A total of 40 IBS-D patients, who were treated at the Department of Gastroenterology of the China-Japan Friendship Hospital from September 2017 to April 2018, and 18 age- and sex-matched healthy controls were recruited for the study. All IBS-D patients were diagnosed based on the Rome IV criteria. Healthy controls consisted of patient spouses, medical examiners and volunteers. The patients and healthy controls were included if they did not take the following drugs within 2 wk of the study: Antispasmodics, analgesics, microecological preparations, antidepressants, anti-inflammatory drugs, antibiotics, other drugs and antacids that can affect gastrointestinal motility. The exclusion criteria were as follows: (1) Gastrointestinal organic diseases, endocrine system diseases, metabolic diseases, connective tissue diseases and other organ diseases; (2) A history of gastrointestinal or abdominal surgery; and (3) A previous or current diagnosis of mental illness.

All subjects were informed of the study details and provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of China-Japan Friendship Hospital.

Clinical symptoms and psychosocial assessment: The severity of clinical symptoms was evaluated using the IBS symptom severity scale[3], the VAS pain scale and the frequency course scale. The visceral sensitivity index was used to evaluate visceral sensitivity and gastrointestinal-specific anxiety. Psychiatric symptoms were assessed using the validated Hospital Anxiety and Depression Scale[21]. The IBS-specific quality of life was used to evaluate the quality of life of IBS patients in this disease state.

Visceral sensitivity test: The visceral sensitivity test was performed in the Gastroenterology Kinetic Laboratory by the same investigator. Before the examination, the subjects were given glycerin to eliminate feces. The subjects were placed in the left lateral decubitus position and were asked to relax. After the mass was eliminated by digital rectal examination, the lubricating pressure measuring catheter was slowly inserted into the rectum, with an insertion depth of 15 cm and a balloon placed above the anal margin of 8 cm. After the subject had adapted for 3 min, the examination began. Air was slowly injected (at a rate of 10 mL/5 s) into the balloon by a 100 mL syringe. The subject's feeling after every 10 mL gas injected was recorded. When the subject felt the initial swelling, continuous defecation sensation, pain discomfort or intolerance, the amount of gas injected (in mL) was recorded, which reflected the initial feeling threshold, continuous defecation threshold and maximum tolerance threshold, respectively.

Specimen collection: Participants were not allowed to take gastrointestinal motility drugs, laxatives, antidiarrheal drugs, microecological agents, antidepressants or other drugs that affect gastrointestinal motility one week prior to the examination. The participants underwent colonoscopy after standard bowel preparation with polyethylene glycol electrolyte powder (Fortrans, BEAUFOUR IPSEN Industrie, Dreux, France), and four mucosal pinch biopsies were taken from the rectosigmoid junction. Two specimens were immediately fixed in 10% formalin for at least 72 h, embedded in paraffin and sectioned (4 μm) for routine hematoxylin and eosin staining and immunohistochemistry. The other specimens were immediately immersed in storage reagent (RNA-Be-Locker A; Sangon, Shanghai, China) and stored at -80 °C for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Venous blood was centrifuged at 2000 r/min for 10 min within 30 min after collection, and the supernatant was taken for analysis.

Histology and immunohistochemistry: Paraffin sections were stained with hematoxylin eosin and processed for immunohistochemistry. The latter section was incubated with primary antibody (rabbit polyclonal anti-SERT antibody, 1:400, Affinity Biosciences, United States; rat monoclonal anti-serotonin antibody, 1:100, Santa, United States; rabbit polyclonal anti-CCK antibody, 1:100, Abcam, United Kingdom) overnight at 4 °C after dewaxing, antigen retrieval, endogenous peroxidase inhibition, and nonspecific antigen blocking. After washing with phosphate buffered saline, the sections were incubated with anti-rat and anti-rabbit secondary antibodies (1/200, Zhongshan Golden Bridge, Beijing, China) combined with horseradish peroxidase at room temperature for 1 h and then visualized using diaminobenzidine. Finally, the sections were counterstained with hematoxylin and observed under a light microscope to examine the average optical density (OD value) of positive expression of the stain.

