Protective Effect Of Previous Heat Shock Treatment On Intestinal Injury Following Mesenteric Ischemia/reperfusion In Rats

OBJECTIVES: To investigate the protective effect of previous heat shock treatment on intestinal injury following mesenteric ischemia/reperfusion (I/R) and the potential mechanisms of I/R injury in rats.METHODS: Forty adult, male Sprague-Dawley rats, weighing 200 to 230 g were divided into 4 gr-oups with 10 rats in each, eg, control group(CTRL), ischemia/reperfusion group (IR), previous heat shock treatment group(42C) and previous heat shock treatment and then ischemia reperfusion group(42IR). Previous heat shock treatment underwent increasing whole body hyperthermia to a rectal temperature of 41.5-42 degrees C for 10 min. Both CTRL and IR groups experienced unheated pretreatment. In both IR and 42IR groups, ischemia was induced by occlusion of the superior mesenteric artery (SMA) for 30 min, followed by 60 min reperfusion. The SMA was separated but not closed for animals in CTRL and 42C groups. The expression of HSP72 was examined in the small intestinal mucosa using Western blotting and pathological changes of the ileal mucosal tissue was evaluated under microscope. The apoptosis of intestinal mucosal epithelial cells was examined by Terminal deoxylnucleotidy-1 transferase mediated-dUTP nick end labeling(Tunel).The enzymatic activity of casapse-3 in mucosal cells was assayed using colorimetric. The apoptotic percentage of peripheral blood leukocyte and polymorphonuclear neutrophil (PMN) was measured by flow cytometry using Annexin-V/PI double staining assay. The peripheral blood leukocyte number in each animal was examined at 4 times: before SMA was closed, after SMA closed for 30min, reperfused for 30min, reperfused for 60min. Statistical was analyzed with SPSS10.0, differences between groups were assessed by using Factorial design ANOVA, the differences of peripheral blood leukocyte number between groups in difference times was assessed by using analysis variance of repeated measure, the correlate analysis between factors was using linear correlate. P< 0.05 was considered significant.RESULTS: (1)Compared with CTRL, 42C and 42IR groups animals, IR group had most severe mucosal injury; The number of mucosal epithelia cells that underwent apoptosis increased obviously in IR group and was significantly higher than that in 42IR, 42C and CTRL groups (P<0.05)), respectively; The enzymatic activity of caspase-3 was significantly higher in IR group than 42IR, 42C and CTRL groups (P<0.01); The apoptotic percentage of peripheral blood leukocyte so as polymorphonuclear neutrophil (PMN) was significantly decreased in IR group than 42IR, 42C and CTRL groups(P<0.01); The peripheral blood leukocyte number in IR group was increased obviously after SMA closed for 30min, and was higher after reperfused, and was significantly higher than that in 42IR, 42C and CTRL groups (P<0.05), respectively. (2) The expression of heat-shock protein 72 significantly increased and ischemia reperfusion-induced mucosal injury significantly relieved in rats subjected to previous heat shock treatment. (3)There was not a significant difference in the histology, the number of apoptosis mucosal epithelial cells, the enzymatic activity of caspase-3, the apoptotic percentage of peripheral blood leukocyte and PMN, and the peripheral blood leukocyte number between 42IR, 42C and CTRL group.(4)There was a positive correlation between the expression of heat-shock protein 72 and the apoptotic percentage of peripheral blood leukocyte (r=0.514, P<0.05) (5) There was a negative correlation between the enzymatic activity of mucosal caspase-3 and the apoptotic percentage of peripheral blood leukocyte (r=-0.764, P<0.05),so as that of PMN (r=-0.845, P<0.05)(6) The expression of heat-shock protein 72 was uncorrelated with the index of apoptosis mucosal epithelia cells (r=-0.278, P>0.05) and the enzymatic activity of mucosal caspase-3 (r=-0.393, P>0.05) .CONCLUSIONS Mesenteric ischemia/reperfusion (I/R) can induce severe intestinal mucosal injury, the mucosal cells apoptosis increased, the apoptotic percentage of peripheral blood leukocyte and PMN decreased. Whole body hyperthermia pretreatment can protect against subsequent mesenteric ischemia reperfusion injury by increased induction of heat shock protein 72 and inhibiting of peripheral blood leukocyte activation in rats.