The cyclopentenone prostaglandin 15-deoxy-delta(12,14)- PGJ2 attenuates the development of colon injury caused by dinitrobenzene sulphonic acid in the rat

Abstract

1. Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leukocyte infiltration, and increased expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) in the colon. Recent evidence also suggests that the cyclopentenone prostaglandin (PG) 15-deoxy-delta(12,14)-PGJ(2) (15d- PGJ(2)) functions as an early anti-inflammatory signal. 2. The aim of the present paper is to investigate the effects of 15d-PGJ(2) in rats subjected to experimental colitis. 3. Colitis was induced in rats by intra-colonic instillation of dinitrobenzene sulphonic acid (DNBS). 15d-PGJ(2) was administered daily as intraperitoneal injection (20 or 40 microg kg(-1)). On day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. 4. 15d-PGJ(2) significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. 15d-PGJ(2) also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase (MPO) activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde (MDA) and (iv) of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). 5. Furthermore, 15d-PGJ(2) reduced the increase in immunohistochemical staining for (i) inducible nitric oxide synthase (iNOS), (ii) nitrotyrosine and (iii) poly (ADP-ribose) polymerase (PARP), as well as (iv) the increased expression of ICAM-1 caused by DNBS in the colon. 6. Electrophoresis mobility shift assay (EMSA) of inflamed colon revealed that 15d- PGJ(2) also caused a substantial reduction of the activation of nuclear factor-kappaB (NF-kappaB). Furthermore, 15d-PGJ(2) stimulates the activation of heat shock protein 72 (hsp72) in the inflamed colon, as assessed by Western blot analysis. 7. In conclusion, 15d-PGJ(2) reduces the development of experimental colitis.

Figure 1

Effect of 15d-PGJ₂ treatment on the colon damage score and neuthophil infiltration. Macroscopic damage score (A), histological damage score (B) and myeloperoxidase (MPO) activity in the colon from DNBS-treated rats. Macroscopic damage score, histological damage score and MPO activity were significantly increased in DNBS-treated rats in comparison to sham. 15d-PGJ₂-treated rats show a significant reduction of macroscopic damage score, histological damage score and MPO activity. Macroscopic damage score (n=10 rats for each group), and histological damage score (n=6 section for each animals) were scored by two independent observers blinded to the experimental protocol MPO values are mean±s.e.mean of 10 rats for each group. ^*P<0.01 vs SHAM; ○P<0.01 vs DNBS.

Figure 2

Effect of 15d-PGJ₂ on colon injury. No histological modification was observed in mucosal from sham-operated rats (A). Mucosal injury was produced after DNBS administration characterized by absence of epithelium and a massive mucosal and submucosal infiltration of inflammatory cells (B). Treatment with 15d-PGJ₂ (40 μg kg⁻¹) (C) corrected the disturbances in morphology associated with DNBS administration. Original magnification: ×125. Figure is representative of at least three experiments performed on different experimental days.

Figure 3

Effects of 15d-PGJ₂ on the levels of cytokines in the colon. Colon levels of TNF-α and IL-1β were significantly increased at 4 days after DNBS administration. 15d-PGJ₂ treatment significantly reduced in a dose dependent manner the cytokine levels. Values are mean±s.e.mean of 10 rats for each group. ^*P<0.01 vs SHAM; ○P<0.01 vs DNBS.

Figure 4

Immunohistochemical localization for iNOS in the colon. No positive staining for iNOS (A) was found in the colon section from sham-administered rats. Immunohistochemical analysis for iNOS (B) show positive staining localized in the injured area from DNBS-treated rats. The intensity of the positive staining for iNOS (C) was significantly reduced in the colon from 15d-PGJ₂ (40 μ kg⁻¹)-treated rats. Original magnification: ×125. Figure is representative of at least three experiments performed on different experimental days.

Figure 5

Immunohistochemical localization for nitrotyrosine and PAR in the colon. No positive staining for nitrotyrosine (A) and for PAR (D) was found in the colon section from sham-operated rats. Immunohistochemical analysis for nitrotyrosine (B) and for PAR (E) show positive staining localized in the injured area from a DNBS-treated rats. The intensity of the positive staining for nitrotyrosine (C) and for PAR (F) was significantly reduced in the colon from 15d-PGJ₂ (40 μg kg⁻¹)-treated rats. Original magnification: ×125. Figure is representative of at least three experiments performed on different experimental days.

Figure 6

Effect of 15d-PGJ₂ on lipid peroxidation. Malondialdehyde (MDA) in the colon from DNBS-treated rats. MDA levels were significantly increased in DNBS-treated rats in comparison to sham. 15d-PGJ₂ -treated rats show a significant reduction of MDA levels. Values are mean±s.e.mean of 10 rats for each group. ^*P<0.01 vs SHAM; ○P<0.01 vs DNBS.

Figure 7

Immunohistochemical localization of ICAM-I in the colon. Staining of colon tissue sections obtained from sham-operated rats with anti-ICAM-1 antibody showed a specific staining along vessels, demonstrating that ICAM-1 is constitutively expressed (A). Section obtained from DNBS-treated rats showed intense positive staining for ICAM-1 (B) on endothelial cells. The degree of endothelial staining for ICAM-1 (C) was markedly reduced in tissue section obtained from 15d-PGJ₂ (40 μg kg⁻¹)-treated rats. Original magnification: ×125. Figure is representative of at least three experiments performed on different experimental days.

Figure 8

Effect of 15d-PGJ₂ on NF-κB/DNA binding activity in rat colon. Whole extracts from inflamed (control) or non-inflamed (naïve) rat colon were prepared as described in Methods and incubated with ³²P-labelled NF-κB probe. Representative EMSA of NF-κB show the effect of 15d-PGJ₂ (20 μg kg⁻¹) on NF-κB/DNA binding activity evaluated in tissue from colon rat 4 days after induction of colitis. In competition reaction whole cell extracts were incubated with radiolabelled NF-κB probe in absence or presence of identical but unlabelled oligonucleotides (W.T. 50×), mutated non-functional κB probe (Mut. 50×) or unlabelled oligonucleotide containing the consensus sequence for Sp-1 (Sp-1 50×). Data illustrated are from a single experiment and are representative of three separate experiments.

Figure 9

Effect of 15d-PGJ₂ on hsp72 protein expression in rat colon. Representative Western blot of hsp72 (A) as well as the densitometric analysis (B) shows the effect of 15d-PGJ₂ (20 μg kg⁻¹) on hsp72 protein expression evaluated in rat colon tissue 4 days after colitis induction. Immunoblotting in panel A is representative of one colon out of five to six analysed. The results in panel B are expressed as mean±s.e.mean from five to six colons. ^***P<0.001, vs control group.

Figure 10

Proposed scheme of some of the delayed inflammatory pathways in DNBSinduced colitis, and potential sites of the anti-inflammatory actions of 15d-PGJ₂. DNBS, at least in part via activation of nuclear factor-κB. (NF-κB), triggers the expression of inducible NO synthase (iNOS), NO, in turn, combines with superoxide to form ONOO-. 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 PARS, leading to cellular dysfunction. Expression of the adhesion molecules (ICAM-1) is also dependent on the activation of NF-κB. In addition, endothelial dysfunction can directly induced the upregulation of ICAM-1, leading to the enhanced neutrophils infiltration. We propose that the anti-inflammatory effects of 15d-PGJ₂I may include (1) inhibition of the activation of NF-κB and prevention of the expression of iNOS and ICAM-1, (3) inhibition of ONOO formation, (4) prevention of the activation of PARP and (5) reduction of neutrophils infiltration. See Discussion for further explanations.