Water Avoidance Stress Research Articles

Berberine prevents stress-induced gut inflammation and visceral hypersensitivity and reduces intestinal motility in rats.

BACKGROUNDIrritable bowel syndrome (IBS) is a common chronic non-organic disease of the digestive system. Berberine (BBR) has been used to treat patients with IBS, but the underlying therapeutic mechanism is little understood. We believe that BBR achieves its therapeutic effect on IBS by preventing stress intestinal inflammation and visceral hypersensitivity and reducing bowel motility.AIMTo test the hypothesis that BBR achieves its therapeutic effect on IBS by preventing subclinical inflammation of the intestinal mucosa and reducing visceral hypersensitivity and intestinal motility.METHODSIBS was induced in rats via water avoidance stress (WAS). qRT-PCR and histological analyses were used to evaluate the levels of cytokines and mucosal inflammation, respectively. Modified ELISA and qRT-PCR were used to evaluate the nuclear factor kappa-B (NF-κB) signal transduction pathway. Colorectal distention test, gastrointestinal transit measurement, Western blot, and qRT-PCR were used to analyze visceral sensitivity, intestinal motility, the expression of C-kit (marker of Cajal mesenchymal cells), and the expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB.RESULTSWAS led to mucosal inflammation, visceral hyperalgesia, and high intestinal motility. Oral administration of BBR inhibited the NF-κB signal transduction pathway, reduced the expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α], promoted the expression of anti-inflammatory cytokines (IL-10 and transforming growth factor-β), and improved the terminal ileum tissue inflammation. BBR inhibited the expression of BDNF, TrkB, and C-kit in IBS rats, leading to the reduction of intestinal motility and visceral hypersensitivity. The therapeutic effect of BBR at a high dose (100 mg/kg) was superior to than that of the low-dose (25 mg/kg) group.CONCLUSIONBBR reduces intestinal mucosal inflammation by inhibiting the intestinal NF-κB signal pathway in the IBS rats. BBR reduces the expression of BDNF, its receptor TrkB, and C-kit. BBR also reduces intestinal motility and visceral sensitivity to achieve its therapeutic effect on IBS.

Effects of thermized donkey milk with lysozyme activity on altered gut barrier in mice exposed to water-avoidance stress

Nutrition plays a crucial role in human gut health through the improvement of gut barrier functionality. Donkey milk represents an interesting source of natural antimicrobial factors such as lysozyme. Recently, anti-inflammatory properties of donkey milk lysozyme activity were described in a mouse model of ileitis. The current increase of donkey milk consumption highlights the necessity to propose a healthy milk compliant with microbiological standards. This study aims to define a heat treatment of donkey milk, retaining its high lysozyme activity, and to evaluate its beneficial effects on a gut barrier impairment model due to chronic stress in mice. To perform this experiment, samples of raw donkey milk were collected in 15 distinct French farms. Microbiological analysis and lysozyme content and activity were evaluated for each sample. Then, several heat treatments were carried out to define a time and temperature combination that allowed for both a reduction in the number of total micro-organisms, increasing the shelf-life of the product, and preservation of lysozyme activity. The beneficial effect of heated donkey milk on the gut barrier of mice was evaluated and compared with raw donkey milk. We found that samples of raw donkey milk showed low total mesophilic microbial counts, and no pathogens were detected. Among the different heat-treatment procedures tested, a 2-min, 72°C combination was determined to be the most optimal time and temperature combination to preserve lysozyme activity and increase the shelf-life of donkey milk. Oral administration of this heat-treated donkey milk in mice counteracted chronic stress-induced intestinal damage, illustrated by gut hyper-permeability and low-grade inflammation, similar to raw donkey milk. We have demonstrated for the first time that oral intervention with donkey milk, optimally heat-treated to retain enzymatic lysozyme activity, improves intestinal barrier damage linked to psychological stress in mice.

Vagal Transient Receptor Potential Ankyrin 1 Mediates Stress-exacerbated Visceral Mechanonociception After Antral Cold Exposure.

