Inflammatory bowel disease (IBD) mainly includes Crohn’s disease (CD) and ulcerative colitis (UC), which belongs to a class of chronic recurrent gastrointestinal inflammatory diseases and immune dysfunction. Patients usually experience abdominal pain, diarrhea, bloody stool, and severe weight loss. According to the disease activity, it can be divided into the period of activity and remission.1 Irritable bowel syndrome (IBS) is a kind of functional bowel disorder characterized by recurrent abdominal pain, disordered defecation. IBD and IBS have overlapping symptoms. Patients with IBD in clinical remission (IBDR) suffer from IBS-like symptoms such as abdominal discomfort, bellyache, diarrhea, and abnormal defecation, which are called “IBD-IBS.”2,3 According to the latest report, the prevalence of IBS-like symptoms among IBDR patients is generally between 25.0% and 60.0%4 due to the different definition of IBD in remission, variation of inclusion criteria, and the small samples of some studies. A meta-analysis found that 35.0% of the inactive patients had IBS-like symptoms.3 Approximately 45.0% of IBD patients with normal inflammatory markers developed IBS-like symptoms.5
In recent years, health-related quality of life (QOL) in chronic diseases has played a predominant role in patients’ mental health, social adaptation and utilization of health resources. The QOL of IBD patients was seriously affected because of the prolonged and recurrent course.6-9 The etiology of IBD-IBS remains unclear. It may have some similar pathogenesis as IBS, such as psychological factors, visceral hypersensitivity, impaired intestinal barrier, and so on. Hypersensitivity of viscera is a principal element underlying abdominal analgesia in IBS. Visceral hypersensitivity refers to increased visceral perception to chemical, temperature, mechanical, PH, and other stimuli, as well as decreased sensory threshold during rectal dilation stimulation. The mechanism of visceral hypersensitivity is not entirely understood. It may occur via a disturbance of the different levels of sensitization pathways: increased alertness of central system, hypersensitivity of sensory nerve endings, disturbance of autonomic nervous system, endocrine, and neurotransmitter abnormalities.10 Mast cells (MCs) may have a critical effect on visceral hypersensitivity in IBS,11,12 through releasing neuroendocrine substances such as 5-hydroxytryptamine (5-HT), nerve growth factor (NGF), protease, and triggering relevant receptors on nerve endings.
Currently, some studies are available on the prevalence of IBD-IBS mainly from Euro-American studies. In China, the prevalence of IBD-IBS has not yet been investigated. In addition, studies on the relationship between IBD-IBS and psychological factors, visceral hypersensitivity are not fully verified, and the formation mechanism of IBD-IBS remains incompletely clear. The purposes of this study are: (1) to evaluate the prevalence, clinical symptoms, QOL, and psychological status of Chinese IBD-IBS patients, (2) to assess the changes of visceral sensitivity in patients with IBD-IBS, and (3) to preliminarily investigate the roles of MC, 5-HT and NGF in formation of visceral hypersensitivity in IBD-IBS patients.
The patients who visited the First Affiliated Hospital of Nanjing Medical University were recruited from August 2017 to September 2018. We enrolled 71 IBDR patients (including 24 ulcerative colitis in remission [UCR] patients and 47 Crohn’s disease in remission [CDR] patients), 26 IBS patients, and 20 healthy controls (HCs) for a cross-sectional study. Each IBD patient met the diagnostic criteria drafted by the European Crohn’s and Colitis Organization.13,14 The definition of IBD in remission was (1) Mayo index (UC) ≤ 1 with no single subitem score > 1, (2) Crohn’s disease activity index (CDAI) was < 150, (3) C-reflection protein (CRP) was < 10 mg/L, and (4) erythrocyte sedimentation rate (ESR) was < 20 mm/hr.8,15,16 HCs were selected from Physical Examination Center. Patients with cardio-cerebrovascular diseases, tumors, infectious diseases, use of steroids, and NSAIDs within 6 months, motility drugs with 1 month or smoking, and alcohol intake with 1 year were excluded. Peripheral blood of all patients and HCs were collected. The rectosigmoid biopsies were performed for later analysis. The Ethics Committee of the First Affiliated Hospital of Nanjing Medical University had approved this medical research and related procedures (Ethics No. 2018-SR-061).
