The variation of tryptase expression in the colonic mucosa of patients with diarrhea-predominant irritable bowel syndrome and its effect on the visceral sensitivity

Abstract

Objective To observe the variation of tryptase expression in the colonic mucosa of patients with diarrhea-predominant irritable bowel syndrome(IBS-D) and the effects of their colonic mucosa cultured supernatants on mice visceral sensitivity. Methods The IBS-D patients study: of twenty-four patients with IBS-D and 12 healthy volunteers, four mucosal biopsies of each individual were collected at ascending colon under colonoscopy. The quantity of mucosal mast cells were detected by toluidine blue stains, and the tryptase concentration of colonic mucosa cultured supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Animal experiments: fifty-six male six to eight weeks old C57BL/6 mice were divided into 7 groups and then were administrated with healthy volunteer's ascending colonic mucosa-cultured supernatants 100 μl (group A), IBS-D patient' s ascending colonic mucosa-cultured supernatants 100 μl (group B), IBS-D ascending colonic mucosacultured supernatants 100 μl with Fut-175(50 μg/ml, group C), tryptase with different concentration:0.6 ng (group D), 2.2 ng (group E), tryptase 2.2 ng with Fut-175 (group F), and 0.9% sodium chloride as control (group G) respectively. After six hours, the abdominal myoelectric activities (AMAs) were recorded during colorectal balloon distension (CRD), and the expressions of substance P (SP), Calcitonin gene-related peptide (CGRP) in spinal dorsal horn of lumbosaeral region were detected by immunohistochemistry. Results The number of mast cells in the ascending colon of patients with IBS-D [6.33 ± 1.64) / HP] was higher than that in controls [(3.08 ± 0.77 ) / HP], the difference was statistical significant (P = 0.000). The tryptase concentration of ascending colonic mucosa cultured supernatants in IBS-D patients [(0. 072 ± 0. 023) ng/mg] was higher than that in controls [(0.026 ± 0.014) ng/mg], the difference was statistical significant ( P = 0. 000). In animal experiments, when the inside pressure of balloon was 30, 45, 60 mmHg, respectively, the AMAs value in group B [(27.50±5.23), (41.69±5.80), (54. 03±6.13) μV, respectively] and group E [(27. 05±1.66), (40. 66±3.39), (54. 38± 4.68) μV, respectively] were significantly higher than those in group A, group C, group D, group F and group G (P＜0. 05, respectively). The average integrated option densities (IOD) of SP and CGRP in spinal dorsal horn in groupB (121.72 ± 4.67,123.54±4.32)and E (124.69±5.06,126.32±5.09) were higher than in group A, group C, group D,group F and group G (P＜0.05, respectively). Conclusion The quantity of mast cell and the content of tryptase in the ascending colonic mucosa of IBS-D patients increased significantly. And their colonic mucosa cultured supernatants caused the mice visceral hypersensitivity, which indicated that tryptase may involved in the variation of visceral sensitivity in IBS-D patients. Key words: Irritable bowel syndrome; Mast cells; Tryptases; Visceral sensitivity; Substance P; Calcitonin gene-related peptide