Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS.

OBJECTIVE: The intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS. DESIGN: 39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states. RESULTS: Patients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota. CONCLUSION: A subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.

Close association between abnormal expressed enzymes of energy metabolism and diarrhea-predominant irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most common functional intestinal diseases, but its pathogenesis is still unknown. The present study aimed to screen the differentially expressed proteins in the mucosa of colon between IBS with diarrhea (IBS-D) patients and the healthy controls. METHODS: Forty-two IBS-D patients meeting the Rome III diagnostic criteria and 40 control subjects from July 2007 to June 2009 in Chinese PLA General Hospital were enrolled in the present study. We examined the protein expression profiles in mucosa of colon corresponding to IBS-D patients (n = 5) and controls (n = 5) using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Secondly, Western blot and immunohistochemical analysis were carried out to validate the screened proteins in 27 IBS-D patients and 27 controls. Thirdly, high-performance liquid chromatography (HPLC) was further carried out to determine ATP concentration in the mucosa of colon between 10 IBS-D patients and 8 controls. Comparisons between 2 groups were performed by Student's t-test or Mann-Whitney U-test. RESULTS: Twelve differentially expressed proteins were screened out. The α-enolase (ENOA) in the sigmoid colon (0.917 ±â€Š0.007 vs. 1.310 ±â€Š0.100, t = 2.643, P = 0.017) and caecum (0.765 ±â€Š0.060 vs. 1.212 ±â€Š0.122, t = 2.225, P = 0.023), Isobutyryl-CoA dehydrogenase (ACAD8) in the sigmoid colon (1.127 ±â€Š0.201 vs. 1.497 ±â€Š0.392, t = 7.093, P = 0.008) of the IBS-D group were significantly lower while acetyl-CoA acetyltransferase (CT) in the caecum (2.453 ±â€Š0.422 vs. 0.931 ±â€Š0.652, t = 8.363, P = 0.015) and ATP synthase subunit d (ATP5H) in the sigmoid (0.843 ±â€Š0.042 vs. 0.631 ±â€Š0.042, t = 8.613,P = 0.007) of the IBS-D group was significantly higher, compared with the controls. The ATP concentration in the mucosa of the sigmoid colon in IBS-D group was significantly lower than that of control group (0.470 [0.180, 1.360] vs. 5.350 [2.230, 7.900], U = 55, P < 0.001). CONCLUSIONS: Many proteins related to energy metabolism presented differential expression patterns in the mucosa of colon of the IBS-D patients. The abnormalities in energy metabolism may be involved in the pathogenesis of IBS which deserves more studies to elucidate.

Exploring the Potential of RET Kinase Inhibition for Irritable Bowel Syndrome: A Preclinical Investigation in Rodent Models of Colonic Hypersensitivity.

Abdominal pain represents a significant complaint in patients with irritable bowel syndrome (IBS). While the etiology of IBS is incompletely understood, prior exposure to gastrointestinal inflammation or psychologic stress is frequently associated with the development of symptoms. Inflammation or stress-induced expression of growth factors or cytokines may contribute to the pathophysiology of IBS. Here, we aimed to investigate the therapeutic potential of inhibiting the receptor of glial cell line-derived neurotrophic factor, rearranged during transfection (RET), in experimental models of inflammation and stress-induced visceral hypersensitivity resembling IBS sequelae. In RET-cyan fluorescent protein [(CFP) RetCFP/+] mice, thoracic and lumbosacral dorsal root ganglia were shown to express RET, which colocalized with calcitonin gene-related peptide. To understand the role of RET in visceral nociception, we employed GSK3179106 as a potent, selective, and gut-restricted RET kinase inhibitor. Colonic hyperalgesia, quantified as exaggerated visceromotor response to graded pressures (0-60 mm Hg) of isobaric colorectal distension (CRD), was produced in multiple rat models induced 1) by colonic irritation, 2) following acute colonic inflammation, 3) by adulthood stress, and 4) by early life stress. In all the rat models, RET inhibition with GSK3179106 attenuated the number of abdominal contractions induced by CRD. Our findings identify a role for RET in visceral nociception. Inhibition of RET kinase with a potent, selective, and gut-restricted small molecule may represent a novel therapeutic strategy for the treatment of IBS through the attenuation of post-inflammatory and stress-induced visceral hypersensitivity.

