The Protective Role And Mechanism Of Ischemic Postconditioning Against Ischemia-reperfusion Injury In Rat Intestine

Background and objective Among the internal organs, the intestine is probablythe most sensitive to ischemia-reperfusion injury. Many studies have demonstrated that ischemia-reperfusion injury of the intestine could result in an alteration of absorptive function of the intestine, intestinal mucosal barrier dysfunction, intestinal flora ecosystem imbalance and lead to translocation of bacterial and endotoxin from gut into systemic circulation. Translocated bacteria/endotoxin will excessively activate mono-macrophage to release a series of inflammatory mediators and cytokines that induced a second peak of cytokines in systemic circulation. Subequently cytokines lead to the septic process and promote development of systemic inflammatory response syndrome (SIRS) and even multiple organ dysfunction syndrome (MODS), and result in significant morbidity and mortality. Ischemic preconditioning refers to a phenomenon whereby exposure of a tissue to brief periods of ischemia protects them from the deleterious effects of prolonged ischemia-reperfusion injury. This phenomenon was first described in 1986 by Murry et al. in the canine heart. Subequently benefical effects have been demonstrated in the liver, brain, kidney, and intestine in various animal models. However, clinical application of ischemic preconditioning has been disappointingly limited. The implementation of protective therapy at the time of reperfusion is clinically feasible because the onset of reperfusion is more predictable and is under the clinician's control. In 2003, Vinten-Johnsen and his colleagues reported that brief episodes of myocardial ischemia and reperfusion employed during reperfusion after a prolonged ischemic insult have been demonstrated to play a cardioprotective role. This phenomenon was termed "ischemic postconditoning (IPOST)". In a dog model postconditioning limits infarct size, reduces tissue edema and polymorphonuclear neutrophil accumulation in the area at risk myocardium, and improves endothelial function. It has been suggested that postconditioning is as effective as preconditioning in limiting infarct size and preserving postischemic endothelial function. Kin et al proposed that postconditioning might be related to attenuation of oxygen-derived free radical production in the early minutes of reflow. Ischemic postconditioning is an alternative, more amenable approach. However, the mechanism of this new method remains unknown and there are no any studies about the protection of ischemic postconditioning against ischemia-reperfusion intestine. Therefore, we investigated the protective role and mechanism of ischemic postconditioning against intestinl ischemia-reperfusion injury in rat and compared with ischemic preconditioning. This study may not only provide a novel approach for clinic use but also provide useful theoretical and practical evidences for reducing ischemia-reperfusion injury.Methods Using rat model of intestine I/R injury, Male Sprague-Dawley ratsweighing 220-250 g were divided into 4 groups which were Sham operation group (S), ischemia-reperfusion group (I/R), ischemic preconditioning group (IPC) and ischemic postconditioning group (IPOST), respectively. In these groups, an I/R procedure was performed by occlusion of the superior mesenteric artery (SMA) for 45 min followed by reperfusion for 1h, 6h or 24h. In Sham group, there was no intervention. In IPC group, the SMA was occluded by two cycles of ischemia for 5 min and reperfused for 5 min before the prolonged occlusion. In IPOST group, three cycles of 30-s reperfusion and 30-s reocclusion were preceded at the start of reperfusion. After reperfusion, the small intestines were resected for experimental detection. The following four aspects were investigated.1. The protective role of ischemic postconditioning against ischemia-reperfusion injury in rat intestine. After reperfused for 1 h, 6 h, or 24 h, the changes of intestinal mucosal pathomorphology, intestine tissue water (wet weight/dry weight), the intestinal bacteria/endotoxin translocation rate and viable bacterial counts, the endotoxin contents in portal and cava vein, the contents of TNF-αin cava venous serum, and the hepatic functions were determined.2. The antioxidative mechanism of ischemic postconditioning against ischemia-reperfusion injury in rat intestine. The contents of malondialdehyde (MDA) , the activity of superoxide dismute (SOD) and the activity of myeloperoxidase (MPO) were determined respectively by spectrophotometer.3. The mechanism of ischemic postconditioning against apoptosis of intestinal mucosal cells in