Article Search
검색
검색 팝업 닫기

Metrics

Help

  • 1. Aims and Scope

    Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE

  • 2. Editorial Board

    Editor-in-Chief + MORE

    Editor-in-Chief
    Yong Chan Lee Professor of Medicine
    Director, Gastrointestinal Research Laboratory
    Veterans Affairs Medical Center, Univ. California San Francisco
    San Francisco, USA

    Deputy Editor

    Deputy Editor
    Jong Pil Im Seoul National University College of Medicine, Seoul, Korea
    Robert S. Bresalier University of Texas M. D. Anderson Cancer Center, Houston, USA
    Steven H. Itzkowitz Mount Sinai Medical Center, NY, USA
  • 3. Editorial Office
  • 4. Articles
  • 5. Instructions for Authors
  • 6. File Download (PDF version)
  • 7. Ethical Standards
  • 8. Peer Review

    All papers submitted to Gut and Liver are reviewed by the editorial team before being sent out for an external peer review to rule out papers that have low priority, insufficient originality, scientific flaws, or the absence of a message of importance to the readers of the Journal. A decision about these papers will usually be made within two or three weeks.
    The remaining articles are usually sent to two reviewers. It would be very helpful if you could suggest a selection of reviewers and include their contact details. We may not always use the reviewers you recommend, but suggesting reviewers will make our reviewer database much richer; in the end, everyone will benefit. We reserve the right to return manuscripts in which no reviewers are suggested.

    The final responsibility for the decision to accept or reject lies with the editors. In many cases, papers may be rejected despite favorable reviews because of editorial policy or a lack of space. The editor retains the right to determine publication priorities, the style of the paper, and to request, if necessary, that the material submitted be shortened for publication.

Search

Search

Year

to

Article Type

Original Article

Split Viewer

Tryptase and Protease-Activated Receptor 2 Expression Levels in Irritable Bowel Syndrome

Wen-Jing Liang*, Guo Zhang*, He-Sheng Luo, Lie-Xin Liang*, Dan Huang*, and Fa-Can Zhang*

*Department of Gastroenterology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China, Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, China

Correspondence to: Fa-Can Zhang, Department of Gastroenterology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China, Tel: +86-0771-2186186, Fax: +86-0771-2186184, E-mail: zhangfacan@126.com

Received: August 18, 2014; Revised: March 16, 2015; Accepted: April 23, 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gut Liver 2016;10(3):382-390. https://doi.org/10.5009/gnl14319

Published online October 12, 2015, Published date May 15, 2016

Copyright © Gut and Liver.

Background/Aims

Previous studies have revealed that mast cells (MCs) may activate the protease-activated receptors and release of neuropeptides involved in the pathogenesis of irritable bowel syndrome (IBS). The levels of protease-activated receptor 2 (PAR-2) and tryptase can contribute to understanding the pathogenesis of IBS.

Methods

Colonoscopic biopsies were performed of 38 subjects (20 with IBS-diarrhea [IBS-D], eight with IBS-constipation [IBS-C], and 10 healthy volunteers). The mRNA and protein levels of tryptase and PAR-2 were assessed by real-time PCR and Western blot. The levels of vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP) were measured by immunohistochemistry, and MCs were counted by toluidine blue staining.

Results

Significant increases in the mRNA expression of tryptase (p<0.05, IBS-D, IBS-C vs control) and PAR-2 (p<0.05, IBS-D, IBS-C vs control) and in the tryptase protein level (p<0.05, IBS-D, IBS-C vs control) were detected in IBS. Elevations of MCs, CGRP, VIP and SP (p<0.05, IBS-D vs control) were observed for IBS-D only.

Conclusions

Tryptase levels may upregulate the function of PAR-2, resulting in the release of neuropeptide and they were correlated with clinical symptoms associated with IBS.

Keywords: Irritable bowel syndrome, Inflammation, PAR-2, Tryptases

Irritable bowel syndrome (IBS) is a multifactorial disease and the mechanisms associated with the onset and progression of IBS still remain unclear. It is generally considered that altered motility of gastrointestinal cells, neural immune endocrine balance, inflammatory response and visceral hypersensitivity are involved in pathophysiology of IBS.

Elevation of mast cell has been observed in IBS patients as well as animal models of the disease.1 Vivinus-N?bot et al.2 have reported an elevation of mast cells in the mucosa of IBS patients and have found a correlation of increased mast cells with altered mucosal permeability. Degranulation of mast cells releases tryptase,3 which is suggested to generate low grade inflammation, visceral hypersensitivity and high permeability4 in IBS. Mast cell tryptase is proposed to activate proinflammatory pathways and influences neuronal signaling by activation of protease-activated receptor 2 (PAR-2).5

PAR-2 belongs to a family of four G protein-coupled receptors which are activated by trypsin, mast cell tryptase, coagulation factors VIIa and Xa, and so forth.6 Previous studies have shown that PAR-2 is linked to visceral pain in the colon and results in an increased recruitment of inflammatory cells.7 Some studies have reported that tryptase can result in hypersensitivity symptoms via activation of PAR-2,8 present on epithelial cells, immune cells, and the terminal afferent nerves.9 However, Fiorucci et al.10 have reported that the activation of PAR-2 has a protective effect against the development of colitis. Activation of PAR-2 produces an inflammatory response and hypersensitivity reaction11 by a neurogenic mechanism involving afferent neurons and release of the calcitonin gene-related peptide (CGRP) and substance P (SP).12 CGRP, SP and vasoactive intestinal peptide (VIP) are neuropeptides present in the central nervous system and the gastrointestinal system and play an important role in the regulation of visceral sensation and gastrointestinal motility. Numerous reports now indicate alterations of neuropeptide in the IBS. However, most investigations demonstrating an association between PAR-2 and IBS are limited to investigations in animal models.8,13,14

Few studies have reported the correlation between tryptase, PAR-2, neuropeptides and clinical symptoms; while several studies on the expression levels of tryptase and PAR-2 report contradictory results. Moreover, there are more studies focusing on IBS-diarrhea (IBS-D) subtype when compared to the IBS-constipation (IBS-C) subtype.

In this study, we investigate the mRNA and protein level expression of mast cell tryptase, PAR-2 and neuropeptides in the colon of IBS patients and their correlation with clinical symptoms of IBS patients.

1. Subjects

Biopsy specimens of the ascending colon were drawn from colonoscopies of 20 patients with IBS-D, eight patients with IBS-C, and 10 healthy controls at the Guangxi Zhuang Autonomous Region People’s Hospital from May 1, 2012 to November 29, 2012. All the IBS patients exhibited symptoms that meet the Rome-III criteria (Table 1). Subjects in the control group had no gastrointestinal symptoms, and no history of chronic medical disease. None of the IBS patients had asthma and other allergic diseases, history of abdominal surgery, inflammatory bowel disease, or postinfectious IBS. Informed consent was signed by all patients, and this study was approved by the hospital’s local ethics committee.