GUCY2C Signaling Opposes the Acute Radiation-Induced GI Syndrome.

High doses of ionizing radiation induce acute damage to epithelial cells of the gastrointestinal (GI) tract, mediating toxicities restricting the therapeutic efficacy of radiation in cancer and morbidity and mortality in nuclear disasters. No approved prophylaxis or therapy exists for these toxicities, in part reflecting an incomplete understanding of mechanisms contributing to the acute radiation-induced GI syndrome (RIGS). Guanylate cyclase C (GUCY2C) and its hormones guanylin and uroguanylin have recently emerged as one paracrine axis defending intestinal mucosal integrity against mutational, chemical, and inflammatory injury. Here, we reveal a role for the GUCY2C paracrine axis in compensatory mechanisms opposing RIGS. Eliminating GUCY2C signaling exacerbated RIGS, amplifying radiation-induced mortality, weight loss, mucosal bleeding, debilitation, and intestinal dysfunction. Durable expression of GUCY2C, guanylin, and uroguanylin mRNA and protein by intestinal epithelial cells was preserved following lethal irradiation inducing RIGS. Oral delivery of the heat-stable enterotoxin (ST), an exogenous GUCY2C ligand, opposed RIGS, a process requiring p53 activation mediated by dissociation from MDM2. In turn, p53 activation prevented cell death by selectively limiting mitotic catastrophe, but not apoptosis. These studies reveal a role for the GUCY2C paracrine hormone axis as a novel compensatory mechanism opposing RIGS, and they highlight the potential of oral GUCY2C agonists (Linzess; Trulance) to prevent and treat RIGS in cancer therapy and nuclear disasters. Cancer Res; 77(18); 5095-106. ©2017 AACR.

[Disaccharidase deficiency and functional bowel diseases]. / Disakharidaznaia nedostatochnost' i funktsional'nye zabolevaniia kishechnika.

AIM: To elucidate the role of intestinal carbohydrases (glucoamylase, maltase, sucrose, and lactase) in the etiology and pathogenesis of functional bowel diseases (FBD). SUBJECTS AND METHODS: 74 patients (36 men and 38 women) aged 18 to 50 years with FBD were examined. According to Rome IV criteria (2016), there was diarrhea-predominant irritable bowel syndrome (IBS) in 21 patients, functional diarrhea (FD) in 33, constipation-predominant IBS in 6, functional constipation (FC) in 4, and mixed IBS in 10. The activity of carbohydrases in the small intestine mucosa (SIM) was investigated by the Dahlquist method modified by Trinder in the duodenal biopsy specimens obtained during esophagogastroduodenoscopy. RESULTS: Lactase deficiency was identified in 87.8% of the patients; maltase deficiency in 48.6%; sucrose deficiency in 51.3%; and glucoamylase deficiency in 85.1%. The activity of all the investigated enzymes was reduced in 23 (31.1%) patients with FBD; deficiency of 1-3 carbohydrases was found in 47 (63.5%). Normal enzymatic activity was established in 4 (5.4%) patients. CONCLUSION: In the majority of patients with FBD, the intestinal symptoms are caused by the decreased activity of SIM carbohydrases. Therefore, disaccharidase deficiency associated with an established damaging agent (nonsteroidal anti-inflammatory drugs, antibiotics, acute intestinal infections, etc.) should be considered to be a more precise diagnosis.

Epithelial expression and function of trypsin-3 in irritable bowel syndrome.

