Protective effects of a new stable, highly active SOD mimetic, M40401 in splanchnic artery occlusion and reperfusion

Abstract

1. Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of M40401, a new S:,S:-dimethyl substituted biscyclohexylpyridine Mn-based superoxide dismutase mimetic (SODm, k(cat)=1.2x10(+9) M(-1) s(-1) at pH=7.4), in rats subjected to SAO shock. 2. Treatment of rats with M40401 (applied at 0.25, 2.5 or 25 microg kg(-1), 15 min prior to reperfusion), attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. M40401 also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. 3. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SAO-shocked rats which had received M40401. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40401 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue sections from SAO-shocked rats. M40401 treatment significantly improved survival. 4. Additionally, the very high catalytic activity of this new mimetic (comparable to the native human Cu/Zn SOD enzyme and exceeding the activity of the human Mn SOD enzyme) translates into a very low dose ( approximately microg kg(-1)) required to afford protection in this SAO model of ischemia reperfusion injury. 5. Taken together, our results clearly demonstrate that M40401 treatment exerts a protective effect, and part of this effect may be due to inhibition of the expression of adhesion molecules and peroxynitrite-related pathways with subsequent reduction of neutrophil-mediated cellular injury.

Figure 1

Structure of M40401.

Figure 2

The fall in mean arterial blood pressure (MAP) in SAO rats (n=6) is blocked in a dose dependent manner by M40401. ^*P<0.05 versus sham, °P<0.05 versus IR.

Figure 3

Reperfusion of the ischemic splanchnic circulation leads to profound increase in plasma (A) and ileum (B) levels of MDA and this is inhibited in a dose-dependent manner by M40401. Each point is the mean±s.e.mean for n=10 experiments. ^*P<0.05 versus sham, °P<0.05 versus IR.

Figure 4

Reperfusion of the ischemic splanchnic circulation leads to profound increase in plasma TNFα (A) and IL1β (B) and this is inhibited in a dose-dependent manner by M40401. Each point is the mean±s.e.mean for n=10 experiments. ^*P<0.05 versus sham, °P<0.05 versus IR.

Figure 5

Reperfusion of the ischemic splanchnic circulation leads to the infiltration of neutrophils in the lung (A) and ileum (B). M40401 significantly inhibit in a dose-dependent manner the neutrophils infiltration. Each point is the mean±s.e.mean for n=10 experiments. ^*P<0.05 versus sham, °P<0.05 versus IR.

Figure 6

Plasma NOx levels (A); Plasma peroxynitrite production assessed by oxidation of dihydrorhodamine 123 to rhodamine (B). There was no change in the plasma levels of nitrate/nitrite during occlusion or 60 min of reperfusion period. Peroxynitrite production in the SAO-shocked rats were significantly increased versus sham group. M40401 significantly inhibit in a dose-dependent manner the elevation of the plasma peroxynitrite production. Each point is the mean±s.e.mean for n=10 experiments. ^*P<0.05 versus sham, °P<0.05 versus IR.

Figure 7

No positive staining for nitrotyrosine (A) was found in the ileum section from sham-administered rats. Sixty minutes after reperfusion immunohistochemical for nitrotyrosine (B) show positive staining localised in the vascular wall and in the inflammatory cells (arrows) in the injured area from a SAO-shocked rats. The intensity of the positive staining for nitrotyrosine (C) was significantly reduced in the ileum from M40401-treated rats. Original magnification: ×145. Figure is representative of at least three experiments performed on different experimental days.

Figure 8

Staining of ileum tissue sections obtained from sham-operated rats with anti-ICAM-1 antibody showed a specific staining along vessels (arrow), demonstrating that ICAM-1 is constitutively expressed (A), no P-selectin staining was seen in sham animals (B). Section obtained from SAO shocked-rats showed intense positive staining (see arrows) for ICAM-1 (C) and for P-selectin (D) on vascular wall. The degree of endothelial staining for ICAM-1 (E) and for P-selectin (F) was markedly reduced in tissue section obtained from M40401-treated rats. Original magnification: ×145. Figure is representative of at least three experiments performed on different experimental days.

Figure 9

Distal ileum section from a sham rat demonstrating the normal architecture of the intestinal epithelium and wall (A). Distal ileum section from a SAO shocked-rats mice demonstrating oedema of the distal portion of the villi (B). Distal ileum from M40401-treated rats shows reduced SAO-induced organ injury (C) Original magnification: ×125. Figure is representative of at least three experiments performed on different experimental days.