2. Reagents

Trizol and cDNA reverse transcription kit was from Roche (Roche, Mannheim, Germany). Polymerase chain reaction (PCR) primers were synthesized by Sangon Biotechnology (Shanghai, China). SuperReal PreMix Plus was purchased from Tiangen Biotechnology (Beijing, China). Rabbit anti-GAPDH and anti-PAR-2 polyclonal antibody for Western blot were from Cell Signaling Technology (Danvers, MA, USA). Antitryptase polyclonal antibody for Western blot was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All horseradish peroxidase-conjugated secondary antibodies were from Beyond Time (Beijing, China). Anti-SP, anti-CGRP, and anti-VIP antibody for immunohistochemistry was from Bioss Biotechnology (Beijing, China). SP-9000/9001/9002Histostain-Plus Kits was from ZSGB Biotechnology (Beijing, China). Toluidine blue dye was purchased from Solarbio Biotechnology (Beijing, China).

3. Staining process of H&E and toluidine blue method

Tissues were treated with a series of fixation, dehydration, embedding, and staining. Samples were deparaffinized in xylene, hematoxylin and eosin staining, dehydrated in 95%, 90%, 80% ethanol, cleared in xylene, checked under microscope to observe morphological change of human colonic membrane (Fig. 1). Sample was selected in the descending colon and cleaned with normal saline, fixed with 10% in formalin, dehydrated, paraffin embedded, continuously slided, toluidine blue method stained, deparaffinized and rehydrated, dipped in toluidine blue for 30 minutes, 2 to 3 drops of glacial acetic acid, until the nucleus and granulation were pretty clear, dried with cold air, cleared in xylene mounted in checked under microscope, randomly selected three high-power fields (×200) and counted the number of cells, expressing mast cell counts.

4. Real-time polymerase chain reaction

Tissue expression of tryptase and PAR-2 mRNA was assessed by quantitative real-time PCR (RT-PCR). After thawing, tissue was homogenized in Trizol (800 μL added to 5 mg tissue). Total RNA was extracted, and reversed by using reverse transcription kit after measuring concentration of total RNA with Nano drop. Total RNA is quantitatived to 1 μg. The information of primers of tryptase, PAR-2 and actin is listed as Table 2. The PCR reactions were set up in a volume of 20 μL, containing 2 μL of the cDNA diluted 1:5 with PCR grade water, 10.4 μL of SYBR Green Supermix, and 300 nmol/L of the forward and reverse primer each. The PCR conditions consisted of preliminary denaturation at 95°C for 15 minutes, followed by 50 cycles of 95°C for 10 seconds, 60°C for 32 seconds. Melting curve analysis was performed to confirm the specificity as well as the integrity of the PCR products by the presence of a single peak. Products were subjected to agarose gel electrophoresis (Fig. 2). Expression levels of the mRNA were quantified by calculating threshold cycle values compare with the β-actin endogenous control using 2-DDCt.

5. Western blot

Total protein was extracted from colon musocal tissues and lysed in RIPA lysate (50 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 [v/v], 1× protease inhibitor cocktail, 1 mM sodium vanadate, and 10 mM NaF). Equal amount of total protein were separated by SDS-PAGE with 12% resolving layer and 4% stacking layer and then transferred to PVDF membranes with 200 mmA for 1 hour. After blocked in 5% notfat milk diluted with TBS-T for 1 hour in room temperature with shaking, membranes were incubated with primary antibodies (working dilution tryptase 1:3,000; PAR-2 1:100) with gentle shaking overnight at 4°C, washed with Tris-buffered saline and then incubated with species-appropriate secondary antibodies for 90 minutes at 37°C. After washing in TBS-T, membranes were incubated with ECL Developer Kit in dark room. The efficiency of protein loading and transfer was assessed by reprobing membranes with an anti-GAPDH antibody. Density of each band was analysis using Image J (http://rsb.info.nih.gov/ij/). Expression of the target protein band was compared with the corresponding control band and the overall trend in protein expression.

6. Immunohistochemistry

Colonoscopy was done using a standard bowel preparation and all colonic biopsy samples from 28 IBS patients and five control subjects were taken from the descending colon. One tissue sample (3 to 5 mg) from each patient and control subject was immediately fixed in buffered 10% formalin and processed for immunohistochemistry. For immunohistochemistry, paraffin-embedded specimens were cut with a microtome at 4 μm. The process ran as follows. Briefly, sections antigen retrieval were performed by boiling in sodium citrate buffer solution for 2 minutes and 30 seconds, and nonspecific antigens were blocked for 15 minutes with normal goat serum fluid. Slides were incubated overnight at 4°C with primary antibodies (working dilution anti-SP, 1:300; anti-CGRP, 1:300; anti-VIP, 1:400). Then the peroxidase-conjugated secondary antibodies was added to slides for 30 minutes at 37°C after washing in PBS and then staining with the DAB kits. The negative control was incubated with blocking buffer alone without primary antibody. Photographs were taken on an Olympus microscope at 200× magnification. The difference in the protein expression was evaluated by the Image-pro Plus 6.0 image analysis software (http://www.mediacy.com/index.aspx?page=IPP).

7. Statistical analysis

Experimental data is represented as mean±standard deviation. One-way analysis of variance was used for analysis. Nonparametric test (Kruskal-Wallis H test) was used for the analysis of nonnormal distribution data. Spearman analysis was used for correlation analysis. A p<0.05 was considered statistically significant. SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis.

1. Elevated mRNA levels of tryptase and PAR-2

mRNA levels of tryptase (p=0.002, IBS-D vs control; p=0.000, IBS-C vs control) and PAR-2 (p=0.007, IBS-D vs control; p=0.000, IBS-C vs control) were higher in IBS patient group in comparison to the healthy controls (Fig. 3). However, no significant difference was observed between IBS-D and IBS-C patients.

2. Different expression of tryptase and PAR-2

Elevated expression of tryptase was found in IBS group (p=0.002, IBS-D vs control; p=0.041, IBS-C vs control) (Fig. 4). There was no significant difference in the protein levels of PAR-2 between IBS and controls. No difference was detected in IBS-D when compared to the levels in IBS-C group.

3. Elevation of mast cells, CGRP, VIP, and SP in IBS-D

We found markedly different levels of neuropeptide between subgroups of IBS patients. Elevation of mast cells, CGRP, VIP, and SP were observed in IBS-D only (p=0.014, p=0.021, p=0.005, p=0.021 vs control) (Figs 5?8).

4. Correlation analysis

The scoring criteria for abdominal pain is as follows: 1 (mild) slight abdominal pain, does not affect daily activities, 2 (moderate) moderate abdominal pain, not easy to ignore, need to adjust daily activities, 3 (severe) severe abdominal pain, daily activities affected and need to be adjusted. Severity of abdominal bloating was scored using a 1?3 scale (1: Mild; occasional bloating, about half an hour to ease, 2: Moderate; frequent bloating for 1?2 hours cannot be relieved, 3: Severe; bloating persists for more than 2 hours and cannot be relieved except after taking medication). A correlation between mast cell number, tryptase, PAR-2, VIP, SP and CGRP and symptoms associated with IBS was detected. The presence of mast cells and the expression of tryptase, VIP and SP were associated with abdominal pain. A correlation between mast cell number and abdominal bloating was also found (Table 3).

