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. 2001 Apr;48(4):478–488. doi: 10.1136/gut.48.4.478

Calpain inhibitor I reduces colon injury caused by dinitrobenzene sulphonic acid in the rat

S Cuzzocrea 1, M McDonald 1, E Mazzon 1, H Mota-Filipe 1, T Centorrino 1, M Terranova 1, A Ciccolo 1, D Britti 1, A Caputi 1, C Thiemermann 1
PMCID: PMC1728239  PMID: 11247891

Abstract

BACKGROUND AND AIMS—Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leucocyte infiltration, upregulation of expression of intercellular adhesion molecule 1 (ICAM-1), and upregulation of P-selectin in the colon. The aim of the present study was to examine the effects of calpain inhibitor I in rats subjected to experimental colitis.
METHODS—Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS).
RESULTS—Rats experienced haemorrhagic diarrhoea and weight loss. Four days after administration of DNAB, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology as well as by an increase in myeloperoxidase activity in the mucosa) was associated with upregulation of ICAM-1 and P-selectin as well as high tissue levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed intense staining in the inflamed colon. Staining of sections of colon obtained from DNBS treated rats with an anti-cyclooxygenase 2 antibody showed diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase was found mainly in macrophages located within the inflamed colon of DNBS treated rats. Calpain inhibitor I (5 mg/kg daily intraperitoneally) significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. Calpain inhibitor I also caused a substantial reduction in (i) degree of colon injury, (ii) rise in myeloperoxidase activity (mucosa), (iii) increase in tissue levels of malondialdehyde, (iv) increase in staining (immunohistochemistry) for nitrotyrosine and PARP, as well as (v) upregulation of ICAM-1 and P-selectin caused by DNBS in the colon.
CONCLUSION—Calpain inhibitor I reduces the degree of colitis caused by DNBS. We propose that calpain inhibitor I may be useful in the treatment of inflammatory bowel disease.


Keywords: calpain; calpain inhibitor I; cyclooxygenase; nitric oxide; inflammatory bowel disease; rat

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Figure 1  .

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Effects of calpain inhibitor I (CALP-1) on the macroscopic damage score (A) and microscopic histological score (B). Colon damage was scored by two independent observers. Values are mean (SEM) of data obtained from 10 rats in each group. **p<0.01 v sham; ††p<0.01 v dinitrobenzene sulphonic acid (DNBS).

Figure 2  .

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Effects of calpain inhibitor I on colon injury. No histological modification was observed in the mucosa of sham operated rats (A). Mucosal injury was produced after dinitrobenzene sulphonic acid (DNBS) administration, characterised by the absence of epithelium and massive mucosal and submucosal infiltration with inflammatory cells (B). Treatment with calpain inhibitor I (C) corrected the disturbances in morphology associated with DNBS administration. Original magnification ×120. The figure is representative of at least three experiments performed on different experimental days.

Figure 3  .

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Organ weight. A significant increase in weight in the spleen (A) and colon (B) was consistently seen four days after dinitrobenzene sulphonic acid (DNBS) injection. The weight of the organs was significantly reduced in rats who had been treated with calpain inhibitor I (CALP-1). Values are mean (SEM) of 10 rats in each group. **p<0.01 v sham; ††p<0.01 v DNBS.

Figure 4  .

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Effect of calpain inhibitor I (CALP-1) treatment on dinitrobenzene sulphonic acid (DNBS) induced mortality. Survival is significantly improved in CALP-1 treated rats in comparison with the high mortality rate of the DNBS treated rats. n=10 rats in each group. **p<0.01 v DNBS.

Figure 5  .

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Effect of calpain inhibitor I (CALP-1) treatment on changes in body weight four days after dinitrobenzene sulphonic acid (DNBS) intracolonic administration. Body weight was recorded immediately before DNBS administration and at the end of the experimental period. CALP-1 treatment significantly prevented the loss in body weight. Values are mean (SEM) of 10 rats in each group. **p<0.01 v sham; ††p<0.01 v DNBS.

Figure 6  .