OBJECTIVES: Proteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity. DESIGN: Trypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin (PRSS1, 2 and 3) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3. RESULTS: We showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism. CONCLUSIONS: In IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity.

Expression of inducible nitric oxide synthase in mast cells contributes to the regulation of inflammatory cytokines in irritable bowel syndrome with diarrhea.

BACKGROUND: Nitric oxide (NO) and mast cells (MCs) are possibly involved in the development of irritable bowel syndrome (IBS), but details on their role and interactions still remain undetermined. We aimed to investigate the expression of inducible NO synthase (iNOS) in MCs of the colon of IBS with diarrhea (IBS-D), and elucidated a potential role of NO in the differential regulation of cytokines in MCs. METHODS: Colonic mucosal biopsies of 19 IBS-D patients and 16 healthy controls were collected. The expression of tryptase and iNOS was investigated by immunohistochemistry, Western blotting, and real-time PCR. Effects of NO on the expression of cytokines in rat bone marrow MCs (BMMCs) were examined using a cytokine array by NG-nitro-l-arginine methyl ester (L-NAME) treatment. KEY RESULTS: Immunohistochemistry for tryptase revealed an increase in number of MCs with extensive iNOS expression in the colonic mucosa of IBS-D. Tryptase, iNOS and interleukin (IL)-1ß mRNA and protein levels were upregulated in IBS-D compared with healthy controls. Specifically, a positive correlation between tryptase and iNOS protein expression was observed in the colon of IBS-D (r = 0.667, p < 0.05). Supernatant from IBS-D increased iNOS expression in BMMCs. Antibody array showed that agrin, beta-nerve growth factor, fractalkine, granulocyte-macrophage colony-stimulating factor, IL-1ß, IL-1R6, IL-13, leptin, tumor necrosis factor alpha were suppressed, and cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2α, CINC-3, monocyte chemotactic protein-1, matrix metalloproteinase-8 were strongly produced in L-NAME treated BMMCs, comparable to levels in the control group. CONCLUSIONS & INFERENCES: Our findings provide new evidence that NO is able to regulate many cytokines in MCs that may be involved in the development of IBS.

Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases.

Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors (PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein.

TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity.

AIM: To investigate the roles of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB on cystathionine ß synthetase (CBS) expression and visceral hypersensitivity in rats. METHODS: This study used 1-7-wk-old male Sprague-Dawley rats. Western blot analysis was employed to measure the expression of TLR4, NF-κB and the endogenous hydrogen sulfide-producing enzyme CBS in colon dorsal root ganglia (DRG) from control and "irritable bowel syndrome" rats induced by neonatal colonic inflammation (NCI). Colon-specific DRG neurons were labeled with Dil and acutely dissociated to measure excitability with patch-clamp techniques. Immunofluorescence was employed to determine the co-expression of TLR4, NF-κB and CBS in DiI-labeled DRG neurons. RESULTS: NCI significantly upregulated the expression of TLR4 in colon-related DRGs (0.34 ± 0.12 vs 0.72 ± 0.02 for the control and NCI groups, respectively, P < 0.05). Intrathecal administration of the TLR4-selective inhibitor CLI-095 significantly enhanced the colorectal distention threshold of NCI rats. CLI-095 treatment also markedly reversed the hyperexcitability of colon-specific DRG neurons and reduced the expression of CBS (1.7 ± 0.1 vs 1.1 ± 0.04, P < 0.05) and of the NF-κB subunit p65 (0.8 ± 0.1 vs 0.5 ± 0.1, P < 0.05). Furthermore, the NF-κB-selective inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the upregulation of CBS (1.0 ± 0.1 vs 0.6 ± 0.1, P < 0.05) and attenuated visceral hypersensitivity in the NCI rats. In vitro, incubation of cultured DRG neurons with the TLR4 agonist lipopolysaccharide significantly enhanced the expression of p65 (control vs 8 h: 0.9 ± 0.1 vs 1.3 ± 0.1; control vs 12 h: 0.9 ± 0.1 vs 1.3 ± 0.1, P < 0.05; control vs 24 h: 0.9 ± 0.1 vs 1.6 ± 0.1, P < 0.01) and CBS (control vs 12 h: 1.0 ± 0.1 vs 2.2 ± 0.4; control vs 24 h: 1.0 ± 0.1 vs 2.6 ± 0.1, P < 0.05), whereas the inhibition of p65 via pre-incubation with PDTC significantly reversed the upregulation of CBS expression (1.2 ± 0.1 vs 0.6 ± 0.0, P < 0.01). CONCLUSION: Our results suggest that the activation of TLR4 by NCI upregulates CBS expression, which is mediated by the NF-κB signaling pathway, thus contributing to visceral hypersensitivity.