Our study demonstrates an elevated expression of mast cell tryptase in IBS patients when compared with the control group. We also observed increased mRNA levels of PAR-2, as seen from the RT-PCR analysis. Up regulation of CGRP, SP, VIP was detected in IBS-D. No significant difference was found in the protein levels of PAR-2 between IBS patients and control subjects. We found a positive correlation between the levels of mast cell tryptase, PAR-2, neuropeptides and symptoms of IBS such as abdominal pain and bloating.

Previous studies have shown that mast cells and tryptase play an important role in IBS. Some studies have revealed an elevation of mast cell and tryptase, which are consistent with the findings of our study. However, some experiments show an increased level of mast cell tryptase in IBS-D and not in IBS-C, which may be attributed to the differences in experimental methods as well as sample size. Barbara et al.15 revealed that colonic mast cells in proximity to mucosal nerves may contribute to abdominal pain in IBS. PAR-2 is ubiquitously expressed on gastrointestinal mucosal cells, smooth muscle cells and neurons. It has been shown in previous studies that activation of PAR-2 induces a wide range of processes such as triggering intestinal secretion, modulating gatrointestinal motility,16,17 participating in colonic inflammatory reactions, visceral hypersensitivity,18 and high paracellular permeability. Other studies indicate contrasting results regarding the expression of PAR-2 in IBS. Lee et al.19 found no differences in the mRNA expression levels of PAR-2 in IBS-D when compared to healthy controls. Kim et al.20 have reported that PAR-2-positive cells numbers were significantly higher stressed rats when compared to nonstressed rats (where stress was used to mimic visceral hypersensitivity and intestinal permeability related to IBS). The results above focus on the expression of PAR-2 in IBS-D. We were not able to find difference in protein expression levels of PAR-2, which is consistent with the results of Bian et al.21 There is a possibility that PAR-2 contributes to the development of IBS upon its activation, rather than the level of mRNA or protein expression. This may explain no significant increase in the expression levels of PAR-2.

PAR-2 may perform different functions between IBS-D and IBS-C, resulting in different clinical symptoms. Tryptase activated PAR-2-positive neurons release neuropeptides, such as CGRP, SP, and VIP, which further play an important role in regulating intestinal motility. The enteric nerve interacts with intestinal mast cells by neuropeptides such as SP, VIP, and CGRP. Furthermore, enteric nerves are activated by mast cell mediators, interacting with intestinal epithelial cells, vascular cells and multiple inflammatory and immune cells. Hence, neuropeptide is thought to regulate gut motility and secretion and modulate visceral pain. An elevation of CGRP, SP, and VIP was detected in IBS-D compared with control. The mechanism of the involvement of neuropeptides in the pathological process of IBS has not yet been elucidated. The neuropeptide receptor subtypes perform different functions and are differentially distributed in the gut. In summary, our data indicates that mast cell tryptase might be responsible for the activation of PAR-2 and once activated, PAR-2 regulates neuropeptide secretion which is responsible for symptoms associated with IBS. The expression of mast cell tryptase, SP, and VIP in the intestinal mucosa is positively correlated with abdominal pain in patients with IBS. There is a correlation between mast cells and the expression of tryptase, SP and VIP.

Interestingly, there’s a discrepancy between protein and mRNA levels of PAR-2, in contrast to those of tryptase levels. The absence of mRNA?protein correlation for a subset of investigated genes suggests that the relationship between mRNA and protein content is not strictly linear. Different regulatory mechanisms (such as synthesis and degradation rates), acting on both the synthesized mRNA and the synthesized protein can affect the expression levels of the two molecules differentially. Further research investigating the biosynthesis and degradation of mRNA and protein levels of PAR-2 and tryptase may unravel the reasons for the discrepancies observed.

One of the limitations of the current study is the small sample size of patients. And our study focused on only part of colon. RT-PCR, Western blot, and immunohistochemistry were used to detect expression of tryptase, PAR-2 and some neuropeptides. RT-PCR is a very sensitive technique that can detect low copy numbers of RNA by exponential amplification. Western blotting has high sensitivity of detecting protein. Immunohistochemistry help to reveal the location of protein in tissue. These methods are used widely in previous studies of tryptase, PAR-2, and neuropeptides.

Our work evidences that mast cells are up regulated in IBS and mast cell tryptase may activate PAR-2, which regulates the expression of neuropeptides, resulting in symptoms associated with IBD. Thus, we could conclude that increased activation of PAR-2 rather than the increased expression levels may be responsible for symptoms of IBS. Further studies investigating the mechanisms regulating PAR-2 activation will help shed light on the signaling mechanisms associated with the development of IBS and at the same time, targeting the mast cell-PAR-2 signaling might help in developing therapeutic measures against IBS.

The authors thank Dr. Wei Jiao, Dr. Fei Liu, Dr. Jiao Lan and Rui-Ping Xiao from Scientific Research Center of the People’s Hospital of Guangxi Zhuang Autonomous Region for their valuable technological assistance on our project.

This work was supported by grants from the National Natural Science Foundation of China (number: 81160053 to F.C.Z.) the URL of website is http://isisn.nsfc.gov.cn/egrantindex/funcindex/prjsearch-list. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

No potential conflict of interest relevant to this article was reported.

Fig. 1.H&E staining showed a normal structure of the intestinal mucosa; inflammatory cells were observed in the irritable bowel syndrome-diarrhea (IBS-D), irritable bowel syndrome-constipation (IBS-C), and normal groups. (A) Control subjects (×200); (B) IBS-D patients (×200); (C) IBS-C patients (×200).
Fig. 2.Polymerase chain reaction (PCR) products were subjected to agarose gel electrophoresis. All the real-time-PCR products from the three groups migrated as expected (A: actin, 206 bp; T: tryptase, 216 bp; P: protease-activated receptor 2, 108 bp).

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation.


Fig. 3.Quantification of tryptase and protease-activated receptor 2 (PAR-2) mRNA after normalization to the housekeeping gene β-actin. (A) Tryptase mRNA; (B) PAR-2 mRNA. Significant elevation of tryptase and PAR-2 expression was observed in irritable bowel syndrome patients compared with the control group, but no difference was observed in subtypes. The fold change in mRNA expression was calculated relative to the endogenous β-actin control using 2?ΔΔCt. The data are expressed as the mean±SEM.

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation. *p<0.05 compared with control subjects.


Fig. 4.The expression of tryptase and protease-activated receptor 2 (PAR-2) in the colon mucosa. The expression results were compared with healthy controls. The density of each individual band was compared with the corresponding control band and normalized to the GAPDH gene. (A) The level of tryptase increased in the irritable bowel syndrome (IBS) group, but no significant difference was observed in subtypes of IBS. (B) There was no difference in the expression levels of PAR-2 between IBS patients and the control group.

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation. *p<0.05 compared with control group.