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Effect of calpain inhibitor I (CALP-1) treatment on inducible nitric oxide synthase (iNOS) activity four days after dinitrobenzene sulphonic acid (DNBS) intracolonic administration. DNBS administration induced a significant increase in iNOS activity. CALP-1 treatment significantly reduced iNOS activity. Values are mean (SEM) of 10 rats in each group. **p<0.01 v sham; ††p<0.01 v DNBS.

Figure 7  .

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Immunohistochemical localisation for cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in the colon. Immunohistochemical staining for COX (A) and iNOS (B) showed positive staining localised in the injured area from dinitrobenzene sulphonic acid (DNBS) treated rats. The intensity of the observed staining for COX (D) and iNOS (E) was significantly reduced in the colon from calpain inhibitor I treated rats. (C) and (F) represent the staining combination of (A-B) and (D-E), respectively. Original magnification ×145. The figure is representative of at least three experiments performed on different experimental days.

Figure 8  .

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Immunohistochemical localisation of nitrotyrosine and poly (ADP-ribose) polymerase (PARP) in the colon. No positive staining for nitrotyrosine (A) or PARP (B) was found in colon sections from sham operated rats. Immunohistochemical staining for nitrotyrosine (D) and PARP (E) showed positive staining localised in the injured area from a dinitrobenzene sulphonic acid (DNBS) treated rat. The intensity of the staining for nitrotyrosine (G) and PARP (H) was significantly reduced in the ileum from calpain inhibitor I treated rats. (C), (F), and (I) represent the staining combination of (A-B), (D-E), and (G-H), respectively. Original magnification ×145. The figure is representative of at least three experiments performed on different experimental days.

Figure 9  .

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Effect of calpain inhibitor I (CALP-1) on neutrophil infiltration and lipid peroxidation. Myeloperoxidase (MPO) activity (A) and malondialdehyde (MDA) levels (B) in the colon from dinitrobenzene sulphonic acid (DNBS) treated rats. MPO activity and MDA levels were significantly increased in DNBS treated rats in comparison with sham controls. CALP-1 treated rats showed a significant reduction in MPO activity and MDA levels. Values are mean (SEM) of 10 rats in each group. **p<0.01 v sham; ††p<0.01 v DNBS.

Figure 10  .

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Immunohistochemical localisation of P-selectin in the colon. Staining of colon sections obtained from sham operated rats with anti-intercellular adhesion molecule 1 (ICAM-1) antibody showed specific staining along vessels, demonstrating that ICAM-1 is constitutively expressed (A). Ileum sections from sham operated rats revealed no positive staining for P-selectin (B). Sections obtained from dinitrobenzene sulphonic acid (DNBS) treated rats showed intense positive staining for ICAM-1 (D) and P-selectin (E) on endothelial cells. The degree of endothelial staining for ICAM-1 (G) and P-selectin (H) was markedly reduced in tissue sections obtained from calpain inhibitor I treated rats. (C), (F), and (I) represent the staining combination of (A-B), (D-E), and (G-H) respectively. Original magnification ×145. The figure is representative of at least three experiments performed on different experimental days.

Figure 11  .

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Proposed scheme of some of the delayed inflammatory pathways involving nitric oxide (NO*), hydroxyl radical (OH*), and peroxynitrite (ONOO) in dinitrobenzene sulphonic acid (DNBS) induced colitis, and potential sites of the anti-inflammatory actions of calpain inhibitor I. DNBS triggers expression of inducible nitric oxide (NO) synthase (iNOS), at least in part, via activation of nuclear factor κB (NF-κB). NO in turn combines with superoxide to form ONOO-. Hydroxyl radical (produced from superoxide via the iron catalysed Haber-Weiss reaction) and ONOO- or peroxynitrous acid (ONOOH) induce cellular injury. Part of the injury is related to the development of DNA single strand breakage, with consequent activation of poly (ADP-ribose) polymerase (PARP), leading to cellular dysfunction. Expression of the inducible isoform of cyclooxygenase (COX-2) is also dependent on activation of NF-κB. In addition, NO can directly increase the catalytic activity of COX-2, leading to enhanced production of proinflammatory prostaglandin metabolites. We propose that the anti-inflammatory effects of calpain inhibitor I may include (1) inhibition of activation of NF-κB and prevention of expression of iNOS and COX-2, (2) inhibition of ONOO formation, and (3) prevention of activation of PARP (see discussion for further explanations).

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