Correlation between anxiety-depression status and cytokines in diarrhea-predominant irritable bowel syndrome

The aim of this study was to investigate the correlation between anxiety-depression status and cytokines in diarrhea-predominant irritable bowel syndrome (IBS-D). IBS-D patients were divided into an anxiety-depression IBS-D group and a non-anxiety-depression IBS-D group. Patients without IBS, anxiety or depression were selected as the control group. Scoring was performed using the self-rating anxiety scale (SAS) and self-rating depression scale (SDS). Levels of IL-1β and IL-10 in the blood and sigmoid colon mucosa were detected, and the proportions of IL-1β- and IL-10-positive cells in the sigmoid colon mucosa were determined. The results demonstrated that the SDS and SAS scores in the IBS-D group were significantly higher than those in the control group (P<0.05). The levels of IL-1β in the blood and sigmoid colon mucosa and the proportion of IL-1β-positive cells in the sigmoid colon mucosa in the IBS-D group were significantly higher than those in the control group (P<0.05). The levels of IL-10 in the blood and sigmoid colon mucosa and the proportion of IL-10-positive cells in the IBS-D group were significantly lower than those in the control group (P<0.05). The levels of IL-1β in the blood and sigmoid colon mucosa and the proportion of IL-1β-positive cells in the anxiety-depression IBS-D group were significantly higher than those in the non-anxiety-depression IBS-D group, and the levels of IL-10 and the proportion of IL-10-positive cells in the anxiety-depression IBS-D group were significantly lower than those in the non-anxiety-depression IBS-D group (P<0.05). Anxiety-depression status may cause the IL-1β and IL-10 levels in IBS patients to change and result in an imbalance of the proinflammatory and anti-inflammatory cytokines, leading to the occurrence or aggravation of IBS.


Introduction
Irritable bowel syndrome (IBS) refers to a group of symptoms that are persistent or intermittent but are not associated with clear morphological changes or biochemical abnormalities. IBS manifests as abdominal pain and abdominal distension, as well as abnormalities in bowel evacuation habit and stool characteristics for unknown reasons (1). IBS is a heterogeneous disease caused by numerous factors that are interelated with each other. Among these factors, psychology, emotional state, society and environment play important roles in the development of IBS. Furthermore, nervous, immune and endocrine systems also contribute to the development of this medical condition, leading to intestinal smooth muscle movement disorders and visceral paresthesia (2). The view that an inflammatory reaction exists in IBS patients has attracted extensive attention (3). The possible interaction between psychological abnormalities and the immune system has also been emphasized. In the current study, we explored the psychological status of IBS patients, as well as the mechanism of action of cytokines.

Materials and methods
General data. IBS patients who received treatment at Xingtai People's Hospital (Hebei, China) between March 2008 and September 2010 were enrolled. This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Xingtai People's Hospital. Written informed consent was obtained from all participants. The participants were grouped based on the following standards: i) Rome Ш standards: recurrent abdominal pain or indisposition that started no less than six months before diagnosis and occurred on at least three days a month within the most recent three months. This phenomenon had to be accompanied by at least two out of the following three situations (not necessarily occurring in succession): a) improvement following defecation; b) disease accompanied by a change in defecation frequency (3/day or <3/week); and c) disease accompanied by changes in stool characteristics; ii) IBS typing standards based on the stool characteristic typing proposed by the Rome Ш team. Diarrhea-predominant IBS (IBS-D) is defined as pasty/watery stools >25% and massive/hard stools <25%.
Patients meeting the following standards were excluded: i) organic diseases, including ulcer, erosion and mass according Correlation between anxiety-depression status and cytokines in diarrhea-predominant irritable bowel syndrome to endoscopy; ii) organic disease in the liver, gall, pancreas or intestinal tract according to laboratory, ultrasonic and X-ray examination; iii) heart disease, diabetes, thyroid disease, connective tissue disease or psychosis; iv) administration of cathartics, antidiarrheals, prokinetic drugs or digestive enzyme drugs within one month; and v) history of abdominal surgery. Patients' anxiety and depression were respectively evaluated using the self-rating anxiety scale (SAS) and self-rating depression scale (SDS). Patients meeting the diagnostic criteria of IBS-D were randomized into a normal psychological status IBS-D group and an anxiety-depression IBS-D group. The former was composed of 12 patients, including 6 males and 6 females. Their ages ranged from 17 to 61 years with an average age of 39 years. The latter consisted of 16 patients, including 6 males and 10 females. Their ages ranged from 17 to 58 years with an average age of 37.5 years. In addition, 15 patients with colonic polyps and internal hemorrhoids who received a health examination at the same hospital during the same period were selected as the control group (non-anxiety-depression non-IBS patients). The 15 patients of the control group contained 6 males and 9 females. Their ages ranged from 20 to 57 years with an average age of 38.5 years. The control subjects had no digestive symptoms, including abdominal pain, diarrhea and constipation, or nervous symptoms. Additionally, they had not recently been treated with any drugs.
Specimens. Venous blood (2 ml) was extracted on an empty stomach in the morning for supernatant determination. Two mucous membranes from the sigmoid colon were obtained from each participant for the preparation of biopsy specimens. One was fixed in neutral formalin for interleukin (IL)-1β and IL-10 immunohistochemistry and the other was immediately frozen in liquid nitrogen for radioimmunoassay.
Radioimmunoassay. The sigmoid colon mucosa specimen was placed in a homogenizer, 0.1 M acetic acid was added and the speciment was then fully homogenized. The homogenate liquid was transferred to a plastic tube and left to stand overnight. An equal volume of 0.1 M sodium hydroxide solution was added, followed by centrifuging at 1409 x g for 15 min. The supernatant was obtained for detection. The sigmoid colon mucosa and blood specimens were analyzed using a SN-695B radioimmunoassay intelligent measuring instrument (Shanghai Institute of Nuclear Research, Shanghai, China) using a sequential saturation sampling approach, and a standard curve was automatically drawn.
Immunohistochemistry. Mucosal specimens were fixed in 10% neutral formalin, routinely dehydrated and embedded in paraffin, then serially sectioned. The obtained sections were dewaxed, hydrated and then incubated with 0.3% methanol-hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. The sections were rinsed with distilled water and saturated in phosphate-buffered saline (PBS; pH 6.0) for 5 min for antigen repair. The sections were incubated with IL-1β and IL-10 primary antibodies (1:700 and 1:50 in dilution, respectively) at 37˚C for 1 h. PBS replaced the primary antibodies for the control. Following PBS washing, the sections were incubated with 50 µl biotinylated secondary antibody working solution at 37˚C for 30 min. After rinsing with PBS for 10 min, 100 µl freshly-prepared diaminobenzidine solution (containing a drop of 3% H 2 O 2 ) was added to each section for coloration. Then, the sections were stained with hematoxylin, dehydrated and mounted.
Outcome evaluation. All the specimens were observed using a Motic Med 6.0 digital medical image analysis system (Shanghai Xinzhen Equipment Co., Ltd., Shanghai, China) under the same amplifying power. For each section, ten visual fields were selected. The average optical density (OD) of positively stained cells was used to represent the level of antigen expression. The expression was semiquantitated through computed image analysis. A higher than average OD value indicated a higher expression level of IL-1β and IL-10 in the cells.
Statistical analysis. All data are presented as mean ± standard deviation and analyzed by SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA. The t-test was performed for comparisons between groups. P<0.05 was considered to indicate a statistically significant difference.

