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. 2018 Sep;175(17):3516-3533.
doi: 10.1111/bph.14396. Epub 2018 Jul 23.

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome

Hannah Ceuleers  1 , Nikita Hanning  1 , Jelena Heirbaut  1 , Samuel Van Remoortel  2 , Jurgen Joossens  3 , Pieter Van Der Veken  3 , Sven M Francque  4 , Michelle De Bruyn  5 , Anne-Marie Lambeir  5 , Joris G De Man  1 , Jean-Pierre Timmermans  2 , Koen Augustyns  3 , Ingrid De Meester  5 , Benedicte Y De Winter  1
Affiliations

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome

Hannah Ceuleers et al. Br J Pharmacol. 2018 Sep.

Abstract

Background and purpose: Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS).

Experimental approach: Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates.

Key results: VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αβ-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals.

Conclusions and implications: Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain.

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Figures

Figure 1
Figure 1
Overview of the experimental design. On day 0, rats received an intrarectal treatment with TNBS (colitis) or saline (control). The severity of colitis and the mucosal healing were monitored in vivo using colonoscopy: on day 3 to confirm the presence of colitis and from day 10 onwards every 4 days until complete mucosal healing. Hereafter, EMG electrodes were implanted, and 3 days later, all experiments were conducted after a single i.p. injection with vehicle/serine protease inhibitor.
Figure 2
Figure 2
The effect of nafamostat mesylate (0.01–10 mg·kg−1) and its vehicle (water for injection) on VMRs (n = 8 per group and n = 7 per group for 10 mg·kg−1) and colonic compliance (n = 6 per group) in post‐colitis and control rats. The statistical analysis was performed on the complete dataset, but separate graphs were made for each dose for greater clarity. Thus, in rats post‐colitis, the effects of 0.01 mg·kg−1 nafamostat are shown in A; 0.1 mg·kg−1 in B, 1.0 mg·kg−1 in C and 10 mg·kg−1 in D. In E, 0.1 mg·kg−1 of nafamostat was given to control rats, i.e. without colitis and did not change the VMR responses. In F, 0.1 mg·kg−1 nafamostat did not change colonic compliance in either control or post‐colitis rats. Data are presented as mean ± SEM. *P < 0.05; significantly different from control + vehicle; # P < 0.05; significantly different from post‐colitis + vehicle; generalized estimating equations + least significant difference post hoc test.
Figure 3
Figure 3
The effect of UAMC‐00050 (0.01–1 mg·kg−1) and its vehicle (5% DMSO) on VMRs. In A, results presented are from post‐colitis rats + 0.01 mg·kg−1 (n = 9); in B, post‐colitis + 0.1 mg·kg−1 (n= 7); in C, post‐colitis + 1 mg·kg−1, (n = 7); in D, results are from control rats treated with 1 mg·kg−1, UAMC‐00050 (n = 6). In E, the lack of effects of 1 mg·kg−1 UAMC‐00050 on colonic compliance are shown, for control rats and post colitis rats (n = 6). The statistical analysis was performed on the complete dataset, but separate graphs were made for each dose for greater clarity. Data are presented as mean ± SEM. *P < 0.05; significantly different from control + vehicle. # P < 0.05, significantly different from post‐colitis + vehicle; generalized estimating equations + least significant difference post hoc test.
Figure 4
Figure 4
The effect of UAMC‐01162 (1–2.5 mg·kg−1) and its vehicle (5% DMSO) on VMRs and colonic compliance in post‐colitis and control rats (n = 10 per group). In A, results presented are from post‐colitis rats + 1 mg·kg−1; in B, post‐colitis + 2.5 mg·kg−1. In C, the results are from control rats treated with 2.5 mg·kg−1 UAMC‐01162. In D, the results show the lack of effects of 2.5 mg·kg−1 UAMC‐01162 on colonic compliance are shown, for control rats and post‐colitis rats. The statistical analysis was performed on the complete dataset, but separate graphs were made for each dose for greater clarity. Data are presented as mean ± SEM. *P < 0.05; significantly different from control + vehicle. # P < 0.05; significantly different from post‐colitis + vehicle; generalized estimating equations + least significant difference post hoc test.
Figure 5
Figure 5
Immunohistochemistry with mast cell tryptase antibody in rat colon. (A) Representative images of the colonic mucosa of a control and a post‐colitis animal with mast cell tryptase (arrow). (B–D) The number of tryptase‐positive mast cells mm−2 in the colonic mucosa of control and post‐colitis rats with or without treatment with a serine protease inhibitor. Data shown are means +SEM; n = 8 per group. *P < 0.05; significant effect of the factor ‘group’; no significant effect of the factor ‘drug’; no significant interaction between the factors ‘group’ and ‘drug’; two‐way ANOVA.
Figure 6
Figure 6
Immunohistochemical localization of PAR2, PAR4 and TRPA1 channels in sensory nerve fibres of the distal colon. (A–C) Representative images showing co‐localization of PAR2 (red) in CGRP‐immunopositive nerve fibres (green). (D–F) Representative images showing the presence of PAR4 immunoreactivity (red) in the colonic epithelium and in enteric nerve plexuses (arrowheads) but not in the CGRP‐immunoreactive nerve fibre population (inset). (G–I) Representative images showing co‐localization of TRPA1 protein (red) in CGRP‐immunopositive nerve fibres (green).
Figure 7
Figure 7
Proteolytic activities determined in colon samples. Trypsin‐like activity was measured using Boc‐Gln‐Ala‐Arg‐AMC (in A) and n‐Tos‐Gly‐Pro‐Arg‐AMC (in B). To determine the neutrophil elastase (in C), pancreas elastase (in D), KLK (in E) and chymotrypsin‐like activity (in F), Suc‐Ala‐Ala‐Pro‐Val‐AMC, Suc‐Ala‐Ala‐Ala‐AMC, H‐Pro‐Phe‐Arg‐AMC and Suc‐Ala‐Ala‐Pro‐Phe‐AMC were used respectively. Data are presented as mean ± SEM (n = 12). *P < 0.05; Mann–Whitney U‐test.
Figure A1
Figure A1
Proteolytic activities determined in faecal samples. Trypsin‐like activity was measured using Boc‐Gln‐Ala‐Arg‐AMC (in A) and n‐Tos‐Gly‐Pro‐Arg‐AMC (in B). To determine the neutrophil elastase (in C), pancreas elastase (in D), KLK (in E) and chymotrypsin‐like activity (in F), Suc‐Ala‐Ala‐Pro‐Val‐AMC, Suc‐Ala‐Ala‐Ala‐AMC, H‐Pro‐Phe‐Arg‐AMC and Suc‐Ala‐Ala‐Pro‐Phe‐AMC were used respectively. Data are presented as mean ± SEM. Mann–Whitney U‐test; n = 12.
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