IBS-like symptom and IBS was evaluated by utilizing the Rome III diagnostic questionnaire for IBS17: (1) symptoms occur at least 6 months before diagnosis; (2) onset of abdominal analgesia or discomfort more than 3 days of each month within the previous 3 months; and (3) at least 2 of the following characteristics: improvement after defecation, relationship with changes in stool frequency or stool form. Abdominal pain was detected utilizing a visual analogue scale (VAS; 0 = no pain and 10 = extreme pain).18 The general QOL was evaluated with 36-items short-form health survey (SF-36), and the disease-specific QOL (inflammatory bowel disease questionnaire [IBDQ]) were adopted as well.19 Assessment of anxiety and depression was respectively conducted with the health 7 item generalized anxiety disorder scale (GAD-7) and the 9 item patient health questionnaire (PHQ-9).20,21
Two experienced gastroenterologists assessed the endoscopic scores for each IBD patient utilizing the Crohn’s disease endoscopic index of severity (CDEIS)22 and ulcerative colitis endoscopic index of severity (UCEIS).23 The CDEIS evaluates 5 colonic segments: rectum, sigmoid and left colon, traverse colon, right colon, and ileum. The presence of ulceration, the percentage of ulcerated surface and the percentage of surface involved by CD are measured for each colonic segment. In addition, the presence of stenoses is assessed. These items are weighted and summed to a total score. UCEIS uses 3 variables: vascular pattern (normal, patchy obliteration, or obliterated); bleeding (none, mucosal, luminal mild, luminal moderate, or severe); and erosions and ulcers (none, erosions, superficial ulcer, or deep ulcer). These variables are summed to a total score.
A high-resolution anorectal manometric electrode catheter with a balloon (MSC-2195; Medtronic, Inc, USA) was used to detect rectal perception. Each person underwent a gradual distension procedure through injecting gas into the balloon (rate: 2 mL/sec) in the lateral position. Volumes (mL) were continuously monitored. Patients were asked to rate sensations of initial perception, defecation distress, and maximum tolerance.
All colon tissues were stored overnight in 4% paraformaldehyde and cut into sections, then dewaxed and rehydrated. Endogenous peroxidase was blocked by 3% H2O2 for 15 minutes. The antigen of tryptase and NGF were retrieved by boiling in the buffer (sodium citrate, 10 mmol/L, pH = 6.0) in a microwave for 15 minutes. Antigen retrieval of 5-HT was performed by incubating in protease K liquid (20 mg/mL) for 30 minutes. Then the slides were incubated with diluted antibody at 4°C overnight (trypsin-like ab2378, Abcam, Cambridge, MA, USA,1:100; 5-HT NB120-16007, Novus, San Diego, CA, USA,1:50; NGF ab6199, Abcam, Cambridge, MA, USA, 1:200) and followed by incubating in the secondary antibody (TL-125-QPH; Thermo, Waltham, MA, USA) for 50 minutes and the DAB solution (G1211; Service bio, Wuhan, Hubei, China), then counterstained with hematoxylin. The Image Pro-Plus software (Media Cybernetics, Inc, Sarasota, FL, USA) was used to obtain a mean density of 5 random fields per slide. Tryptase-positive MCs were counted as the average of 5 random non-over- lapped high-powered fields (final magnification, ×400) for each slide.
Protein samples obtained from human colon tissue were dissected, then transferred to the nitrocellulose membrane and sealed with 5% skim milk for 1 hour. Incubate membranes with primary antibodies (5-hydroxytryptamine 3 [5-HT3] receptor: bs-4289R, Bioss, Beijing, China, 1:500; transient receptor potential vanilloid 1 [TRPV1]: bs-23926R, Bioss, Beijing, China, 1:500; tropomyosin receptor kinase A [TrkA]: bs-10210R, Bioss, Beijing, China, 1:500) overnight at 4℃, then incubated for 1 hour with the secondary antibody (ab6721, Abcam, Cambridge, Mass, USA, 1:10 000) at room temperature. The membrane was dropped in ECL Solution and exposure to autoradiography film (Kodak XAR film, Rochester, NY, USA). The obtained image was analyzed by Image-Pro-Plus (version 6.0).
Enzyme-linked immunosorbent assay was applied to detect the serum levels of 5-HT (E-EL-0033c; Elabscience, Wuhan, Hubei, China) and NGF (EK1141-24; Mutiscience, Hangzhou, Zhejiang, China) according to the manufacturer’s protocol.
