Protective effects of relaxin in ischemia/reperfusion-induced intestinal injury due to splanchnic artery occlusion

Abstract

1. Splanchnic artery occlusion (SAO) followed by reperfusion causes endothelial injury and inflammation which contribute to the pathophysiology of shock. We investigated the effects of relaxin (RLX), known to afford protection against the deleterious effects of cardiac ischemia/reperfusion, given to rats subjected to splanchnic artery occlusion and reperfusion (SAO/R)-induced splanchnic injury. 2. RLX (30 ng kg(-1), 15 min. before reperfusion) significantly reduced the drop of blood pressure and high mortality rate caused by SAO/R. RLX also reduced histopathological changes, leukocyte infiltration (myeloperoxidase) and expression of endothelial cell adhesion molecules in the ileum. RLX counteracted free radical-mediated tissue injury, as judged by significant decrease in the tissue levels of peroxidation and nitration products (malondialdehyde, nitrotyrosine), DNA damage markers (8-hydroxy-2'-deoxyguanosine, poly-ADP-ribosylated DNA) and consumption of tissue antioxidant enzymes (superoxide dismutase). As a result, RLX led to a reduction of ileal cell apoptosis (caspase 3, terminal deoxynucleotidyltransferase-mediated UTP end labeling). The effects of RLX appear specific, as inactivated RLX substituted for the bioactive hormone had no effects. 3. In conclusion, these results show that RLX exerts a clear-cut protective effect in SAO/R-induced splanchnic injury, likely due to endothelial protection, decreased leukocyte recruitment and hindrance of free radical-mediated tissue injury leading to cell death, lethal complications and high mortality rate. Thus, RLX could be used therapeutically in intestinal ischemia.

Figure 1

Trend of mean arterial pressure in SAO/R rats. RLX, but not iRLX, prevents the fall in mean arterial pressure in SAO/R rats. Arrows point at start and withdrawal of RLX, iRLX or vehicle. Significance of differences (two-way ANOVA): ^*P<0.001 vs sham-operated animals; ^§P<0.001 vs SAO/R+vehicle; ^#P<0.001 vs SAO/R+RLX.

Figure 2

(a) MPO activity is significantly increased in the ileum of SAO/R rats. RLX, but not iRLX, significantly reduces MPO activity. Significance of differences (one-way ANOVA): ^*P<0.01 vs SAO/R+vehicle and SAO/R+iRLX. (b) Representative histopathological features of the ileum in sham-operated rats and SAO/R rats treated with vehicle or RLX. Severe mucosal abnormalities, consisting of truncated, edematous villi, hemorrhage foci and loss of surface and gland epithelium, can be seen in SAO/R+vehicle rats. These abnormalities are absent in SAO/R+RLX rats.

Figure 3

Immunostaining for ECAMs of ileal tissue sections from SAO/R rats treated with vehicle or RLX. RLX reduces the overexpression of the adhesion molecules investigated.

Figure 4

MDA (a) and 8-OHdG (b) are increased, while MnSOD activity (c) is decreased in ileal tissue of SAO/R+vehicle rats. RLX, but not iRLX, significantly prevents the occurrence of the above changes. ^*P<0.01 vs sham-operated, ^§P<0.01 vs SAO/R+vehicle, ^#P<0.01 vs SAO/R+RLX.

Figure 5

Immunocytochemical detection of Poly-ADP-ribosylated DNA (PAR) and NT in ileal tissue sections from SAO/R rats treated with vehicle or RLX. RLX reduces the expression of both PAR and NT.

Figure 6

(a) Caspase-3 activity is increased in ileal tissue of SAO/R+vehicle rats. This effect is prevented by RLX, but not by iRLX. ^*P<0.01 vs sham-operated animals, P<0.01 vs SAO/R+vehicle, ^#P<0.01 vs SAO/R+RLX. (b) TUNEL-positive cells in ileal tissue sections from sham-operated rats and SAO/R rats treated with vehicle or RLX. Numerous apoptotic cells can be seen in the ileum of SAO/R+vehicle as well as in the SAO/R+iRLX rats. In contrast, only few apoptotic cells can be detected at the villum tips in the samples from SAO/R+RLX rats.