Results
Comparison of anxiety and depression between the IBS and control groups. As shown in Table I

Comparison of IL-1β and IL-10 levels between the IBS groups.
As shown in Table III, the levels of IL-1β in the blood and sigmoid colon mucosa in the anxiety-depression IBS-D group   Table IV show that the proportion of IL-1β-positive cells in the IBS-D group (0.298±0.312) was significantly higher than that in the control group (0.091±0.036; P<0.05). The proportion of IL-10-positive cells in the IBS-D group (0.083±0.045) was significantly lower than that in the control group (0.159±0.138; P<0.05). Table V, the proportion of IL-1β-positive cells in the anxiety-depression IBS-D group (0.310±0.223) was significantly higher than that in the non-anxiety-depression IBS-D group (0.234±0.124; P<0.05). However, the proportion of IL-10-positive cells in the anxiety-depression IBS-D group (0.075±0.097) was significantly lower than that in the non-anxiety-depression IBS-D group (0.104±0.257; P<0.05).

Discussion
IBS refers to a group of symptoms that are persistent or intermittent but without noticeable morphological changes or biochemical abnormalities, represented by abdominal pain and abdominal distension, as well as abnormalities in bowel evacuation habit and stool characteristics for unknown reasons (4,5). The fundamental mechanism underlying these symptoms is based on abnormalities in intestinal movement dysfunction due to intestinal hypersensitivity and hyperreactivity (6). In the current study, the results demonstrated that the IBS patients had significantly increased anxiety and depression scores compared with the control group. This finding indicates that IBS patients are subjected to noticeable psychological abnormalities. Mental stress may lead to basic immune dysfunction. The effect of mental stress on gastrointestinal sensation and movement may involve the Table II. Comparison of IL-1β and IL-10 levels between the IBS group and the control group.
In the current study, IBS-D patients that met the anxiety-depression criteria were selected according to the criteria of the anxiety-depression scales and then compared with the normal control and non-anxiety-depression IBS patients to determine differences in the levels of IL-1β and IL-10 in the blood and sigmoid mucous membranes, as well as mucosal positive cell expression. The results demonstrated that the IL-1β level in the colonic mucous membrane and blood of the IBS-D group were significantly increased compared with those of the control group, whereas the levels of IL-10 were markedly reduced. The levels of IL-1β and IL-10 in the sigmoid colon mucosal membranes and blood of the anxiety-depression IBS-D group were significantly different from those of the non-anxiety-depression IBS group. Furthermore, immunohistochemistry revealed significant differences in the positive expression of IL-1β and IL-10 between the anxiety-depression and non-anxiety-depression IBS-D groups. These findings suggest that psychological status affects the levels of cytokines and induces or aggravates the digestive symptoms in IBS-D patients. Furthermore, chronic somatic symptoms aggravate patients' psychological burden, thereby leading to the aggravation of anxiety and depression. Thus, somatic symptoms and psychological status interact with or even promote each other (18)(19)(20)(21). Cytokines, inflammatory reactions and psychological factors all play important roles in the development of IBS. Psychological factors lead to variations in the levels of cytokines and the balance between anti-inflammatory and pro-inflammatory factors, resulting in the aggravation of IBS or digestive symptoms; whereas the aggravation of somatic symptoms exacerbates psychological burden and anxiety-depression symptoms, exhibiting a close interactive correlation (22)(23)(24).
IBS is a heterogeneous disease caused by numerous factors. Anxiety-depression status may cause changes in the IL-1β and IL-10 levels of IBS patients resulting in an imbalance of the proinflammatory and anti-inflammatory cytokines, leading to the occurrence or aggravation of IBS.