We recruited 42 IBS-D patients (15 women and 27 men; mean age, 29.4 years; age range, 22-40 years) and 20 healthy controls (8 women and 12 men; mean age, 28.9 years; age range, 20-38 years). All patients were treated at the gastroenterology department of the China-Japan Friendship Hospital from January 2016 to July 2016. IBS-D was diagnosed according to the Rome III criteria. Controls were recruited through public advertisement or from asymptomatic patients undergoing colonoscopy for colorectal cancer screening or polyposis follow-up. Exclusion criteria included the use of anti-spasmodics, analgesics, antibiotics, nonsteroidal anti-inflammatory drugs, corticosteroids, mast cell stabilizers, and anti-depressants or a history of organic diseases (documented by patient history, appropriate consultation, and laboratory testing), celiac disease (documented by celiac serology), allergic disease, major abdominal surgery, and severe psychiatric disorders.

All participants underwent venous blood sampling and colonoscopy. Before colonoscopy, all subjects underwent a standard bowel preparation in the form of 3 L of dissolved polyethylene glycol electrolyte powder (Fortrans, Beaufour Ipsen Industrie, Dreux, France). Four biopsies were obtained in each subject from the rectosigmoid junction in order to standardize the site of sampling. Two biopsy specimens were used for routine hematoxylin and eosin (H and E) staining, mast cell staining, and immunohistochemistry. The other two biopsy specimens were used for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).

IBS-D patients and control patients both gave informed consent in a written form before entry into the study. The study protocol was approved by the China-Japan Friendship Hospital Ethics Committee (No. 2015-33) and the study was conducted in accordance with the Declaration of Helsinki.

Questionnaires: Clinical status was evaluated with the use of validated questionnaires. The IBS Symptom Severity Scale[22,23], a 5-item self-reporting questionnaire designed to measure disease severity, was given to each participant to complete. Questionnaire items included information regarding abdominal pain, bloating, satisfaction with bowel habits, and overall interference with quality of life. The total score of the questionnaire ranges from 0 to 500.

The IBS-specific Quality of Life questionnaire[24,25] was used to evaluate changes in patients’ quality of life. This scale evaluates 34 broad well-being factors based on variables including feelings of dysphoria, social interactions, body image, and health worries. The Hamilton Anxiety Scale[26,27] and Hamilton Depression Scale[27,28] were used to measure anxiety and depression, respectively.

Visceral sensitivity testing: Participants were placed in the left lateral decubitus position. After a digital rectal examination, a lubricated catheter with a latex balloon at the tip (Medical Measurement Systems, Enschede, the Netherlands) was inserted into the rectum such that the balloon was 8 cm proximal to the anal verge. Then, the balloon was inflated at a speed of 10 mL/5 s. During balloon inflation, feelings of initial perception, defecation urge, and discomfort/pain were reported by the participants. The balloon volumes were recorded at the first sensation threshold, defecating sensation threshold, and maximum tolerable threshold. All the tests were performed by the same investigator between 1 and 3 PM in a blinded fashion.

Serum leptin level: All fasting blood samples from both IBS-D patients and controls were collected between 11 AM and 1 PM to avoid diurnal variation. Whole blood was centrifuged immediately at 1151 g for 10 min, and stored at -80 °C until assay. Serum leptin levels were measured in duplicate using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Human Leptin Instant ELISA; Bender MedSystems GmbH, Vienna, Austria).

Histology, immunohistochemistry, and immunofluorescence: H and E sections from formalin-fixed and paraffin-embedded tissue samples were blindly assessed by independent observers. Mast cells were stained with toluidine blue. The slides were first soaked in 0.5% toluidine blue, and then differentiated with acetone. Afterwards, five 400 × magnification fields (field area, 0.237 mm²) were chosen randomly and scanned. Mast cells were identified using light microscopy by their metachromatic cytoplasmic granules. Finally, mast cell degranulation was assessed based on unclear or irregular cell membranes and the presence of extruded secretory granules. The mean mast cell number per millimetre square of the mucosal area (/mm²) was calculated. The percentage of degranulated mast cells (mast cell activation rate) was also calculated in each section (degranulated mast cells/the total number of mast cells × 100%).

Immunohistochemistry and immunofluorescence experiments were performed on paraffin-embedded, 4-mm-thick sections. The following primary antibodies were used: rabbit polyclonal anti-leptin antibody (1:100; Abcam, Cambridge, United Kingdom), rabbit polyclonal anti-leptin receptor antibody (1:50; Abcam, Cambridge, United Kingdom), mouse monoclonal anti-mast cell tryptase antibody (1:50; Abcam, Cambridge, United Kingdom), and mouse monoclonal anti-PGP9.5 antibody (1:50; Abcam, Cambridge, United Kingdom).