[Small intestinal bacterial overgrowth as a cause of lactase deficiency].

AIM: To establish the rate of lactase deficiency (LD) in patients with post-infectious irritable bowel syndrome (PI-IBS), to define a role of enteric bacteria in the pathogenesis of hypolactasia, and to evaluate the efficiency of probiotic therapy. SUBJECTS AND METHODS: Examinations were made in 386 patients with PI-IBS, including 112 (79.4%) women; mean age 33.9 ± 9.1 years; disease duration 2.6 ± 1.4 years. Rapid tests of small intestinal mucosa (SIM) biopsy specimens obtained from the duodenal retrobulbar segment were used to diagnose LD. Bacterial growth was estimated by a hydrogen breath test using a H2 MICRO gas analyzer. RESULTS: The patients with PI-IBS were revealed to have moderate and severe LD in 25.6 and 10.9%, respectively. All the patients with LD were detected to have small intestinal (SI) bacterial overgrowth (BOG). An inverse correlation was found between LD and the degree of SI BOG (r = -0.53; p < 0.001). 73.7% of the patients with moderate LD showed a positive effect of probiotic therapy as regression of clinical symptoms of LD, a decrease of hydrogen levels in expired air from 72.4 ± 25.1 to 16.41 ± 3.2 ppm (p < 0.05), an increase of lactate activity in the SIM biopsy specimens and an improvement of quality of life from 2.69 ± 0.53 to 5.53 ± 0.64 scores according to the GCI scale. No improvement occurred in 73.8% of the patients with severe LD. CONCLUSION: LD was identified in 36.5% of the patients with PI-IBS. There was an inverse correlation between the degree of LD and SI BOG. The good therapeutic effect of probiotics in LD suggests that the symbiotic gut microflora positively affects the activity of lactase in the human SIM. No therapeutic effect of probiotics in patients with severe LN serves as the basis for a search for more active probiotic therapy.

[The role of small bowel microflora in the development of secondary lactase deficiency and the possibilities of its treatment with probiotics].

AIM: To estimate the incidence of secondary lactase deficiency (SLD) in patients with postinfectious irritable bowel syndrome (PIBS) and the value of the small bowel microflora in its development and to elaborate treatment options for SLD. SUBJECTS AND METHODS: One hundred and thirty-eight patients with PIBS, including 112 (81.2%) women and 26 (18.8%) men, were examined. The patients' mean age was 33.9 +/- 9.1 years. The duration of the disease was 2.6 +/- 1.4 years. Lactase deficiency (LD) was diagnosed using the color scale to test biopsy specimens from the duodenal retrobulbar region. The bacterial overgrowth syndrome (BOS) was identified by a 2-hour lactulose (20 ml) hydrogen breath test. Sixty patients with moderate SLD were randomized to 2 groups: 1) 41 patients received basic therapy (mesim forte as one tablet t.i.d., no-spa, 40 mg, t.i.d.) and combined probiotic bifiform (Ferrosan) containing Bifidobacterium longum 107, Enterococcus faecium 107 as one capsule t.i.d. for 14 days. Group 2 patients (n = 19) had basic therapy in combination with placebo. RESULTS: SLD was detected in 59.4% of the patients with PIBS, including 43.5 and 15.9% with moderate and severe forms, respectively. In all cases, SLD was accompanied by BOS in the small bowel lumen, as confirmed by the results of a hydrogen breath test [101 +/- 37 ppm (a normal value of < 20 ppm)]. After a 14-day course of therapy with the combined probiotic bifiform, restoration of eubiosis in the small bowel lumen was achieved in 70.8% of the patients, as shown by the lesser degree of BOS (86.9 +/- 40.9 and 17.4 +/- 6.6 ppm before and after treatment, respectively; p < 0.01) and by normalization of the lactase test (p < 0.01). In the comparative placebo group, 68.4% showed no clear positive changes, SLD and BOS remained. CONCLUSION: The changes in the small bowel intraluminal microflora, which developed after prior intestinal infection, played a great role in the development of SLD. Bifiform belongs to the currently available probiotics and may be recommended to correct SLD in patients with PIBS resulting from the impaired microbiota of the small bowel and to prevent BOS.