The data was analyzed using the IBM SPSS 21.0 software (IBM Corp, Armonk, NY, USA). The continuous data were presented as mean ± standard deviation, and the categorical data was expressed as number (percentage). Independent
Seventy-one IBDR patients (CDR 47 and UCR 24) were included in this study (Table 1). IBS-like symptoms were found in 14 out of 47 patients with CDR (29.8%) and in 12 out of 24 patients with UCR (50.0%). The CDAI and CDEIS in CD-IBS+ group were significantly higher than CD-IBS– group (CDAI
VAS scores of CD-IBS+, UC-IBS+, and IBS groups were 5.2 ± 2.1, 3.8 ± 1.2, and 3.8 ± 2.2, respectively. The VAS score of CD-IBS+ group was increased compared to IBS (
General QOL (total SF-36 score) of IBD-IBS patients was significantly lower than those without IBS-like symptoms (CD-IBS+ vs CD-IBS–,
Disease-specific QOL (IBDQ) scores of IBD-IBS patients were obviously decreased (CD-IBS+ vs CD-IBS–,
The prevalence of anxiety in CD-IBS+, CD-IBS–, UC-IBS+, UC-IBS–, and IBS groups was 64.3%, 27.3%, 75.0%, 25.0%, and 30.8%, respectively. Meanwhile, the prevalence of depression in the above 5 groups respectively was 57.1%, 42.4%, 58.7%, 16.3%, and 42.3%. IBD-IBS patients had a higher prevalence of anxiety than IBS patients and those without IBS-like symptoms (GAD-7: CD-IBS+ vs CD-IBS–,
The threshold of initial perception, defecation distress and maximum tolerance in UCR patients with IBS-like symptoms showed fewer values versus those with no such symptoms (initial perception: 35.75 ± 13.36 vs 50.00 ± 5.29,
There was a positive association between abdominal pain and anxiety/depression in IBD-IBS patients (GAD-7: CD-IBS+,
The threshold values of initial perception, defecation distress, and maximum tolerance in CD-IBS+ group negatively correlated with anxiety and depression scores (Fig. 1F and 1G). Moreover, there was an inverse relationship between defecation distress/maximum volume threshold values and anxiety/depression scores in UC-IBS+ group (Fig. 1H and 1I).
MC tryptase is expressed in the MC cytoplasm, representing the degranulation and activation of MCs. The results showed MCs was mainly distributed in colonic lamina propria of IBD-IBS and IBS, while MCs was not detected in HCs (Fig. 2A). The mean number for tryptase-positive MCs was higher in IBD-IBS patients than those in IBS patients (CD-IBS+ vs IBS,
MCs can release 5-HT and NGF after activation. In IBD-IBS and IBS patients, 5-HT is mainly distributed in colonic glandular cells and lamina propria, and NGF is expressed in colonic submucosa. The expressions of 5-HT, as well as NGF, were not detected in normal colonic tissues (Fig. 3A and 3B). The expressions of 5-HT and NGF in colonic tissues of IBDR patients with IBS-like symptoms were upregulated compared to the patients without such symptoms (5-HT: CD-IBS+ vs CD-IBS–,
The levels of 5-HT in serum CD-IBS+, CD-IBS–, UC-IBS+, UC-IBS–, IBS, and HCs groups were 200.2 ± 49.8, 141.5 ± 15.5, 189.7 ± 37.7, 117.8 ± 15.2, 155.7 ± 36.6, and 117.7 ± 23.4 (ng/mL), respectively. Serum levels of NGF in these groups were 44.9 ± 25.3, 14.4 ± 10.9, 33.2 ± 14.3, 6.1 ± 3.8, 22.4 ± 11.2, and 9.04 ± 6.5 (pg/mL), respectively. A significantly higher serum 5-HT and NGF levels was found in IBD-IBS patients (5-HT: CD-IBS+ vs CD-IBS–,
The expressions of 5-HT and NGF in IBD-IBS patients positively related with VAS scores and anxiety/depression scores, while negatively associated with rectal perception (Table 4). The results suggested that 5-HT and NGF may be correlated with abdominal pain, anxiety, depression, and visceral hypersensitivity.
Western blot showed that the expression of 5-HT3, tropomyosin receptor kinase A (TrkA) and TRPV1 in IBD-IBS and IBS patients were upregulated compared with the HCs. Furthermore, the expressions of 5-HT3, TrkA and TRPV1 in the colonic tissue of IBDR patients with IBS-like symptoms were notable higher than those of patients without such symptoms (Fig. 4).
In this investigation, the prevalence of IBS-like symptoms in CDR patients was 29.8% and 50.0% in UCR patients with remission, similar to the previous studies.5,7,8,24 The present study confirmed that IBDR patients with IBS-like symptoms had higher VAS scores than those without such symptoms, especially for CD-IBS + patients, which may be due to the long course, a wide range of lesions, and transmural inflammation. Additionally, we found that IBDR patients with IBS-like symptoms had worse QOL than those without such symptoms, which were consistent with previous studies.8,25-27 UCR patients with IBS-like symptoms were predisposed to suffer from worsened physical role, general health, and vitality. The general health of CDR patients with IBS-like symptoms was impacted as well. Through a questionnaire, we confirmed that patients with IBS-like symptoms had important differences in the dimensions of general symptom and emotional function compared to patients without such symptoms. These results indicated that IBD-IBS patients are not only affected by physical symptoms, but also by emotional, social function, and mental state. We showed that the prevalence of anxiety and depression in IBD-IBS patients was 57.1-75.0%, which was consistent with the results previously reported.28,29 IBD-IBS patients were more likely to have anxiety and depression than those who had no IBS-like symptoms. Further analysis indicated that there was a tight relationship between anxiety, depression, and abdominal pain. Therefore, detection of anxiety and depression in IBDR patients and the early psychological intervention for patients with IBS-like symptoms may be conducive to improving the physical condition and quality of life.