Quantitative real-time PCR detection: The expression of CCK and SERT was analyzed by real-time quantitative PCR. Total RNA in the tissue was extracted by TRIzol Reagent (Invitrogen Life Technologies, Waltham, MA, United States) and reverse transcribed into cDNA. Real-time quantitative PCR was then carried out in a StepOnePlus Real-Time PCR system (Applied Biosystems, Waltham, MA, United States) using the FastStart Universal SYBR Green Master Rox Kit (Roche, Shanghai, China). Finally, the mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. The primers were designed and synthesized by Beijing Aoke Biotechnology Co., Ltd. The upstream region of SERT was 5'-CAGCGTGTGAAGATGGAGAAG-3', and the downstream region of SERT was 5'-TGGGATAGAGTGCCGTGTGT-3'. The upstream region of CCK was 5'-CAGAGGAGGCAGAATAAGAA-3', and the downstream region of CCK was 5'-CAGGAGTCACAGATGAAGAA-3'. The upstream housekeeping gene GAPDH (internal reference) was 5'-GGAAGCTTGTCATCAATGGAAATC-3', and the downstream housekeeping gene GAPDH was 5'-TGATGACCCTTTTGGCTCCC-3'.

The above were used for quantitative real-time PCR. The total reaction volume was 10 μL, including 5 μL of SYBR master mix (Bio-Rad, United States), 0.2 μL of upstream and downstream primers, 1 μL of template cDNA and 3.6 μL of ddH₂O. The reaction conditions consisted of 5 min at 95 °C, 10 s at 94 °C, 10 s at 59 °C, 10 s at 72 °C for 40 cycles, and the temperature change rate was 20 °C/s, followed by 10 s at 95 °C, 15 s at 65 °C and then increased to 95 °C/10 s at a rate of 0.1 °C/s.

The fluorescence signal was continuously monitored for dissolution curve analysis, and GAPDH was used as an internal reference for relative quantitative analysis. The 2-ΔCt method was used to determine the expression level of the SERT gene relative to the internal reference (ΔCt = Ct target gene - Ct internal reference).

Plasma SERT and CCK levels: The levels of SERT and CCK in plasma were determined by double antibody sandwich ELISA (SERT/CCK kit).

Data were analyzed using SPSS version 24.0 (SPSS Inc, Chicago, IL, United States), and the significance level was set at P < 0.05 (two-tailed). The Shapiro-Wilk test was used to test the normal distribution of continuous variables. Comparisons between IBS patients and healthy controls regarding sociodemographic and clinical characteristics were examined using the independent sample t-test, Mann-Whitney U test, and χ² test as appropriate. The correlations between continuous variables were examined using Pearson correlation analyses if normally distributed; otherwise, Spearman correlation analyses were conducted.

The 40 IBS-D patients had a mean age of 44.50 ± 9.27 years, and 75% (n = 30) of these patients were male. The mean age of the 18 healthy controls was 42.33 ± 12.81 years, and 72.2% (n = 13) were male. The comparisons between IBS-D patients and healthy controls with regard to clinical characteristics are shown in Table 1. There were significant differences in the visceral sensitivity index (75.78 ± 9.31 vs 53.39 ± 4.49, P < 0.001), initial sensation threshold (31.00 ± 8.41 mL vs 52.22 ± 8.09 mL, P < 0.001), defecating sensation threshold (51.75 ± 13.57 mL vs 89.44 ± 8.73 mL, P < 0.001) and maximum tolerable threshold (97.25 ± 23.64 mL vs 171.11 ± 20.83 mL, P < 0.001) between patients and healthy controls. Anxiety (7.78 ± 2.62 vs 2.89 ± 1.02, P < 0.001) and depressive (6.38 ± 2.43 vs 2.06 ± 0.73, P < 0.001) symptoms were also significantly different between IBS-D patients and healthy controls.