Fig. 5.The number of mast cells in the colon was detected by the toluidine blue method (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) Evaluation of the density (mean) showed an elevation of mast cell numbers in IBS-D patients compared to control patients, and IBS-D patients showed an increased level of tryptase compared to IBS-C patients. *p<0.05 compared with control subjects.
Fig. 6.The expression of calcitonin gene-related peptide (CGRP) in the colon as evaluated by immunohistochemistry (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) An increase of CGRP was observed in the IBS-D group compared to the control group, and there was a significant difference between the IBS-D and IBS-C groups. *p<0.05 compared with control subjects; p<0.05 compared with IBS-C subjects.
Fig. 7.The expression of vasoactive intestinal peptide (VIP) in the colon as evaluated by immunohistochemistry (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) An increase in VIP was found in IBS-D patients compared to control subjects, and there was a significant difference between the IBS-D and IBS-C groups. *p<0.05 compared with control subjects; p<0.05 compared with IBS-C patients.
Fig. 8.The expression of substance P (SP) in the colon as evaluated by immunohistochemistry (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) An increase in SP was found in IBS-D patients compared to control subjects, and there was a significant difference between the IBS-D and IBS-C groups. *p<0.05 compared with control subjects; p<0.05 compared with IBS-C subjects.

Clinical Characteristic of Irritable Bowel Syndrome Patients and Control Subjects

CharacteristicControl (n=10)IBS-D (n=20)IBS-C (n=8)
Age, mean (range), yr45.9 (30?63)41.8 (24?64)34.1 (24?54)
Sex, female/male5/513/76/2
Abdominal discomfort04.15±0.764.38±0.64
Bloating03.15±1.714.19±1.53
Altering diarrhea/constipation000
History of infection000

Primers for Real-Time Polymerase Chain Reaction

GeneForward primer/reverse primerPCR product, bpGene bank no.
ActinTgACgTggACATCCgCAAAg/CTggAAggTggACAgCgAgg205NM_001101.3
TryptaseATCgTgCACCCACAgTTCTAC/CTTCACCTgCTTCAgAggAAAT216NM_024164.5
PAR-2CCCTgAACATCACgACCTg/AggCTgggAACAgAAAgACC108NM_005242.4

Relationship between Tryptase/Protease-Activated Receptor 2 and Abdominal Pain and Bloating

Abdominal painAbdominal bloating
Mast cellr=0.391*r=0.405*
Tryptaser=0.498*r=?0.073
PAR-2r=0.123r=0.120
VIPr=0.399*r=?0.148
SPr=?0.445*r=?0.037
CGRPr=0.119r=?0.188

PAR-2, protease-activated receptor 2; r, correlation coefficient; VIP, vasoactive intestinal peptide; SP, substance P; CGRP, calcitonin gene-related peptide.

*p<0.05.


  1. Mart?nez, C, Lobo, B, and Pigrau, M (2013). Diarrhoea-predominant irritable bowel syndrome: an organic disorder with structural abnormalities in the jejunal epithelial barrier. Gut. 62, 1160-1168.
    CrossRef
  2. Vivinus-N?bot, M, Dainese, R, and Anty, R (2012). Combination of allergic factors can worsen diarrheic irritable bowel syndrome: role of barrier defects and mast cells. Am J Gastroenterol. 107, 75-81.
    CrossRef
  3. Miller, HR, and Pemberton, AD (2002). Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology. 105, 375-390.
    Pubmed KoreaMed CrossRef
  4. Wilcz-Villega, EM, McClean, S, and O’Sullivan, MA (2013). Mast cell tryptase reduces junctional adhesion molecule-A (JAM-A) expression in intestinal epithelial cells: implications for the mechanisms of barrier dysfunction in irritable bowel syndrome. Am J Gastroenterol. 108, 1140-1151.
    Pubmed CrossRef
  5. Cenac, N, Andrews, CN, and Holzhausen, M (2007). Role for protease activity in visceral pain in irritable bowel syndrome. J Clin Invest. 117, 636-647.
    Pubmed KoreaMed CrossRef
  6. Oikonomopoulou, K, Hansen, KK, and Saifeddine, M (2006). Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs). Biol Chem. 387, 817-824.
    Pubmed
  7. Cenac, N, Coelho, AM, and Nguyen, C (2002). Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2. Am J Pathol. 161, 1903-1915.
    Pubmed KoreaMed CrossRef
  8. Reed, DE, Barajas-Lopez, C, and Cottrell, G (2003). Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons. J Physiol. 547, 531-542.
    Pubmed KoreaMed CrossRef
  9. Jacob, C, Yang, PC, and Darmoul, D (2005). Mast cell tryptase controls paracellular permeability of the intestine: role of protease-activated receptor 2 and beta-arrestins. J Biol Chem. 280, 31936-31948.
    Pubmed CrossRef
  10. Fiorucci, S, Mencarelli, A, and Palazzetti, B (2001). Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis. Proc Natl Acad Sci U S A. 98, 13936-13941.
    Pubmed KoreaMed CrossRef
  11. Demir, IE, Sch?fer, KH, Tieftrunk, E, Friess, H, and Ceyhan, GO (2013). Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity. Acta Neuropathol. 125, 491-509.
    Pubmed CrossRef
  12. Steinhoff, M, Vergnolle, N, and Young, SH (2000). Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med. 6, 151-158.
    Pubmed CrossRef
  13. Nozdrachev, AD, Akoev, GN, Filippova, LV, Sherman, NO, Lioudyno, MI, and Makarov, FN (1999). Changes in afferent impulse activity of small intestine mesenteric nerves in response to antigen challenge. Neuroscience. 94, 1339-1342.
    CrossRef
  14. Bueno, L, Fioramonti, J, Delvaux, M, and Frexinos, J (1997). Mediators and pharmacology of visceral sensitivity: from basic to clinical investigations. Gastroenterology. 112, 1714-1743.
    Pubmed CrossRef
  15. Barbara, G, Stanghellini, V, and De Giorgio, R (2004). Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology. 126, 693-702.
    Pubmed CrossRef
  16. Corvera, CU, D?ry, O, and McConalogue, K (1997). Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. J Clin Invest. 100, 1383-1393.
    Pubmed KoreaMed CrossRef
  17. Duerksen, DR, Wilhelm-Boyles, C, and Parry, DM (2005). Intestinal permeability in long-term follow-up of patients with celiac disease on a gluten-free diet. Dig Dis Sci. 50, 785-790.
    Pubmed CrossRef
  18. Coelho, AM, Vergnolle, N, Guiard, B, Fioramonti, J, and Bueno, L (2002). Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats. Gastroenterology. 122, 1035-1047.
    Pubmed CrossRef
  19. Lee, JW, Park, JH, and Park, DI (2010). Subjects with diarrhea-predominant IBS have increased rectal permeability responsive to tryptase. Dig Dis Sci. 55, 2922-2928.
    Pubmed CrossRef
  20. Kim, DH, Cho, YJ, Kim, JH, Kim, YB, and Lee, KJ (2010). Stress-induced alterations in mast cell numbers and proteinase-activated receptor-2 expression of the colon: role of corticotrophin-releasing factor. J Korean Med Sci. 25, 1330-1335.
    Pubmed KoreaMed CrossRef
  21. Bian, ZX, Li, Z, and Huang, ZX (2009). Unbalanced expression of protease-activated receptors-1 and -2 in the colon of diarrhea-predominant irritable bowel syndrome patients. J Gastroenterol. 44, 666-674.
    Pubmed CrossRef

Article

Original Article

Gut and Liver 2016; 10(3): 382-390

Published online May 15, 2016 https://doi.org/10.5009/gnl14319

Copyright © Gut and Liver.