Background/AimsAbdominal pain can be evoked or exacerbated after gastrointestinal cold stimulation in some patients with diarrhea-predominant irritable bowel syndrome (IBS-D), indicating a low temperature-induced sensitization of visceral perception. We investigated the role of vagal transient receptor potential ankyrin 1 (TRPA1, a cold-sensing ion channel) in cold-aggravated visceral mechanonociception in a stress-induced IBS animal model.MethodsTRPA1 expression was examined in antral biopsies of healthy controls and IBS-D patients. Abdominal symptoms were assessed before and after warm or cold water intake. The visceromotor response (VMR) to colorectal distention (CRD) following intra-antral infusion of cold saline was measured in animals undergoing sham or chronic water avoidance stress. TRPA1 expression, extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation, and neuronal calcium influx in vagal afferents were assessed.ResultsCompared to healthy controls, IBS-D patients displayed elevated antral TRPA1 expression, which was associated with symptom scores after cold (4°C) water intake. Intra-antral infusion of cold saline increased VMR to CRD in naive rats, an effect dependent on vagal afferents. In stressed rats, this effect was greatly enhanced. Functional blockade and gene deletion of TRPA1 abolished the cold effect on visceral nociception. TRPA1 expression in vagal (but not spinal) afferents increased after stress. Moreover, the cold-induced, TRPA1-dependent ERK1/2 activation and calcium influx in nodose neurons were more robust in stressed rats.ConclusionsStress-exaggerated visceral mechanonociception after antral cold exposure may involve up-regulation of TRPA1 expression and function on vagal afferents. Our findings reveal a novel mechanism for abnormal gastrointestinal cold sensing in IBS.

Multi-omics Analysis of Gut Microbiota and Metabolites in Rats With Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a common gastrointestinal dysfunctional disease. The pathophysiology of IBS is, however, largely unknown. This study aimed to determine whether evaluation of fecal metabolite and microbiota profiles may offer an opportunity to identify a novel pathophysiological target for IBS, and to reveal possible gut microbe–metabolite associations. By using gas chromatography coupled to time-of-flight mass spectrometry (GC-TOFMS) and 16S rRNA gene sequencing, we measured fecal metabolites and microbiota of the control and water avoidance stress (WAS)-induced IBS rats. We found a significantly differential metabolite profile between the IBS and control groups; a cluster of metabolites was also found to be significantly associated with the amount of defecations. Moreover, the WAS group exhibited a decreased alpha diversity of the microbial population as compared to the control group. However, the characteristics of gut microbiota could not differentiate the IBS group from the control group. Correlation of the metabolite level with the number of microbial genera showed no significant association between the control and IBS groups. This study provides a global perspective on metabolomics and microbiota profiling in WAS-induced IBS model and a theoretical basis for research on the pathophysiology of IBS.

Down-regulation of HTR1A-modulated ACC activation contributes to stress-induced visceral hyperalgesia in rats.

Long-term stress was suggested to cause visceral hypersensitivity and promote functional gastrointestinal disorders (FGIDs). Some brain regions such as the anterior cingulate cortex (ACC) may play an important role for generating visceral hypersensitivity; however, its molecular mechanisms are not clear. This study aimed to explore the role of 5-HT1A receptors (HTR1As) in activating ACC and corresponding mechanism, in stress-induced visceral hyperalgesia rats. The VH rat model was established by chronic water avoidance stress (WAS), and the visceral sensitivity was measured by electromyogram. Rat's anxiety-like behaviors were evaluated by the open field test (OFT) and elevated plus maze (EPM). To overexpress or down-regulate HTR1A expression, HTR1A-specific lentivirus expressing the green fluorescent protein was administered into the ACC. Protein expression levels were observed by Western blot. The protein expression of HTR1A in bilateral ACC in WAS group was significantly lower than that in normal control (NC) and Sham-WAS groups, while the levels of c-fos in the ACC of WAS rats were significantly higher. Down-regulation of HTR1As could induce VH in control rats with the increased expression of c-fos, p-ERK, and p-Akt in ACC, while up-regulation of HTR1As in the ACC could partly inhibit ACC sensitization and stress-induced visceral hyperalgesia. Down-regulation of HTR1As modulates ACC activation probably through activating ERK and Akt pathways, thus contributes to the formation of stress-induced visceral hyperalgesia.