For immunohistochemistry studies, the sections were first incubated with the primary antibody overnight. Next, they were incubated at room temperature with a universal secondary antibody (EnVision Detection Systems, Dako, Denmark) for 60 min and then visualized using diaminobenzidine. Finally, the nuclei were labeled by counterstaining the sections with Mayer’s hematoxylin. The quantification of immunoreactivity was performed by two operators in a blinded fashion. Three randomly selected fields from each section were scanned under a Nikon Eclipse 80i microscope (Nikon Instruments Co., Ltd., Tokyo, Japan). Photographs were taken with a Nikon DS-Ri1 camera (Nikon Instruments Co., Ltd., Tokyo, Japan). The images were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, United States). During the analysis, the average integrated option density (IOD) of positive staining substances of three non-overlapping fields chosen randomly was measured for the final analysis.

For immunofluorescence studies, the sections were prepared sequentially as follows: incubated with primary antibody (double labeling) overnight, rinsed with phosphate-buffered saline, and incubated at room temperature with goat anti-mouse IgG H and L (Alexa Fluor 647) (1/300; Abcam, Cambridge, United Kingdom) or goat anti-rabbit IgG H and L (Alexa Fluor^® 488) (1/400; Abcam, Cambridge, United Kingdom) for 2 h. Specimens were examined by two operators in a blinded fashion using a Nikon Eclipse 90i microscope (Nikon Instruments Co., Ltd., Tokyo, Japan). Representative photomicrographs were taken using a Nikon DS-Ri1 camera.

Leptin gene expression analysis using qRT-PCR: The gene expression of leptin and the leptin receptor in the colonic mucosa was analyzed using qRT-PCR. Each supernatant was assayed in duplicate, and altogether three independent experiments were conducted. TRIzol reagent (Invitrogen Life Technologies, Waltham, MA, United States) was used to isolate total RNA. Then, the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, United States) was used to perform reverse transcription. Next, real-time PCR was carried out using the FastStart Universal SYBR Green Master Rox kit (Roche, Shanghai, China) in the StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, United States). Finally, leptin and leptin receptor mRNA expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The following primers were used for PCR amplification: forward, 5’-ACTTTGGTATCGTGGAAGGACTCAT-3’ and reverse, 5’-GTTTTTCTAGACGGCAGGTCAGG-3’ for GAPDH; forward, 5’-ACACGCAGTCAGTCTCCTCCAA-3’ and reverse, 5’-CTGGAAGGCATACTGGTGAGGA-3’ for leptin; and forward, 5’-GCCAACAGCCAAACTCAACG-3’ and reverse, 5’-GGTGGGCTGGACCAAGAAATC-3’ for leptin receptor. The results were first normalized to GAPDH expression using the 2^-ΔΔCt method[29]. Then, the leptin and leptin receptor mRNA levels in each sample are expressed as the fold-change relative to the average level of healthy controls, in whom the mean value was used as the reference and considered as 1.

All statistical analyses were performed using SPSS for Windows software, version 24.0 (SPSS Inc, Chicago, IL). All data are reported as values (mean ± SD) or medians [interquartile range (IQR)]. Independent sample t-tests or nonparametric Mann-Whitney U-tests were used to analyze quantitative data. The χ² test or Fisher’s exact test were used to analyze qualitative data. Correlations between two parameters were performed using Spearman’s correlation coefficient, followed by Bonferroni correction to adjust multiple comparisons, with a corrected significance level of 0.0038 (0.05/13). Two-tailed P-values < 0.05 were considered significant.

There were no statistically significant differences in age, gender, or body mass index between the IBS-D group and the control group. The median IBS Symptom Severity Scale score in the IBS-D group was 225.0 (range, 100-475). There was no significant difference between male and female IBS-D patients in terms of IBS Symptom Severity Scale score (P = 0.053). The median duration of disease was 3.5 years (range, 1-13 years) in the IBS-D patients. The IBS-specific Quality of Life score was significantly lower in IBS-D patients (P < 0.001), and the differences in the scores of both the Hamilton Anxiety Scale and the Hamilton Depression Scale were also statistically significant between the IBS-D group and the control group (P < 0.001) (Table 1).

The first sensation threshold, defecation sensation threshold, and maximum tolerable threshold were significantly lower in the IBS-D patients compared with those in the control group (P < 0.001) (Table 1).

Serum leptin levels in the IBS-D patients (median, 1.28 ng/mL; IQR, 1.63 ng/mL) and healthy controls (median, 1.27 ng/mL; IQR, 0.83 ng/mL) were not significantly different (P = 0.657).