Tryptase and Protease-Activated Receptor 2 Expression Levels in Irritable Bowel Syndrome

Wen-Jing Liang*, Guo Zhang*, He-Sheng Luo, Lie-Xin Liang*, Dan Huang*, and Fa-Can Zhang*

*Department of Gastroenterology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China, Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, China

Correspondence to:Fa-Can Zhang, Department of Gastroenterology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China, Tel: +86-0771-2186186, Fax: +86-0771-2186184, E-mail: zhangfacan@126.com

Received: August 18, 2014; Revised: March 16, 2015; Accepted: April 23, 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims

Previous studies have revealed that mast cells (MCs) may activate the protease-activated receptors and release of neuropeptides involved in the pathogenesis of irritable bowel syndrome (IBS). The levels of protease-activated receptor 2 (PAR-2) and tryptase can contribute to understanding the pathogenesis of IBS.

Methods

Colonoscopic biopsies were performed of 38 subjects (20 with IBS-diarrhea [IBS-D], eight with IBS-constipation [IBS-C], and 10 healthy volunteers). The mRNA and protein levels of tryptase and PAR-2 were assessed by real-time PCR and Western blot. The levels of vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP) were measured by immunohistochemistry, and MCs were counted by toluidine blue staining.

Results

Significant increases in the mRNA expression of tryptase (p<0.05, IBS-D, IBS-C vs control) and PAR-2 (p<0.05, IBS-D, IBS-C vs control) and in the tryptase protein level (p<0.05, IBS-D, IBS-C vs control) were detected in IBS. Elevations of MCs, CGRP, VIP and SP (p<0.05, IBS-D vs control) were observed for IBS-D only.

Conclusions

Tryptase levels may upregulate the function of PAR-2, resulting in the release of neuropeptide and they were correlated with clinical symptoms associated with IBS.

Keywords: Irritable bowel syndrome, Inflammation, PAR-2, Tryptases

INTRODUCTION

Irritable bowel syndrome (IBS) is a multifactorial disease and the mechanisms associated with the onset and progression of IBS still remain unclear. It is generally considered that altered motility of gastrointestinal cells, neural immune endocrine balance, inflammatory response and visceral hypersensitivity are involved in pathophysiology of IBS.

Elevation of mast cell has been observed in IBS patients as well as animal models of the disease.1 Vivinus-N?bot et al.2 have reported an elevation of mast cells in the mucosa of IBS patients and have found a correlation of increased mast cells with altered mucosal permeability. Degranulation of mast cells releases tryptase,3 which is suggested to generate low grade inflammation, visceral hypersensitivity and high permeability4 in IBS. Mast cell tryptase is proposed to activate proinflammatory pathways and influences neuronal signaling by activation of protease-activated receptor 2 (PAR-2).5

PAR-2 belongs to a family of four G protein-coupled receptors which are activated by trypsin, mast cell tryptase, coagulation factors VIIa and Xa, and so forth.6 Previous studies have shown that PAR-2 is linked to visceral pain in the colon and results in an increased recruitment of inflammatory cells.7 Some studies have reported that tryptase can result in hypersensitivity symptoms via activation of PAR-2,8 present on epithelial cells, immune cells, and the terminal afferent nerves.9 However, Fiorucci et al.10 have reported that the activation of PAR-2 has a protective effect against the development of colitis. Activation of PAR-2 produces an inflammatory response and hypersensitivity reaction11 by a neurogenic mechanism involving afferent neurons and release of the calcitonin gene-related peptide (CGRP) and substance P (SP).12 CGRP, SP and vasoactive intestinal peptide (VIP) are neuropeptides present in the central nervous system and the gastrointestinal system and play an important role in the regulation of visceral sensation and gastrointestinal motility. Numerous reports now indicate alterations of neuropeptide in the IBS. However, most investigations demonstrating an association between PAR-2 and IBS are limited to investigations in animal models.8,13,14

Few studies have reported the correlation between tryptase, PAR-2, neuropeptides and clinical symptoms; while several studies on the expression levels of tryptase and PAR-2 report contradictory results. Moreover, there are more studies focusing on IBS-diarrhea (IBS-D) subtype when compared to the IBS-constipation (IBS-C) subtype.

In this study, we investigate the mRNA and protein level expression of mast cell tryptase, PAR-2 and neuropeptides in the colon of IBS patients and their correlation with clinical symptoms of IBS patients.

MATERIALS AND METHODS

1. Subjects

Biopsy specimens of the ascending colon were drawn from colonoscopies of 20 patients with IBS-D, eight patients with IBS-C, and 10 healthy controls at the Guangxi Zhuang Autonomous Region People’s Hospital from May 1, 2012 to November 29, 2012. All the IBS patients exhibited symptoms that meet the Rome-III criteria (Table 1). Subjects in the control group had no gastrointestinal symptoms, and no history of chronic medical disease. None of the IBS patients had asthma and other allergic diseases, history of abdominal surgery, inflammatory bowel disease, or postinfectious IBS. Informed consent was signed by all patients, and this study was approved by the hospital’s local ethics committee.

2. Reagents

Trizol and cDNA reverse transcription kit was from Roche (Roche, Mannheim, Germany). Polymerase chain reaction (PCR) primers were synthesized by Sangon Biotechnology (Shanghai, China). SuperReal PreMix Plus was purchased from Tiangen Biotechnology (Beijing, China). Rabbit anti-GAPDH and anti-PAR-2 polyclonal antibody for Western blot were from Cell Signaling Technology (Danvers, MA, USA). Antitryptase polyclonal antibody for Western blot was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All horseradish peroxidase-conjugated secondary antibodies were from Beyond Time (Beijing, China). Anti-SP, anti-CGRP, and anti-VIP antibody for immunohistochemistry was from Bioss Biotechnology (Beijing, China). SP-9000/9001/9002Histostain-Plus Kits was from ZSGB Biotechnology (Beijing, China). Toluidine blue dye was purchased from Solarbio Biotechnology (Beijing, China).

3. Staining process of H&E and toluidine blue method

Tissues were treated with a series of fixation, dehydration, embedding, and staining. Samples were deparaffinized in xylene, hematoxylin and eosin staining, dehydrated in 95%, 90%, 80% ethanol, cleared in xylene, checked under microscope to observe morphological change of human colonic membrane (Fig. 1). Sample was selected in the descending colon and cleaned with normal saline, fixed with 10% in formalin, dehydrated, paraffin embedded, continuously slided, toluidine blue method stained, deparaffinized and rehydrated, dipped in toluidine blue for 30 minutes, 2 to 3 drops of glacial acetic acid, until the nucleus and granulation were pretty clear, dried with cold air, cleared in xylene mounted in checked under microscope, randomly selected three high-power fields (×200) and counted the number of cells, expressing mast cell counts.