Stress‐induced Effects on Endometriosis and Tight Junction Protein Expression are Counteracted by VSL#3 Administration in an Animal Model

IntroductionEndometriosis is a gynecological disorder in which tissue that normally lines the endometrium grows outside the uterus and commonly results in peritoneal inflammation and infertility. Our previous studies show that stress exacerbates the development of endometriosis symptoms with worsening of colonic damage, and probiotic administration can decrease lesion size. Tight junction proteins are important to maintain the integrity of the intestinal barrier, and their disturbance can contribute to a variety of pathological conditions. It is possible that in our model stress contributes to the intestinal effects via modulation of tight junction proteins, but it is unclear whether probiotic use could counteract any of these effects. In this project we focused on tight junction protein expression intensity using the specific integral membrane proteins occludin and claudin‐2 to investigate how probiotics might alleviate endometriotic damage in the colon. Downregulation of occludin can enhance paracellular permeability, while claudin‐2 overexpression has been linked to intestinal pathophysiology by decreasing barrier function of other claudins.HypothesisProbiotic administration will reverse the effects of stress on colonic tight junction protein expression in an animal model of endometriosis.MethodsEndometriosis was induced by suturing uterine horn tissue next to the intestinal mesenteries in female Sprague‐Dawley rats (11–12/group) on day 0. One week later, rats were given the probiotic mixture VSL#3 or placebo in their drinking water before subjecting to 7 days of water avoidance stress (60 mins/day; days 14–20). Non‐stressed controls also received VSL#3 or placebo. On day 60 the rats were sacrificed, the vesicles were retrieved and measured, and the tissues collected before paraffin embedding. Colonic damage was measured and immunofluorescence for occludin and claudin‐2 was performed.ResultsThe vesicle area from rats exposed to stress were significantly larger in size than non‐stressed controls (p<0.05), while after probiotic administration the size was reduced (p<0.01). Colonic macroscopic damage had a trend to increase in endometriosis animals and was reduced after probiotic administration (p<0.05). Colonic occludin expression showed a trend to increase when probiotic was administered after stress, while claudin‐2 expression was downregulated after stress (p<0.01) and probiotic administration (p<0.01).ConclusionStress aggravates endometriosis symptoms causing larger vesicles and increased colonic damage. After probiotic administration, these symptoms are reversed and integrity of the tight junctions in colonic tissue is increased. This tendency provides further support for the potential use of probiotics as a complementary treatment to reduce the effects of inflammation in this condition.Support or Funding InformationSupported in part by P20 GM103475‐15, R25GM096955 & R25GM082406This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dehydroepiandrosterone sulfate improves visceral sensation and gut barrier in a rat model of irritable bowel syndrome

Stress-induced altered visceral sensation and impaired gut barrier play an important role in the pathophysiology of irritable bowel syndrome (IBS). These responses were demonstrated to be peripheral corticotropin-releasing factor (CRF) dependent and also mediated via proinflammatory cytokine in animal IBS model. Dehydroepiandrosterone sulfate (DHEA-S) is known to have anti-inflammatory properties by suppressing proinflammatory cytokine release. We hypothesized that DHEA-S improves stress-induced visceral changes and is beneficial for IBS treatment. We explored the effects of DHEA-S on lipopolysaccharide (LPS)- or repeated water avoidance stress (WAS)-induced visceral allodynia and increased colonic permeability (rat IBS models). The threshold of visceromotor response, i.e. abdominal muscle contractions induced by colonic balloon distention was electrophysiologically measured. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue. DHEA-S abolished visceral allodynia and colonic hyperpermeability induced by LPS in a dose-dependent manner. It also blocked repeated WAS- or peripheral injection of CRF-induced visceral changes. These effects by DHEA-S in LPS model were reversed by bicuculline, a γ-aminobutyric acid (GABA)A receptor antagonist, NG-nitro-L-arginine methyl ester, a nitric oxide (NO) synthesis inhibitor, naloxone, an opioid receptor antagonist, or sulpiride, a dopamine D2 receptor antagonist. However, domperidone, a peripheral dopamine D2 receptor antagonist did not modify the effects. Peripheral injection of astressin2-B, a selective CRF receptor subtype 2 (CRF2) antagonist also reversed these effects. In conclusion, DHEA-S blocked stress-induced visceral changes via GABAA, NO, opioid, central dopamine D2 and peripheral CRF2 signaling. DHEA-S may be useful for IBS treating.