On H and E histology, all colonic mucosal biopsies were reported as normal (Figure 1A and B). Toluidine blue staining revealed that mast cells were often scattered in the lamina propria and submucosa (Figures 1C and D). The mast cell count was not significantly different between the two groups (IBS group: median, 9.62/mm²; IQR, 1.61/mm²vs control group: median, 10.75/mm²; IQR, 4.52/mm²; P = 0.164) (Figure 1E). However, the percentage of degranulated mast cells was significantly increased in IBS-D patients (IBS group: median, 71.2%; IQR, 12.9% vs control group: median, 59.4%; IQR, 18.88%; P < 0.001) (Figure 1F).

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Figure 1 Intestinal mucosal histology (hematoxylin and eosin staining), mast cell toluidine blue staining, mast cell count (/mm²), and mast cell activation rate (%) in irritable bowel syndrome patients and controls. A: Colonic mucosal histology of IBS-D patients (× 200); B: Colonic mucosal histology of healthy controls (× 200); C: Mast cell toluidine blue staining of IBS-D tissue (× 400, arrows represent mast cells); D: Mast cell toluidine blue staining of normal control tissue (× 400, arrows represent mast cells); E: Mast cell count was not significantly different between the two groups (P = 0.164); F: Mast cell activation rate significantly increased in IBS-D patients (P < 0.001). Line in the scatter plots means the median. IBS-D: Iirritable bowel syndrome with diarrhea.

In the colonic mucosa, leptin and leptin receptor staining was mostly scattered in the epithelium and lamina propria (Figure 2A-D). The leptin protein level in mucosal biopsies was significantly increased in the IBS-D patients (IOD median, 4424.71; IQR, 4533.63) vs the control group (IOD median, 933.65; IQR, 888.10; P < 0.001) (Figure 2E). The leptin receptor expression was not significantly increased in the colonic mucosa obtained from IBS-D patients (12454.27 ± 6946.06) compared to controls (12508.07 ± 8088.46) (P = 0.979) (Figure 2F).

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Figure 2 Immunohistochemical staining for leptin and leptin receptor in irritable bowel syndrome patients and healthy controls. A: Colonic mucosal leptin expression in IBS-D patients (× 400); B: Colonic mucosal leptin expression in healthy controls (× 400); C: Colonic mucosal leptin receptor expression in IBS-D patients (× 400); D: Colonic mucosal leptin receptor expression in healthy controls (× 400); E: Colonic mucosal leptin immunoreactivity significantly increased in IBS-D patients than in the healthy controls; F: Colonic mucosal leptin receptor immunoreactivity did not increase significantly in IBS-D patients than in healthy controls. Line in the scatter plots means the median. IBS-D: Iirritable bowel syndrome with diarrhea; IOD: Integral optical density.

Leptin and tryptase double-labeling immunofluorescence experiments recorded colocalization of leptin and tryptase, indicating that mast cells were producers of leptin (Figure 3A and B). Moreover, leptin receptor and tryptase colocalization was found to exist in the colonic mucosa (Figure 4A and B). Double-staining experiments confirmed leptin receptor expression in PGP9.5-immunoreactive nerve fibers (Figure 4C and D).

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Figure 3 Double-labeling immunofluorescence of leptin and tryptase in irritable bowel syndrome patients and healthy controls. A: Colonic mucosal double-labeling immunofluorescence of leptin and tryptase in IBS-D patients (× 400, arrows represent colocalization of leptin and tryptase); B: Colonic mucosal double-labeling immunofluorescence of leptin and tryptase in healthy controls (× 400, arrows represent colocalization of leptin and tryptase). IBS-D: Iirritable bowel syndrome with diarrhea.

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Figure 4 Double-labeling immunofluorescence of leptin receptor with tryptase and PGP9. 5 in irritable bowel syndrome patients and healthy controls (×400 magnification). A: Colonic mucosal double-labeling immunofluorescence of leptin receptor and tryptase in IBS-D patients (× 400, arrows represent colocalization of leptin receptor and tryptase); B: Colonic mucosal double-labeling immunofluorescence of leptin receptor and tryptase in healthy controls (× 400, arrows represent colocalization of leptin receptor and tryptase); C: Colonic mucosal double-labeling immunofluorescence of leptin receptor and PGP9.5 in IBS-D patients (× 400, arrows represent colocalization of leptin receptor and PGP9.5); D: Colonic mucosal double-labeling immunofluorescence of leptin receptor and PGP9.5 in healthy controls (× 400, arrows represent colocalization of leptin receptor and PGP9.5). PGP9.5: Protein gene product 9.5; IBS-D: Iirritable bowel syndrome with diarrhea.