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.

Linaclotide, a synthetic guanylate cyclase C agonist, for the treatment of functional gastrointestinal disorders associated with constipation.

Chronic constipation (CC) and irritable bowel syndrome with constipation (IBS-C) are two functional gastrointestinal disorders that are associated with constipation. CC and IBS-C affect approximately 20% of the general population including the elderly, impairing quality of life. Patients not responding to over-the-counter treatments require effective and safe long-term therapies. Some treatments introduced in the last decade have been associated with side effects that led to withdrawal from the US market (e.g., tegaserod) or intolerance to treatment (e.g., nausea in patients treated with lubiprostone). Linaclotide is a novel drug, with a unique mechanism of action, low bioavailability and local action in the intestinal epithelial cells. It is currently being developed for patients with CC and IBS-C. From animal studies to human pharmacodynamic Phase Ib trials, and a comprehensive program of Phase IIb and III trials in health and disease, linaclotide demonstrates long-term efficacy and safety in CC and IBS-C.

Elevated expression of c-fos in central nervous system correlates with visceral hypersensitivity in irritable bowel syndrome (IBS): a new target for IBS treatment.

BACKGROUND AND AIMS: Although visceral hypersensitivity is a major pathophysiological feature of irritable bowel syndrome (IBS), its molecular mechanisms are still poorly understood. c-fos is a well-established marker of cell activation. Accumulating evidence demonstrates that norepinephrine (NE) system is dysregulated in IBS; however, very little is known on its mechanism. It is our hypothesis that elevated expression of c-fos in central nervous system (CNS) correlates with visceral hypersensitivity in rat model of IBS. Furthermore, we explored the changes of NE system in IBS patients. METHODS: The rat model of IBS was induced by heterotypic chronic and acute stress. Tissues obtained from rat model were analyzed for c-fos levels in CNS (frontal lobe, hippocampus, cornu dorsale) and colon by immunohistochemistry. Real-time reverse transcription polymerase chain reaction was used to detect tyrosine hydroxylase (TH) in the colonic tissues obtained from IBS patients. RESULTS: The rat model of IBS was associated with increased expression of c-fos in different parts of CNS (P = 0.001, P = 0.002, and P = 0.002, respectively), but normal in colon (P = 0.207). The clinical parameters (colonic motility and sensation) of rat model were significantly correlated with elevated c-fos in CNS (P < 0.05). Enterochromaffin cells and serotonin in colon were related to the elevated c-fos in CNS (P < 0.05). The TH messenger ribonucleic acid (mRNA level of IBS-D patients was almost four times as much as that of controls. CONCLUSIONS: Elevated expression of c-fos in CNS might be one of key mechanisms in etiology of IBS. Therefore, regulation of CNS activation could be a major targeting effect when treating IBS patients.

Role for protease activity in visceral pain in irritable bowel syndrome.

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.