MiRNA-29a modulates visceral hyperalgesia in irritable bowel syndrome by targeting HTR7

Some patients with irritable bowel syndrome (IBS) have visceral hypersensitivity, which contributes to their abdominal pain. miRNA-29 was detected to be significantly upregulated in colonic tissues of patients with IBS. However, it is unknown whether miRNA-29a is involved in the visceral hypersensitivity pathogenesis of IBS. This study aimed to investigate whether miRNA-29a participates in visceral hypersensitivity in IBS. We investigated miRNA-29a in intestinal biopsies collected during endoscopy of patients with IBS (n = 10) and healthy volunteers (control) (n = 10). In addition, a water avoidance stress (WAS)-induced visceral hypersensitivity IBS mouse model was established. The abdominal withdrawal reflex (AWR) scores of mice in response to colorectal distention were used to assess visceral sensitivity. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was used to measure miRNA-29a levels. Immunofluorescence, RT-qPCR and western blot were used to measure 5-HT7 receptor (HTR7) levels. Bioinformatic analysis and luciferase reporter assays were used to detect the direct relationship between miRNA-29a and HTR7. Finally, alterations in the levels of HTR7 and miRNA-29a were measured in the human intestinal epithelial cell line NCM460 after transfection with miRNA-29a inhibitor or mimic. Intestinal tissues from patients with IBS and WAS-induced IBS mice had increased levels of miRNA-29a, but reduced levels of HTR7. MiRNA-29a knockout resulted in overexpression of HTR7 and attenuated visceral hyperalgesia in WAS-induced IBS mice. HTR7 was a direct target of miRNA-29a. Based on analyses of intestinal tissue samples from patients with IBS and WAS-induced miRNA-29a–/– mice, miRNA-29a plays a role in the visceral hyperalgesia pathogenesis of IBS, probably through regulating HTR7 expression.

Activation of cannabinoid 2 receptor relieves colonic hypermotility in a rat model of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. The endocannabinoid system is emerging as an important modulator of gastrointestinal (GI) motility in multiple diseases, but its involvement in IBS is unknown. We aimed to determine whether cannabinoid 2 (CB2) receptor modulates intestinal motility associated with stress-induced IBS. A rat IBS model was established by chronic water avoidance stress (WAS). Colonic pathological alterations were detected histologically and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). Real-time PCR, western blot, and immunostaining were performed to identify colonic CB2 receptor expression. Colonic muscle strip contractility was studied by isometric transducers and nitric oxide (NO) was detected by the Griess test. The effects of AM1241, a selective agonist of CB2 receptors, on colonic motility were examined. After 10days of WAS exposure, ITT was decreased and FWC elevated while VMR magnitude in response to CRD was significantly enhanced. Colon CB2 protein and mRNA levels increased and density of CB2-positive macrophages in the mucosa and enteric neurons in the myenteric plexus was higher than in controls. Pharmacological enhancement of CB2 activity by AM1241 relieved colonic hypermotility in WAS rats in a concentration-dependent manner via inhibition of p38 phosphorylation and elevation of NO production. CB2 receptor may exert an important inhibitory effect in stress-induced colonic hypermotility by modulating NO synthesis through p38 mitogen-activated protein kinase signaling. AM1241 could be used as a potential drug to treat disorders with colonic hypermotility.

Chronic Psychological Stress Induces Gastritis in C57BL/6 Mice with Decreased Production of Hydrogen Sulphide (H 2S) and Microbial Dysbiosis

Background: Chronic persistent stress is an important cause of gastritis, but its mechanism still needs research. Methods: Here we have used a mouse model and the water avoidance stress (WAS) test as a model for chronic stress-induced gastritis to investigate underlying mechanisms of this disease; The effect of stress on gastric flora was analyzed based on 16sRNA sequencing; the changes of hydrogen sulfide (H2S) and inflammatory cytokines from gastric tissues were detected by western blotting, Elisa, immunofluorescence and qRT-PCR; H&E (Haemotoxylin and Eosin) staining was used for the gastritis histology score. Findings: Consistent with the results of previous studies, gastric H2S was negatively associated with the inflammatory index and may protect the gastrointestinal tract from inflammation. WAS-induced gastritis was associated with a reduction in H2S release, which appeared to affect the homeostasis of the gastric microbiota of mice. Inflammation and microbial dysbiosis were partially reversed by sodium hydrosulfide (NaHS) and vitamin B6 (VB6) supplementation, suggesting that VB6 supplementation may have a therapeutic potential for stress-induced gastritis. Interpretation: The secretory disease gastritis can have a serious effect on health and quality of life, with an increasing number of people suffering from chronic gastritis that is linked to a high-stress lifestyle. Our research provides clues to the prevention and treatment of strss-induced gastritis. Funding: The present research was supported by the National Natural Science Foundation of China (No. 81671978, 81471991, 81772143, 81670486, and 81374101). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: All animal experiments were approved by the Animal Care Committee and Ethics Commission of Shandong University (LL-201602047).