4. Real-time polymerase chain reaction

Tissue expression of tryptase and PAR-2 mRNA was assessed by quantitative real-time PCR (RT-PCR). After thawing, tissue was homogenized in Trizol (800 μL added to 5 mg tissue). Total RNA was extracted, and reversed by using reverse transcription kit after measuring concentration of total RNA with Nano drop. Total RNA is quantitatived to 1 μg. The information of primers of tryptase, PAR-2 and actin is listed as Table 2. The PCR reactions were set up in a volume of 20 μL, containing 2 μL of the cDNA diluted 1:5 with PCR grade water, 10.4 μL of SYBR Green Supermix, and 300 nmol/L of the forward and reverse primer each. The PCR conditions consisted of preliminary denaturation at 95°C for 15 minutes, followed by 50 cycles of 95°C for 10 seconds, 60°C for 32 seconds. Melting curve analysis was performed to confirm the specificity as well as the integrity of the PCR products by the presence of a single peak. Products were subjected to agarose gel electrophoresis (Fig. 2). Expression levels of the mRNA were quantified by calculating threshold cycle values compare with the β-actin endogenous control using 2-DDCt.

5. Western blot

Total protein was extracted from colon musocal tissues and lysed in RIPA lysate (50 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 [v/v], 1× protease inhibitor cocktail, 1 mM sodium vanadate, and 10 mM NaF). Equal amount of total protein were separated by SDS-PAGE with 12% resolving layer and 4% stacking layer and then transferred to PVDF membranes with 200 mmA for 1 hour. After blocked in 5% notfat milk diluted with TBS-T for 1 hour in room temperature with shaking, membranes were incubated with primary antibodies (working dilution tryptase 1:3,000; PAR-2 1:100) with gentle shaking overnight at 4°C, washed with Tris-buffered saline and then incubated with species-appropriate secondary antibodies for 90 minutes at 37°C. After washing in TBS-T, membranes were incubated with ECL Developer Kit in dark room. The efficiency of protein loading and transfer was assessed by reprobing membranes with an anti-GAPDH antibody. Density of each band was analysis using Image J (http://rsb.info.nih.gov/ij/). Expression of the target protein band was compared with the corresponding control band and the overall trend in protein expression.

6. Immunohistochemistry

Colonoscopy was done using a standard bowel preparation and all colonic biopsy samples from 28 IBS patients and five control subjects were taken from the descending colon. One tissue sample (3 to 5 mg) from each patient and control subject was immediately fixed in buffered 10% formalin and processed for immunohistochemistry. For immunohistochemistry, paraffin-embedded specimens were cut with a microtome at 4 μm. The process ran as follows. Briefly, sections antigen retrieval were performed by boiling in sodium citrate buffer solution for 2 minutes and 30 seconds, and nonspecific antigens were blocked for 15 minutes with normal goat serum fluid. Slides were incubated overnight at 4°C with primary antibodies (working dilution anti-SP, 1:300; anti-CGRP, 1:300; anti-VIP, 1:400). Then the peroxidase-conjugated secondary antibodies was added to slides for 30 minutes at 37°C after washing in PBS and then staining with the DAB kits. The negative control was incubated with blocking buffer alone without primary antibody. Photographs were taken on an Olympus microscope at 200× magnification. The difference in the protein expression was evaluated by the Image-pro Plus 6.0 image analysis software (http://www.mediacy.com/index.aspx?page=IPP).

7. Statistical analysis

Experimental data is represented as mean±standard deviation. One-way analysis of variance was used for analysis. Nonparametric test (Kruskal-Wallis H test) was used for the analysis of nonnormal distribution data. Spearman analysis was used for correlation analysis. A p<0.05 was considered statistically significant. SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis.

RESULTS

1. Elevated mRNA levels of tryptase and PAR-2

mRNA levels of tryptase (p=0.002, IBS-D vs control; p=0.000, IBS-C vs control) and PAR-2 (p=0.007, IBS-D vs control; p=0.000, IBS-C vs control) were higher in IBS patient group in comparison to the healthy controls (Fig. 3). However, no significant difference was observed between IBS-D and IBS-C patients.

2. Different expression of tryptase and PAR-2

Elevated expression of tryptase was found in IBS group (p=0.002, IBS-D vs control; p=0.041, IBS-C vs control) (Fig. 4). There was no significant difference in the protein levels of PAR-2 between IBS and controls. No difference was detected in IBS-D when compared to the levels in IBS-C group.

3. Elevation of mast cells, CGRP, VIP, and SP in IBS-D

We found markedly different levels of neuropeptide between subgroups of IBS patients. Elevation of mast cells, CGRP, VIP, and SP were observed in IBS-D only (p=0.014, p=0.021, p=0.005, p=0.021 vs control) (Figs 5?8).

4. Correlation analysis

The scoring criteria for abdominal pain is as follows: 1 (mild) slight abdominal pain, does not affect daily activities, 2 (moderate) moderate abdominal pain, not easy to ignore, need to adjust daily activities, 3 (severe) severe abdominal pain, daily activities affected and need to be adjusted. Severity of abdominal bloating was scored using a 1?3 scale (1: Mild; occasional bloating, about half an hour to ease, 2: Moderate; frequent bloating for 1?2 hours cannot be relieved, 3: Severe; bloating persists for more than 2 hours and cannot be relieved except after taking medication). A correlation between mast cell number, tryptase, PAR-2, VIP, SP and CGRP and symptoms associated with IBS was detected. The presence of mast cells and the expression of tryptase, VIP and SP were associated with abdominal pain. A correlation between mast cell number and abdominal bloating was also found (Table 3).

DISCUSSION

Our study demonstrates an elevated expression of mast cell tryptase in IBS patients when compared with the control group. We also observed increased mRNA levels of PAR-2, as seen from the RT-PCR analysis. Up regulation of CGRP, SP, VIP was detected in IBS-D. No significant difference was found in the protein levels of PAR-2 between IBS patients and control subjects. We found a positive correlation between the levels of mast cell tryptase, PAR-2, neuropeptides and symptoms of IBS such as abdominal pain and bloating.

