensitivity associated with acute stress by normalizing intestinal epithelial barrier via a synergistic interplay with the different probiotic strains and/or metabolites contained in this product.

Partial Restraint Stress Induces Increased Permeability and Low Grade Inflammation in the Rat Ileum, but Not in the Jejunum

Mixed-lineage kinase-3 (MLK3) activates multiple MAPK pathways and has been shown to initiate apoptosis, proliferation, migration or differentiation in several cell types. MLK3 signaling is also required for breast cancer cell invasion. However, the role of MLK3 signaling in regulation of intestinal epithelial cell sheet migration In Vivo and In Vitro is not known. Given its role in cancer cell migration, we sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility. We compared the healing of jejunal mucosal ulcers induced by topical serosal acetic acid in different age groups of MLK3 null mice with that in age-matched wild type mice and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial migration across type I collagen substrates. In older mice (30-42 weeks of age), ulcer healing at 5 days after the initial insult was reduced by 47% (p<0.05) in MLK3 null and by 29% in MLK3 heterozygous mice when compared with wild-type littermate controls. In younger animals (8-12 weeks), ulcer healing in MLK3 null mice was 25% less at day 3 (n=7, p<0.05) and 23% less at day 5 (n=7, p<0.05) than in wild-type mice. In Vitro, an MLK3 inhibitor significantly decreased closure of circular wounds in Caco-2 cell monolayers after 24 hours compared with to vehicle-treated control by 16.3% and 20.1 % respectively (p<0.05). Proliferative blockade using hydroxyurea (30mM) did not abolish the effect of MLK3 inhibitor on Caco-2 wound closure, suggesting a true effect on cell motility rather than an effect on cell proliferation. The reduction in cell motility by MLK3 inhibitor reduced level of pERK by 79% (p<0.05) and pJNK by 12% (p<0.05) revealing suppression of ERK and JNK signaling. Total MLK3 was also compared in confluent Caco-2 cells to cell that have been migrating for 96 hours. MLK3 was 53% (p<0.05) higher in migrating vs confluent cells further indicating the importance of MLK3 signaling in migrating intestinal epithelial cells. These studies reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration and suggest that MLK3 may be an important target to influence intestinal mucosal healing.