Stress-induced autonomic dysregulation of mitochondrial function in the rat urothelium.

Chronic stress exacerbates the symptoms of most pain disorders including interstitial cystitis/bladder pain syndrome (IC/BPS). Abnormalities in urothelial cells (UTC) occur in this debilitating bladder condition. The sequence of events that might link stress (presumably through increased sympathetic nervous system-SNS activity) to urothelial dysfunction are unknown. Since autonomic dysregulation, mitochondrial dysfunction, and oxidative stress all occur in chronic pain, we investigated whether chronic psychological stress initiated a cascade linking these three dysfunctions. Adult female Wistar Kyoto rats were exposed to 10 days of water avoidance stress (WAS). Bladders were then harvested for Western blot and single cell imaging in UTC cultures. UTC from WAS rats exhibited depolarized mitochondria membrane potential (Ψm ∼30% more depolarized compared to control), activated AMPK and altered UT mitochondria bioenergetics. Expression of the fusion protein mitofusion-2 (MFN-2) was upregulated in the mucosa, suggesting mitochondrial structural changes consistent with altered cellular metabolism. Intracellular calcium levels were elevated in cultured WAS UTC, consistent with impaired cellular function. Stimulation of cultured UTC with alpha-adrenergic (α-AR) receptor agonists increased reactive oxidative species (ROS) production, suggesting a direct action of SNS activity on UTC. Treatment of rats with guanethidine to block SNS activity prevented most of WAS-induced changes. Chronic stress results in persistent sympathetically mediated effects that alter UTC mitochondrial function. This may impact the urothelial barrier and signaling, which contributes to bladder dysfunction and pain. This is the first demonstration, to our knowledge, of a potential autonomic mechanism directly linking stress to mitochondrial dysfunction.

Beneficial effect of butyrate-producing Lachnospiraceae on stress-induced visceral hypersensitivity in rats.

Background and AimEmerging evidence indicates that psychological stress is involved in the pathogenesis of irritable bowel syndrome, which is characterized by visceral hypersensitivity and may be accompanied by gut dysbiosis. However, how such stress contributes to the development of visceral hypersensitivity is incompletely understood. Here, we aimed to investigate the influence that stress‐induced microbial changes exert on visceral sensitivity, as well as the possible underlying mechanisms associated with this effect.MethodsMale Sprague–Dawley rats underwent chronic water avoidance stress (WAS) to induce visceral hypersensitivity. Visceral sensitivity, colonic tight junction protein expression, and short‐chain fatty acids of cecal contents were measured. Fecal samples were collected to characterize microbiota profiles. In a separate study, oral gavage of Roseburia in WAS rats was conducted to verify its potential role in the effectiveness on visceral hypersensitivity.ResultsRepeated WAS caused visceral hypersensitivity, altered fecal microbiota composition and function, and decreased occludin expression in the colon. Stressed rats exhibited reduced representation of pathways involved in the metabolism of butyrate and reduced abundance of several operational taxonomic units associated with butyrate‐producing bacteria, such as Lachnospiraceae. Consistently, supplementation with Roseburia hominis, a species belonging to Lachnospiraceae, significantly increased cecal butyrate content. Moreover, Roseburia supplementation alleviated visceral hypersensitivity and prevented the decreased expression of occludin.ConclusionsReduction in the abundance of butyrate‐producing Lachnospiraceae, which is beneficial for the intestinal barrier, was involved in the formation of visceral hypersensitivity. R. hominis is a potential probiotic for treating stress‐induced visceral hypersensitivity.

Colonic Proteome Signature in Immunoproteasome-Deficient Stressed Mice and Its Relevance for Irritable Bowel Syndrome.