Previous studies have shown that mast cells and tryptase play an important role in IBS. Some studies have revealed an elevation of mast cell and tryptase, which are consistent with the findings of our study. However, some experiments show an increased level of mast cell tryptase in IBS-D and not in IBS-C, which may be attributed to the differences in experimental methods as well as sample size. Barbara et al.15 revealed that colonic mast cells in proximity to mucosal nerves may contribute to abdominal pain in IBS. PAR-2 is ubiquitously expressed on gastrointestinal mucosal cells, smooth muscle cells and neurons. It has been shown in previous studies that activation of PAR-2 induces a wide range of processes such as triggering intestinal secretion, modulating gatrointestinal motility,16,17 participating in colonic inflammatory reactions, visceral hypersensitivity,18 and high paracellular permeability. Other studies indicate contrasting results regarding the expression of PAR-2 in IBS. Lee et al.19 found no differences in the mRNA expression levels of PAR-2 in IBS-D when compared to healthy controls. Kim et al.20 have reported that PAR-2-positive cells numbers were significantly higher stressed rats when compared to nonstressed rats (where stress was used to mimic visceral hypersensitivity and intestinal permeability related to IBS). The results above focus on the expression of PAR-2 in IBS-D. We were not able to find difference in protein expression levels of PAR-2, which is consistent with the results of Bian et al.21 There is a possibility that PAR-2 contributes to the development of IBS upon its activation, rather than the level of mRNA or protein expression. This may explain no significant increase in the expression levels of PAR-2.

PAR-2 may perform different functions between IBS-D and IBS-C, resulting in different clinical symptoms. Tryptase activated PAR-2-positive neurons release neuropeptides, such as CGRP, SP, and VIP, which further play an important role in regulating intestinal motility. The enteric nerve interacts with intestinal mast cells by neuropeptides such as SP, VIP, and CGRP. Furthermore, enteric nerves are activated by mast cell mediators, interacting with intestinal epithelial cells, vascular cells and multiple inflammatory and immune cells. Hence, neuropeptide is thought to regulate gut motility and secretion and modulate visceral pain. An elevation of CGRP, SP, and VIP was detected in IBS-D compared with control. The mechanism of the involvement of neuropeptides in the pathological process of IBS has not yet been elucidated. The neuropeptide receptor subtypes perform different functions and are differentially distributed in the gut. In summary, our data indicates that mast cell tryptase might be responsible for the activation of PAR-2 and once activated, PAR-2 regulates neuropeptide secretion which is responsible for symptoms associated with IBS. The expression of mast cell tryptase, SP, and VIP in the intestinal mucosa is positively correlated with abdominal pain in patients with IBS. There is a correlation between mast cells and the expression of tryptase, SP and VIP.

Interestingly, there’s a discrepancy between protein and mRNA levels of PAR-2, in contrast to those of tryptase levels. The absence of mRNA?protein correlation for a subset of investigated genes suggests that the relationship between mRNA and protein content is not strictly linear. Different regulatory mechanisms (such as synthesis and degradation rates), acting on both the synthesized mRNA and the synthesized protein can affect the expression levels of the two molecules differentially. Further research investigating the biosynthesis and degradation of mRNA and protein levels of PAR-2 and tryptase may unravel the reasons for the discrepancies observed.

One of the limitations of the current study is the small sample size of patients. And our study focused on only part of colon. RT-PCR, Western blot, and immunohistochemistry were used to detect expression of tryptase, PAR-2 and some neuropeptides. RT-PCR is a very sensitive technique that can detect low copy numbers of RNA by exponential amplification. Western blotting has high sensitivity of detecting protein. Immunohistochemistry help to reveal the location of protein in tissue. These methods are used widely in previous studies of tryptase, PAR-2, and neuropeptides.

Our work evidences that mast cells are up regulated in IBS and mast cell tryptase may activate PAR-2, which regulates the expression of neuropeptides, resulting in symptoms associated with IBD. Thus, we could conclude that increased activation of PAR-2 rather than the increased expression levels may be responsible for symptoms of IBS. Further studies investigating the mechanisms regulating PAR-2 activation will help shed light on the signaling mechanisms associated with the development of IBS and at the same time, targeting the mast cell-PAR-2 signaling might help in developing therapeutic measures against IBS.

ACKNOWLEDGEMENTS

The authors thank Dr. Wei Jiao, Dr. Fei Liu, Dr. Jiao Lan and Rui-Ping Xiao from Scientific Research Center of the People’s Hospital of Guangxi Zhuang Autonomous Region for their valuable technological assistance on our project.

This work was supported by grants from the National Natural Science Foundation of China (number: 81160053 to F.C.Z.) the URL of website is http://isisn.nsfc.gov.cn/egrantindex/funcindex/prjsearch-list. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

Fig 1.

Figure 1.H&E staining showed a normal structure of the intestinal mucosa; inflammatory cells were observed in the irritable bowel syndrome-diarrhea (IBS-D), irritable bowel syndrome-constipation (IBS-C), and normal groups. (A) Control subjects (×200); (B) IBS-D patients (×200); (C) IBS-C patients (×200).
Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 2.

Figure 2.Polymerase chain reaction (PCR) products were subjected to agarose gel electrophoresis. All the real-time-PCR products from the three groups migrated as expected (A: actin, 206 bp; T: tryptase, 216 bp; P: protease-activated receptor 2, 108 bp).

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation.

Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 3.

Figure 3.Quantification of tryptase and protease-activated receptor 2 (PAR-2) mRNA after normalization to the housekeeping gene β-actin. (A) Tryptase mRNA; (B) PAR-2 mRNA. Significant elevation of tryptase and PAR-2 expression was observed in irritable bowel syndrome patients compared with the control group, but no difference was observed in subtypes. The fold change in mRNA expression was calculated relative to the endogenous β-actin control using 2?ΔΔCt. The data are expressed as the mean±SEM.

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation. *p<0.05 compared with control subjects.

Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 4.

Figure 4.The expression of tryptase and protease-activated receptor 2 (PAR-2) in the colon mucosa. The expression results were compared with healthy controls. The density of each individual band was compared with the corresponding control band and normalized to the GAPDH gene. (A) The level of tryptase increased in the irritable bowel syndrome (IBS) group, but no significant difference was observed in subtypes of IBS. (B) There was no difference in the expression levels of PAR-2 between IBS patients and the control group.

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation. *p<0.05 compared with control group.

Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 5.

Figure 5.The number of mast cells in the colon was detected by the toluidine blue method (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) Evaluation of the density (mean) showed an elevation of mast cell numbers in IBS-D patients compared to control patients, and IBS-D patients showed an increased level of tryptase compared to IBS-C patients. *p<0.05 compared with control subjects.
Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 6.

Figure 6.The expression of calcitonin gene-related peptide (CGRP) in the colon as evaluated by immunohistochemistry (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) An increase of CGRP was observed in the IBS-D group compared to the control group, and there was a significant difference between the IBS-D and IBS-C groups. *p<0.05 compared with control subjects; p<0.05 compared with IBS-C subjects.
Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 7.

Figure 7.The expression of vasoactive intestinal peptide (VIP) in the colon as evaluated by immunohistochemistry (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) An increase in VIP was found in IBS-D patients compared to control subjects, and there was a significant difference between the IBS-D and IBS-C groups. *p<0.05 compared with control subjects; p<0.05 compared with IBS-C patients.
Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Fig 8.