Visceral hypersensitivity is one of the important pathophysiological changes in IBS, which is related to abdominal pain. The present study revealed that the values of initial sensory threshold, defecation distress threshold, and the maximum tolerance threshold were decreased, which showed the visceral hypersensitivity in IBD-IBS patients. In addition, visceral hypersensitivity was associated with abdominal pain and anxiety/depression in IBD-IBS patients. In murine colitis models, depression may lead to recurrence of colitis by damaging cholinergic nerve function and antidepressant treatment may improve colitis.30 Therefore, we speculate that psychological abnormalities may increase visceral sensitivity and thus participate in the occurrence of IBS-like symptoms in IBDR patients.
This study found that CDAI, CDEIS, CRP, and ESR was higher in CD-IBS+ patients, and low-grade inflammation such as inflammatory cells infiltration was suspected to be related with the development of IBS-like symptoms. Previous studies confirmed that MCs were involved in IBS patients.31-33 Vivinus et al34 found that the number of intraepithelial lymphocytes and MCs were significantly increased in IBD patients with IBS-like symptoms, the number of eosinophils was significantly higher in UC patients compared with HCs, but was not significantly different between patients with or without IBS-like symptoms. Another study determined that the total number of MCs in colonic mucosa of UCR patients as well as the percentage of MCs close to nerve endings increased.18 Visceral hypersensitivity could be reduced by blocking MC activation in IBS-like rat models.35 In this study, we focused on the relationship between MCs and IBS-like symptoms in IBD patients. The present study found that colonic MCs in IBD-IBS patients were significantly more active than those patients without IBS-like symptoms, especially in CD-IBS+ patients. 5-HT and NGF, 2 bioactive substances released from MCs, were reported to be linked to visceral hypersensitivity and intestinal motility dysfunction in IBS patients. The expression of the 5-HT catalytic enzyme, and NGF significantly increased in CDR patients with IBS-like symptoms.36,37 In this study, the expressions of 5-HT and NGF and receptor protein 5-HT3 and TrkA were increased in IBD-IBS patients, expression of NGF in CD-IBS+ patients was obviously upregulated compared to UC-IBS+ patients. Furthermore, serum levels of 5-HT and NGF in IBD-IBS patients positively correlated with abdominal pain, anxiety, depression, and visceral hypersensitivity, which suggest that 5-HT and NGF may be involved in visceral hypersensitivity. On the other hand, anxiety and depression may further stimulate activated MCs to release 5-HT and NGF.
TRPV1 is expressed in primary sensory afferent nerve fibers and can produce pain by activation with capsaicin, NGF, and other substances. Akbar et al38 reported that the number of TRPV1 positive nerve fibers in the colonic mucosal tissues in IBD-IBS patients had a strong correlation with abdominal pain. Lower visceral sensitivity was observed in TRPV1 knockout mice as opposed to wild-type mice during inflammatory recovery.39 In this investigation, we revealed that the expressions of TRPV1 in IBD-IBS patients and IBS patients was obviously upregulated versus those in the healthy controls. Compared with IBDR patients without IBS-like symptoms, TRPV1 expressed highly in those patients with IBS-like symptoms. Generally, TRPV1 may play a vital role in the process of abdominal pain in IBD-IBS.
The present study has several limitations. First, we defined inactive IBD as clinical remission, and the prevalence of IBS-like symptoms in IBD patients with deep remission needs to be assessed. Second, the limited sample size of CD, especially UC, may affect the results. Third, we did not subgroup the IBS-like symptoms. Future studies will have to focus on the correlation between certain IBS subtypes and IBD.
In conclusion, our findings indicated: IBD-IBS patients may have low QOL and psychological abnormalities, as well as visceral hypersensitivity which may be related to increased 5-HT and NGF levels released from activated MCs and upregulated 5-HT3, TrkA, and TRPV1 expressions in the colonic tissue.
This work was supported by the National Natural Science Foundation of China (No. 81770553) and Science and Technology Development Fund of Nanjing Medical University (No. NMUB2018291).
Haiyang Wang and Hongjie Zhang designed the study; Yi Li, Chunhua Jiao, Xiaojing Zhao, and Yan Yang collected specimens; Haiyang Wang, Xiaojing Zhao, and Meifeng Wang performed the research; Haiyang Wang, Jiajia Li, and Xiufang Cui analyzed the data; Haiyang Wang wrote this manuscript; and Hongjie Zhang and Yi Li supervised the report.