Rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2, ligands of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), reduce ischaemia/reperfusion injury of the gut

Abstract

1. The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The thiazolidinedione rosiglitazone and the endogenous cyclopentenone prostaglandin (PG)D2 metabolite, 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2), are two PPAR-gamma ligands, which modulate the transcription of target genes. 2. The aim of this study was to investigate the effect of rosiglitazone and 15d-PGJ2 on the tissue injury caused by ischaemia/reperfusion (I/R) of the gut. 3. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp allowing reperfusion for 2 or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. 4. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 10% of the animals survived for the entire 4 h reperfusion period. Surviving animals were killed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, significant increases in plasma tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels and marked injury to the distal ileum. 5. Increased immunoreactivity to nitrotyrosine was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti-intercellular adhesion molecule (ICAM-1) antibody resulted in diffuse staining. 6. Administration at 30 min prior to the onset of gut ischaemia of the two PPAR-gamma agonists (rosiglitazone (0.3 mg kg-1 i.v.) and 15d-PGJ2 (0.3 mg kg-1 i.v.)) significantly reduced the (i) fall in mean arterial blood pressure, (ii) mortality rate, (iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (iv) lipid peroxidation (MDA levels), (v) production of proinflammatory cytokines (TNF-alpha and IL-1beta) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d-PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM-1 during reperfusion. 7. In order to elucidate whether the protective effects of rosiglitazone and 15d-PGJ2 are related to the activation of the PPAR-gamma receptor, we also investigated the effect of a PPAR-gamma antagonist, bisphenol A diglycidyl ether (BADGE), on the protective effects of rosiglitazone and 15d-PGJ2. BADGE (1 mg kg-1 administered i.v. 30 min prior to the treatment of rosiglitazone or 15d-PGJ2) significantly antagonised the effect of the two PPAR-gamma agonists and thus abolished the protective effect against gut I/R. 8. These results demonstrate that the two PPAR-gamma agonists, rosiglitazone and 15d-PGJ2, significantly reduce I/R injury of the intestine.

Figure 1

No significant alteration of MAP was observed in sham-operated rats (a). Fall in MAP in SAO rats (N=10) is blocked by rosiglitazone (0.3 mg kg⁻¹) but not by BADGE (1 mg kg⁻¹) (b). Coadministration of BADGE and rosiglitazone significantly blocked the effect of the rosiglitazone (b). ^*P<0.01 versus sham, °P<0.01 versus I/R, °°P<0.01 versus rosiglitazone.

Figure 2

No significant alteration of MAP was observed in sham-operated rats (a). Fall in MAP in SAO rats (N=10) is blocked by 15d-PGJ₂ (0.3 mg kg⁻¹) but not by BADGE (1 mg kg⁻¹) (b). Coadministration of BADGE and 15d-PGJ₂ significantly blocked the effect of the 15d-PGJ₂ (b). ^*P<0.01 versus sham, °P<0.01 versus I/R, °°P<0.01 versus 15d-PGJ₂.

Figure 3

Reperfusion of the ischaemic splanchnic circulation leads to profound increase in MDA levels in ileum tissues, which is inhibited by PPAR-γ agonists 15d-PGJ₂ (0.3 mg kg⁻¹ (a)) or by rosiglitazone (0.3 mg kg⁻¹ (b)) but not by BADGE (1 mg kg⁻¹). Coadministration of BADGE and rosiglitazone or 15d-PGJ₂ significantly blocked the effect of the two PPAR-γ agonists. ^*P<0.01 versus sham, °P<0.01 versus I/R, °°P<0.01 versus rosiglitazone or 15d-PGJ₂.

Figure 4

Reperfusion of the ischaemic splanchnic circulation leads to profound increase in plasma TNFα production and this is inhibited by PPAR-γ agonists 15d-PGJ₂ (0.3 mg kg⁻¹ (a)) or by rosiglitazone (0.3 mg kg⁻¹ (b)) but not by BADGE (1 mg kg⁻¹). Coadministration of BADGE and rosiglitazone or 15d-PGJ₂ significantly blocked the effect of the two PPAR-γ agonists. ^*P<0.01 versus sham, °P<0.01 versus I/R, °°P<0.01 versus rosiglitazone or 15d-PGJ₂.

Figure 5

Reperfusion of the ischaemic splanchnic circulation leads to profound increase in plasma IL-1β production and this is inhibited by PPAR-γ agonists 15d-PGJ₂ (0.3 mg kg⁻¹ (a)) or by rosiglitazone (0.3 mg kg⁻¹ (b)) but not by BADGE (1 mg kg⁻¹). Coadministration of BADGE and rosiglitazone or 15d-PGJ₂ significantly blocked the effect of the two PPAR-γ agonists. ^*P<0.01 versus sham, °P<0.01 versus I/R, °°P<0.01 versus rosiglitazone or 15d-PGJ₂.

Figure 6

Reperfusion of the ischaemic splanchnic circulation leads to the infiltration of PMNs into ileum tissues, which is inhibited by PPAR-γ agonists 15d-PGJ₂ (0.3 mg kg⁻¹ (a)) or by rosiglitazone (0.3 mg kg⁻¹ (b)) but not by BADGE (1 mg kg⁻¹). Coadministration of BADGE and rosiglitazone or 15d-PGJ₂ significantly blocked the effect of the two PPAR-γ agonists. ^*P<0.01 versus sham, °P<0.01 versus I/R, °°P<0.01 versus rosiglitazone or 15d-PGJ₂.

Figure 7

Immunohistochemical staining of nitrotyrosine was absent in the ileum section from sham-operated rats (a). After reperfusion nitrotyrosine staining was localised in the injured area from an SAO-shocked rat (b). There was no detectable immunostaining in the ileum from rosiglitazone (c) or from 15d-PGJ₂- (d) treated rats. Original magnification, × 500. The figure is representative of at least three experiments performed on different experimental days.

Figure 8

Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n=5) for ICAM-1 and nitrotyrosine from ileum was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as percentage of total tissue area. ^*P<0.01 versus sham, °P<0.01 versus IR. ND, not determined.

Figure 9

Control tissue from sham-operated rats (a) showed a dark brown staining of endothelium of blood vessels, indicating the presence of constitutive ICAM-1 protein. I/R induced an increase of the positive staining for ICAM-1 along the endothelium wall (b). In rosiglitazone (c) or from 15d-PGJ₂ (d) treated rats subjected to SAO shock, there was no increase of immunostaining for ICAM-1, which was present only along the endothelium wall. Original magnification, × 500. The figure is representative of at least three experiments performed on different experimental days.

Figure 10

Distal ileum section from a sham rat, demonstrating the normal architecture of the intestinal epithelium and wall (a). Distal ileum section from SAO-shocked rats showed inflammatory infiltration by PMNs and lymphocytes extending through the wall and concentrated below the epithelial layer and demonstrating oedema of the distal portion of the villi (b). Distal ileum from rosiglitazone (c) or from 15d-PGJ₂- (d) treated rats shows reduced SAO-induced organ injury. Original magnification, × 125. The figure is representative of at least three experiments performed on different experimental days.

Figure 11

SAO shock-induced mortality. Survival was monitored for 4 h after SAO shock. ^*P<0.01 versus sham, °P<0.01 versus I/R.