Figure 8.The expression of substance P (SP) in the colon as evaluated by immunohistochemistry (×200). (A) Control subjects; (B) irritable bowel syndrome-diarrhea (IBS-D) patients; (C) irritable bowel syndrome-constipation (IBS-C) patients. (D) An increase in SP was found in IBS-D patients compared to control subjects, and there was a significant difference between the IBS-D and IBS-C groups. *p<0.05 compared with control subjects; p<0.05 compared with IBS-C subjects.
Gut and Liver 2016; 10: 382-390https://doi.org/10.5009/gnl14319

Table 1 Clinical Characteristic of Irritable Bowel Syndrome Patients and Control Subjects

CharacteristicControl (n=10)IBS-D (n=20)IBS-C (n=8)
Age, mean (range), yr45.9 (30?63)41.8 (24?64)34.1 (24?54)
Sex, female/male5/513/76/2
Abdominal discomfort04.15±0.764.38±0.64
Bloating03.15±1.714.19±1.53
Altering diarrhea/constipation000
History of infection000

IBS-D, irritable bowel syndrome-diarrhea; IBS-C, irritable bowel syndrome-constipation.


Table 2 Primers for Real-Time Polymerase Chain Reaction

GeneForward primer/reverse primerPCR product, bpGene bank no.
ActinTgACgTggACATCCgCAAAg/CTggAAggTggACAgCgAgg205NM_001101.3
TryptaseATCgTgCACCCACAgTTCTAC/CTTCACCTgCTTCAgAggAAAT216NM_024164.5
PAR-2CCCTgAACATCACgACCTg/AggCTgggAACAgAAAgACC108NM_005242.4

PCR, polymerase chain reaction; PAR-2, protease-activated receptor 2.


Table 3 Relationship between Tryptase/Protease-Activated Receptor 2 and Abdominal Pain and Bloating

Abdominal painAbdominal bloating
Mast cellr=0.391*r=0.405*
Tryptaser=0.498*r=?0.073
PAR-2r=0.123r=0.120
VIPr=0.399*r=?0.148
SPr=?0.445*r=?0.037
CGRPr=0.119r=?0.188

PAR-2, protease-activated receptor 2; r, correlation coefficient; VIP, vasoactive intestinal peptide; SP, substance P; CGRP, calcitonin gene-related peptide.

*p<0.05.


References

  1. Mart?nez, C, Lobo, B, and Pigrau, M (2013). Diarrhoea-predominant irritable bowel syndrome: an organic disorder with structural abnormalities in the jejunal epithelial barrier. Gut. 62, 1160-1168.
    CrossRef
  2. Vivinus-N?bot, M, Dainese, R, and Anty, R (2012). Combination of allergic factors can worsen diarrheic irritable bowel syndrome: role of barrier defects and mast cells. Am J Gastroenterol. 107, 75-81.
    CrossRef
  3. Miller, HR, and Pemberton, AD (2002). Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology. 105, 375-390.
    Pubmed KoreaMed CrossRef
  4. Wilcz-Villega, EM, McClean, S, and O’Sullivan, MA (2013). Mast cell tryptase reduces junctional adhesion molecule-A (JAM-A) expression in intestinal epithelial cells: implications for the mechanisms of barrier dysfunction in irritable bowel syndrome. Am J Gastroenterol. 108, 1140-1151.
    Pubmed CrossRef
  5. Cenac, N, Andrews, CN, and Holzhausen, M (2007). Role for protease activity in visceral pain in irritable bowel syndrome. J Clin Invest. 117, 636-647.
    Pubmed KoreaMed CrossRef
  6. Oikonomopoulou, K, Hansen, KK, and Saifeddine, M (2006). Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs). Biol Chem. 387, 817-824.
    Pubmed
  7. Cenac, N, Coelho, AM, and Nguyen, C (2002). Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2. Am J Pathol. 161, 1903-1915.
    Pubmed KoreaMed CrossRef
  8. Reed, DE, Barajas-Lopez, C, and Cottrell, G (2003). Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons. J Physiol. 547, 531-542.
    Pubmed KoreaMed CrossRef
  9. Jacob, C, Yang, PC, and Darmoul, D (2005). Mast cell tryptase controls paracellular permeability of the intestine: role of protease-activated receptor 2 and beta-arrestins. J Biol Chem. 280, 31936-31948.
    Pubmed CrossRef
  10. Fiorucci, S, Mencarelli, A, and Palazzetti, B (2001). Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis. Proc Natl Acad Sci U S A. 98, 13936-13941.
    Pubmed KoreaMed CrossRef
  11. Demir, IE, Sch?fer, KH, Tieftrunk, E, Friess, H, and Ceyhan, GO (2013). Neural plasticity in the gastrointestinal tract: chronic inflammation, neurotrophic signals, and hypersensitivity. Acta Neuropathol. 125, 491-509.
    Pubmed CrossRef
  12. Steinhoff, M, Vergnolle, N, and Young, SH (2000). Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med. 6, 151-158.
    Pubmed CrossRef
  13. Nozdrachev, AD, Akoev, GN, Filippova, LV, Sherman, NO, Lioudyno, MI, and Makarov, FN (1999). Changes in afferent impulse activity of small intestine mesenteric nerves in response to antigen challenge. Neuroscience. 94, 1339-1342.
    CrossRef
  14. Bueno, L, Fioramonti, J, Delvaux, M, and Frexinos, J (1997). Mediators and pharmacology of visceral sensitivity: from basic to clinical investigations. Gastroenterology. 112, 1714-1743.
    Pubmed CrossRef
  15. Barbara, G, Stanghellini, V, and De Giorgio, R (2004). Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology. 126, 693-702.
    Pubmed CrossRef
  16. Corvera, CU, D?ry, O, and McConalogue, K (1997). Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. J Clin Invest. 100, 1383-1393.
    Pubmed KoreaMed CrossRef
  17. Duerksen, DR, Wilhelm-Boyles, C, and Parry, DM (2005). Intestinal permeability in long-term follow-up of patients with celiac disease on a gluten-free diet. Dig Dis Sci. 50, 785-790.
    Pubmed CrossRef
  18. Coelho, AM, Vergnolle, N, Guiard, B, Fioramonti, J, and Bueno, L (2002). Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats. Gastroenterology. 122, 1035-1047.
    Pubmed CrossRef
  19. Lee, JW, Park, JH, and Park, DI (2010). Subjects with diarrhea-predominant IBS have increased rectal permeability responsive to tryptase. Dig Dis Sci. 55, 2922-2928.
    Pubmed CrossRef
  20. Kim, DH, Cho, YJ, Kim, JH, Kim, YB, and Lee, KJ (2010). Stress-induced alterations in mast cell numbers and proteinase-activated receptor-2 expression of the colon: role of corticotrophin-releasing factor. J Korean Med Sci. 25, 1330-1335.
    Pubmed KoreaMed CrossRef
  21. Bian, ZX, Li, Z, and Huang, ZX (2009). Unbalanced expression of protease-activated receptors-1 and -2 in the colon of diarrhea-predominant irritable bowel syndrome patients. J Gastroenterol. 44, 666-674.
    Pubmed CrossRef
Gut and Liver

Vol.16 No.6
November, 2022

pISSN 1976-2283
eISSN 2005-1212

qrcode
qrcode

Share this article on :

  • line

Popular Keywords

Gut and LiverQR code Download
qr-code

Editorial Office