Leptin mRNA expression was significantly increased in IBS-D patients (median, 1.1226; IQR, 1.6351) vs that seen in the control group (median, 0.8947; IQR, 0.4595; P = 0.009). Similarly, leptin receptor gene expression was also significantly increased in IBS-D patients (median, 1.5491; IQR, 2.0721) vs that seen in the control group (median, 0.9062; IQR, 0.3850; P = 0.001).

In patients with IBS-D, the leptin protein expression level showed a significantly positive correlation with anxiety, depression, and mast cell activation rate, and a negative correlation with the defecation sensation threshold and maximum tolerable threshold (adjusted P < 0.0038). The leptin mRNA level was significantly positively correlated with IBS symptom severity, anxiety, and depression, and negatively correlated with the visceral sensation threshold (adjusted P < 0.0038) (Table 2).

Irritable bowel syndrome (IBS) is a commonly diagnosed functional gastrointestinal disease. Symptoms may worsen over time and significantly impact patient quality of life and work productivity. A major subtype of IBS is IBS with diarrhea (IBS-D). The pathogenesis of IBS-D is complex and poorly understood. Leptin not only exerts significant biological effects, such as appetite control, by signaling satiety and increasing energy expenditure, but also modulates the immune system and gastrointestinal function. Few studies have specifically addressed the role of leptin in the pathogenesis of IBS. To our knowledge, this was the first study to preliminarily investigate the possible role of leptin in the pathophysiology of IBS-D.

The main topics of the present study include evaluating clinical symptoms, psychological characteristics, and visceral sensitivity of IBS-D patients and healthy controls; examining leptin expression, mast cells, and PGP9.5 nerve fibres in IBS-D and healthy controls; and performing correlation analyses between these parameters. Our study proposed a mechanism by which leptin may contribute to the pathophysiology of IBS.

This study aimed to measure leptin expression in both the serum and intestinal mucosa of patients with IBS-D subtype disease and to analyze the relationship of leptin with the clinical features, visceral sensitivity, and number of mast cells and nerve fibers in these patients.

Participants underwent clinical and psychological evaluations using validated questionnaires (including IBS Symptom Severity Scale, IBS-specific Quality of Life, Hamilton Anxiety Scale and Hamilton Depression Scale), along with colonoscopy, colonic mucosal biopsy, and visceral sensitivity testing. Serum leptin levels were assayed using enzyme-linked immunosorbent assay. Mucosal leptin expression and localization were evaluated using immunohistochemistry and immunofluorescence. Mucosal leptin mRNA levels were quantified using quantitative real-time reverse transcription polymerase chain reaction. Mast cell counts and activation rates were investigated by toluidine blue staining. Correlation analyses between these parameters were performed. All statistical analyses were performed using SPSS for Windows software, version 24.0 (SPSS Inc, Chicago, IL).

The authors found that IBS-D patients had significantly increased psychological symptoms and visceral hypersensitivity, and their mucosal leptin expression, leptin mRNA levels, and mast cell activation rates were significantly increased. Also, leptin expression was positively correlated with anxiety, depression, and the mast cell activation rate, but negatively correlated with the defecation sensation threshold and the maximum tolerance threshold during visceral sensitivity testing. Increased levels of mucosal leptin may interact with mast cells and the nervous system to contribute to the pathogenesis of IBS-D.

This study presents evidence that leptin levels are increased in the intestinal mucosa of IBS-D patients, proposes a mechanism by which leptin may contribute to the pathophysiology of IBS, and provides some potential avenues for more specific and effective treatments in these patients. The authors believe that this study makes a significant contribution to the literature because it is an original research that provides information that not only contributes to the understanding of the pathogenesis and pathophysiology of IBS, but also suggests area for future research and therapeutic intervention.

This study preliminarily investigated the possible role of leptin involved in the pathogenesis of IBS-D. Future studies should focus on the following aspects. First, the present study focused on patients with the IBS-D subtype. Therefore, the findings may not be generalizable to constipation-predominant IBS, mixed-type IBS, and unsubtyped IBS patients. Future research should investigate the possible role of leptin in the pathophysiology of the other three subtypes. Second, leptin has been shown to increase during the process of inflammation. However, we cannot be sure that all postinfective IBS patients were excluded from the study. Because these two subgroups may be different in pathogenesis, future experiments should study these two types separately. Finally, we cannot make a cause and effect inference on the basis of our findings, therefore, additional studies of the effects of leptin on mast cells and the nervous system as well as larger clinical studies are needed.