A role for immunoproteasome in the regulation of intestinal permeability has been previously suggested both in mice during water avoidance stress (WAS) and in patients with irritable bowel syndrome (IBS). Here, we provide evidence that the ubiquitin-proteasome system (UPS) contributes to the pathophysiology of IBS. Indeed, we report that colonic proteome is altered in WAS mice and that β2i subunit deficiency modifies the proteome response that is associated with a limitation of colonic hyperpermeability. Interestingly, we show specific alterations of proteins involved in UPS, mitochondrial, and energy metabolism. We also report changes in the pattern of colonic ubiquitome in diarrhea-predominant IBS (IBS-D) patients and particularly a reduced expression of ubiquitinated proteins involved in the nuclear factor-kappa B (NF-κB) inflammatory signaling pathway. All these data suggest that immunoproteasome targeting may represent a new therapeutic strategy for the treatment of IBS patients with increased intestinal permeability.

Altered colonic sensory and barrier functions by CRF: roles of TLR4 and IL-1

Visceral allodynia and increased colonic permeability are considered to be crucial pathophysiology of irritable bowel syndrome (IBS). Corticotropin-releasing factor (CRF) and immune-mediated mechanisms have been proposed to contribute to these changes in IBS, but the precise roles have not been determined. We explored these issues in rats in vivo. The threshold of visceromotor response, i.e., abdominal muscle contractions induced by colonic balloon distention was electrophysiologically measured. Colonic permeability was estimated by quantifying the absorbed Evans blue in colonic tissue. Intraperitoneal injection of CRF increased the permeability, which was blocked by astressin, a non-selective CRF receptor antagonist, but astressin2-B, a selective CRF receptor subtype 2 (CRF2) antagonist did not modify it. Urocortin 2, a selective CRF2 agonist inhibited the increased permeability by CRF. Eritoran, a toll-like receptor 4 (TLR4) antagonist or anakinra, an interleukin-1 receptor antagonist blocked the visceral allodynia and the increased gut permeability induced by CRF. Subcutaneous injection of lipopolysaccharide (immune stress) or repeated water avoidance stress (WAS, psychological stress), 1 h daily for 3 days induced visceral allodynia and increased gut permeability (animal IBS models), which were also blocked by astressin, eritoran or anakinra. In conclusion, stress-induced visceral allodynia and increased colonic permeability were mediated via peripheral CRF receptors. CRF induced these visceral changes via TLR4 and cytokine system, which were CRF1 dependent, and activation of CRF2 inhibited these CRF1-triggered responses. CRF may modulate immune system to alter visceral changes, which are considered to be pivotal pathophysiology of IBS.

Brain corticotropin-releasing factor signaling: Involvement in acute stress-induced visceral analgesia in male rats.

Water avoidance stress (WAS) induces a naloxone-independent visceral analgesia in male rats under non-invasive conditions of monitoring. The objective of the study was to examine the role of brain CRF signaling in acute stress-induced visceral analgesia (SIVA). Adult male Sprague-Dawley rats were chronically implanted with an intracerebroventricular (ICV) cannula. The visceromotor response (VMR) to graded phasic colorectal distension (CRD: 10, 20, 40, 60mmHg, 20seconds, 4minutes intervals) was monitored using manometry. The VMR to a first CRD (baseline) was recorded 5minutes after an ICV saline injection, followed 1hour later by ICV injection of either CRF (30, 100, or 300ng and 1, 3, or 5μg/rat) or saline and a second CRD, 5minutes later. Receptor antagonists against CRF1 /CRF2 (astressin-B, 30μg/rat), CRF2 (astressin2 -B, 10μg/rat), oxytocin (tocinoic acid, 20μg/rat), or vehicle were injected ICV 5minutes before CRF (300ng/rat, ICV) or 15minutes before WAS (1hour). ICV CRF (100 and 300ng) reduced the VMR to CRD at 60mmHg by -36.6%±6.8% and -48.7%±11.7%, respectively, vs baseline (P<0.001), while other doses had no effect and IP CRF (10µg/kg) induced visceral hyperalgesia. Astressin-B and tocinoic acid injected ICV induced hyperalgesia and prevented the analgesic effect of ICV CRF (300ng/rat) and WAS, while astressin2 -B only blocked WAS-induced SIVA. These data support a role for brain CRF signaling via CRF2 in SIVA in a model of WAS and CRD likely mediated by the activation of brain oxytocin pathway.

Chronic stress and intestinal permeability: Lubiprostone regulates glucocorticoid receptor-mediated changes in colon epithelial tight junction proteins, barrier function, and visceral pain in the rodent and human.