The levels of SERT and CCK in plasma were determined by ELISA. CCK and SERT-mRNA expression in colonic mucosa was quantified by qRT-PCR. Immunohisto-chemistry showed that the cytoplasm of cholecystokinin immunoreactive (CCK-IR) cells contained brown granules with a clear background (Figure 1). SERT-positive products were brownish yellow and observed on the membrane (Figure 1). The levels of CCK in the mucosal membrane and plasma were significantly higher in IBS-D patients than in healthy controls, and SERT showed the opposite trend (Table 2).

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Figure 1 Mucosal histology and immunohistochemistry in patients with diarrhea-predominant irritable bowel syndrome. A and B: Mucosal cholecystokinin expression in diarrhea-predominant irritable bowel syndrome patients; C and D: Mucosal serotonin transporter expression in diarrhea-predominant irritable bowel syndrome patients. CCK: Cholecystokinin; SERT: Serotonin transporter.

Spearman correlations between key continuous variables in IBS-D patients are shown in Table 3, Table 4 and Table 5. In IBS-D patients, CCK expression was positively correlated with the severity of abdominal pain. Furthermore, the level of SERT was negatively correlated with the severity of abdominal pain and visceral sensitivity (Table 3, Table 4 and Table 5).

Diarrhea-predominant irritable bowel syndrome (IBS-D) patients have psychosomatic disorders and visceral hypersensitivity. Preclinical studies have shown that the expression of serotonin transporter (SERT) in the gut can affect the mental and psychological symptoms of IBS patients. Patients with lower expression of SERT are more likely to have negative emotions (somatization, anxiety, depression, hostility). An injection of cholecystokinin (CCK) can enhance and induce colonic motility, resulting in abdominal pain. Few studies have reported the relationship between mucous SERT, CCK levels and symptom severity, anxiety, depression and visceral hypersensitivity in patients. It has been suggested that SERT and CCK may be involved in the pathogenesis of IBS-D.

The present research includes three aspects: (1) General demographic characteristics, symptoms, psychological factors and visceral sensitivity; (2) The expression of SERT and CCK in colonic mucosa of IBS-D patients; and (3) Correlations between CCK, SERT and other parameters. Similar to preclinical studies in IBS-D patients, SERT and CCK may be involved in the pathogenesis of IBS-D, which provides a new potential method for identifying a more specific and effective therapeutic target.

The purpose of this study was to determine the expression of SERT and CCK in colonic mucosa of IBS-D patients, and analyze the relationship between SERT, CCK and general demographic characteristics, symptoms, psychological factors and visceral hypersensitivity.

The participants were evaluated by questionnaires (IBS symptom severity scale, visceral sensitivity index, hospital anxiety and Depression Scale) to obtain clinical and psychological characteristics, and underwent colonoscopy and mucosal biopsies. Visceral sensitivity was detected by a high-resolution manometric system, and the expression of SERT and CCK were detected by immunohistochemistry. Mucosal SERT and CCK mRNA levels were measured by real-time quantitative PCR. These parameters were statistically analyzed using SPSS version 24.0.

The results showed that the anxiety and depression symptoms and visceral sensitivity in IBS-D patients were significantly increased. The levels of CCK in the mucosal membrane and plasma were significantly higher in IBS-D patients than in healthy controls, and SERT showed the opposite trend. The expression of CCK was positively correlated with the severity of abdominal pain, and the level of SERT was negatively correlated with the severity of abdominal pain and visceral sensitivity.

IBS-D patients had psychosomatic disorders and visceral hypersensitivity. SERT and CCK might be involved in the pathogenesis of IBS-D by regulating the brain-gut axis and affecting visceral sensitivity. This provides a new potential method for identifying a more specific and effective therapeutic target.

Our study determined the role of SERT and CCK in the pathogenesis of IBS-D. There are some limitations in our study. First, only IBS-D patients were included, and selection bias could limit the generalization to other types of IBS patients. Second, only patients from one hospital were recruited. Third, the sample size was small. According to the existing research results, no causal inferences can be made. Therefore, more carefully designed clinical studies are needed.