Chronic psychological stress is associated with increased intestinal epithelial permeability and visceral hyperalgesia. Lubiprostone, an agonist for chloride channel-2, promotes secretion and accelerates restoration of injury-induced epithelial barrier dysfunction. The mechanisms underlying how lubiprostone regulates colon epithelial barrier function and visceral hyperalgesia in chronic stress remain unknown. Male rats were subjected to water avoidance stress for 10 consecutive days. Lubiprostone was administered daily during the stress phase. Visceromotor response to colorectal distension was measured. Human colon crypts and cell lines were treated with cortisol and lubiprostone. The transepithelial electrical resistance and FITC-dextran permeability were assayed. Chromatin immunoprecipitation was conducted to assess glucocorticoid receptor binding at tight junction gene promoters. Lubiprostone significantly decreased chronic stress-induced visceral hyperalgesia in the rat (P<0.05; n=6). WA stress decreased occludin and claudin-1 and increased claudin-2 in rat colon crypts, which was prevented by lubiprostone. Cortisol treatment induced similar alterations of tight junction protein expression in Caco-2/BBE cells (P<0.05) and significantly changed paracellular permeability in monolayers (P<0.01). These changes were blocked by lubiprostone. Glucocorticoid receptor and its binding at occludin promoter region were decreased in cortisol-treated cells and human colon crypts, which was largely reversed by lubiprostone. In rat colonic cells, glucocorticoid receptor and its co-chaperone proteins were down-regulated after corticosterone treatment and lubiprostone reversed these changes. Lubiprostone preferentially prevents chronic stress-induced alterations of intestinal epithelial tight junctions, barrier function, and visceral hyperalgesia that was associated with modulation of glucocorticoid receptor expression and function.

Role of High-affinity Choline Transporter 1 in Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome.

Background/AimsIrritable bowel syndrome (IBS) is a common disease characterized by intestinal dysmotility, the mechanism of which remains elusive. We aim to determine whether the high-affinity choline transporter 1 (CHT1), a determinant of cholinergic signaling capacity, modulates intestinal motility associated with stress-induced IBS.MethodsA rat IBS model was established using chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically and intestinal motility was assessed by intestinal transit time and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response to colorectal distension. RT-PCR, western blotting, and immunostaining were performed to identify colonic CHT1 expression. Contractility of colonic muscle strips was measured using isometric transducers. enzyme-linked immunosorbent assay was used to measure acetylcholine (ACh). We examined the effects of MKC-231, a choline uptake enhancer, on colonic motility.ResultsAfter 10 days of WAS, intestinal transit time was decreased and fecal water content increased. Visceromotor response magnitude in WAS rats in response to colorectal distension was significantly enhanced. Protein and mRNA CHT1 levels in the colon were markedly elevated after WAS. The density of CHT1-positive intramuscular interstitial cells of Cajal and myenteric plexus neurons in WAS rats was higher than in controls. Ammonium pyrrolidine dithiocarbamate partly reversed CHT1 upregulation and alleviated colonic hypermotility in WAS rats. Pharmacological enhancement of CHT1 activity by MKC-231 enhanced colonic motility in control rats via upregulation of CHT1 and elevation of ACh production.ConclusionUpregulation of CHT1 in intramuscular interstitial cells of Cajal and myenteric plexus neurons is implicated in chronic stress-induced colonic hypermotility by modulation of ACh synthesis via nuclear factor-kappa B signaling.

Upregulation of the high-affinity choline transporter in colon relieves stress-induced hyperalgesia.

BackgroundIrritable bowel syndrome (IBS) is a common disease with hyperalgesia, the mechanisms of which remain elusive. The cholinergic system is known to be involved in pain inhibitory pathways in multiple diseases, and its involvement in IBS is unknown.ObjectiveWe aimed to determine whether high-affinity choline transporter CHT1, a major determinant of the cholinergic signaling capacity, is involved in regulating intestinal sensations associated with stress-induced visceral pain.Materials and methodsAn IBS rat model was established by chronic water avoidance stress (WAS). Colonic pathologic alterations were detected by H&E staining. Visceral sensations were determined by scoring the abdominal withdrawal reflex (AWR) and visceromotor response (VMR) magnitude of the electromyogram in response to colorectal distension (CRD). Abdominal mechanical hyperalgesia was assessed by counting the number of withdrawal events evoked by applying von Frey filaments. Real-time PCR, Western blot, and immunostaining were performed to identify CHT1 expression in the colon. Acetylcholine (ACh) secretion was determined by ELISA. Effects of MKC-231, a choline uptake enhancer, on visceral pain were examined.ResultsAfter 10 days of WAS exposure, AWR score and VMR magnitude in response to CRD were significantly enhanced and the number of withdrawal events was elevated. Protein and mRNA levels of CHT1 were considerably increased in the colon after WAS. CHT1 upregulation in the WAS-exposed group was largely abolished by ammonium pyrrolidinedithiocarbamate. The density of CHT1-positive intramuscular cells and enteric neurons in the myenteric plexus was enhanced in WAS-exposed rats. Pharmacologic enhancement of CHT1 activity by MKC-231 gavage could relieve the visceral pain of WAS rats by upregulating CHT1 protein expression and enhancing ACh production.ConclusionCHT1 may exert an antinociceptive effect in stress-induced visceral pain by modulating ACh synthesis through nuclear factor kappa B signaling. MKC-231 could be used as a potential drug to treat disorders with hyperalgesia.

Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome Is Associated with Upregulation of TMEM16A in Myenteric Plexus

BackgroundIrritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. TMEM16A is a calcium-activated chloride channel (CaCC) involved in intestinal slow-wave generation.AimsTo investigate whether TMEM16A is involved in colonic dysmotility in IBS.MethodsA rat model of IBS was established by chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically, and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). TMEM16A expression was evaluated by RT-PCR, Western blot, and immunofluorescence. Colonic muscle strip contractility was measured by isometric transducers, and the effect of niflumic acid (NFA), a CaCC antagonist, on colonic motility was examined.ResultsAfter 10 days of WAS exposure, ITT was decreased and FWC was elevated. Furthermore, VMR magnitude of WAS rats in response to CRD was significantly enhanced. Protein and mRNA levels of TMEM16A in colon were considerably increased after WAS. The percentage of TMEM16A-positive neurons in myenteric plexus (MP) of WAS rats was significantly higher than controls. Pharmacological blockade of TMEM16A activity by NFA considerably enhanced ITT, with concentration-dependent declines in FWC and VMR magnitude in NFA-treated rats. Further, spontaneous contraction of colonic strips of NFA-treated rats was significantly ameliorated in a concentration-dependent manner in vitro.ConclusionsUpregulation of TMEM16A in MP neurons may play an important role in chronic stress-induced colonic hypermotility, making CaCC-blocking drugs a putatively effective treatment method for colonic hypermotility in IBS.

Transcriptome and methylome profiling in a rat model of irritable bowel syndrome induced by stress.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is associated with psychological stress. However, the full landscape of IBS-related epigenetic factors remains unveiled and needs to be elucidated. The water-avoidance stress (WAS) method was used to induce a rat IBS model. Each rat was monitored, and its defecation and behavior were recorded. Total colon RNA was isolated and subjected to Affymetrix GeneChip analysis. Reduced Representation Bisulfate Sequencing (RRBS) was applied to determine the genome-wide methylation pattern in both IBS and control rats. Rats with IBS egested a significantly increased amount of dry and loose stools compared with the control animals, without significant changes in body weight. Compared with the control group, 309 genes were upregulated and 224 genes were downregulated in the colon of the IBS rats. Notch signaling and focal adhesion were increased in the differentially expressed genes (DEGs). A total of 541 genes had significant lower methylation level and 626 genes had significantly higher methylation level in their promoter regions. Adherens junction and leukocyte transendothelial migration were enriched in the differentially methylated genes (DMGs). Few genes were identified in common in both DEGs and DMGs, suggesting that gene expression was not altered by promoter methylation. Reverse transcription-quantitative polymerase chain reaction validation revealed that the mRNA levels of SSX2IP, PARD3 and VCL were significantly downregulated in the IBS group, in accordance with hypermethylation of their promoters. In summary, the present study used a WAS-induced IBS rat model to provide transcriptome and methylome profiling. Most DEGs were associated with Notch signaling and focal adhesion, and only a few were altered by promoter methylation. The present results demonstrated that psychological stress could influence the integrity of the intestinal mucosa barrier and regulate inflammatory response.