ischemia-reperfusion rats. After reperfused for 6 h, the changes of apoptosis of intestinal mucosal cells were determined with agarose gel electrophoresis and deoxynucleotidy transferase mediated dUTP-biotin nick-end labeling technique (TUNEL). Intestinal tissue samples were taken not only for RT-PCR to detect Bcl-2 and Bax mRNA expression, but also for immunohistochemical analysis of detection Bcl-2 and Bax protein expression and distribution.4. The role of mitochondrion in the ischemic postconditioning against ischemia-reperfusion injury in rats intestine. After reperfused for 1 h, mucosal cellular mitochondrial respiration function was studied by measuring the mitochondrial dehydrogenase-dependent reduction of MTT to its formazan derivative. Intestinal mucosal mitochondrial ultrastructural changes were observed by using transmission electron microscope (TEM), and intestinal mucosal mitochondrial membrane potential was detected by confocal laser scanning microscopy.Results 1. The protective role of ischemic postconditioning againstischemia-reperfusion injury in rat intestine.①The morphological changes in intestinal mucosa: in I/R group, the intestinal mucosa presented dilated and exposed capillaries and denuded villi, some of villi hemorrhage, ulceration. The lamina propria was edema and infiltrated with inflammatory cells. The intestinal mucosal structure maintain intact with lifting of the epithelial layer from the lamina propria and moderate extension of the subepithelial space in IPOST and in IPC group. In IPOST goup, histopathologic scores were significantly lower than that of in I/R group(P<0.05). There were no significant difference between IPC and IPOST group (P>0.05).②The changes of bacterial translocation: 4 kinds of normal intestinal bacteria were determined in I/R group, IPC group and IPOST group. The intestinal bacteria translocation rate and viable bacterial counts in tissues were evidently lower in IPOST group and in IPC group compared with I/R group, there were no significant difference between IPC group and POST group (P>0.05).③The changes of intestine tissue W/D, the endotoxin contents in portal and cava vein, the contents of TNF-αin cava venous serum and the hepatic functions: intestine tissue W/D, the endotoxin contents in portal and cava vein, the contents of TNF-αin cava venous serum and the hepatic functions were obviously lower in I/R group than that of Sham group(P<0.05), but higher than that of IPOST group(P<0.05). There were no significant difference between IPC group and IPOST group (P>0.05).2. The antioxidative mechanism of ischemic postconditioning against ischemia-reperfusion injury in rat intestine. The contents of MDA and activity of MPO was significantly reduced and the activity of SOD enhanced In IPC group and IPOST group versus I/R group (P<0.05). There were no significant difference between IPC group and IPOST group (P>0.05) .3. The mechanism of ischemic postconditioning against the apoptosis of intestinal mucosal cells in ischemia-reperfusion rats.①The changes of apoptosis of intestinal mucosa: The I/R significantly induced fragmentation of the mucosal DNA resulting in an increase in DNA ladders. IPOST and IPC significantly reduced the ladder formation. In I/R group, apoptosis positive cells increase significantly (P<0.05), and distributed from tip to base of villi. The apoptosis was markedly severe in submucosa and lamina propria. It was obviously less in IPOST group and in IPC group compared with I/R group (P<0.05). There were no significant difference between IPC group and IPOST group(P>0.05).②The changes of mRNA and protein expressions of Bcl-2 and Bax: The mRNA and protein expression of Bcl-2 decreased in I/R group(P<0.05), While mRNA and protein expression of Bcl-2 in IPOST group and IPC group were obviously higher than those in I/R group(P<0.05). There were no significant difference between IPC group and IPOST group(P>0.05). The mRNA and protein expression of Bax were siginificantly increased in I/R group(P<0.05), While mRNA and protein expression of Bax in IPOST group and IPC group were obviously higher than those in I/R group(P<0.05). There were no significant difference between IPC group and POST group(P>0.05).4. The role of mitochondrion in the ischemic postconditioning against ischemia-reperfusion injury in rats intestine. (1) The mitochondrial respiratory function was significantly impaired in I/R group rats compared with Sham group (P<0.05). However, ischemic postconditioning significantly ameliorated the mitochondrial respiratory function compared with I/R group (P<0.05). There were no significance between IPOST and IPC group (P>0.05). (2) By TEM examination, intercellular spaces between epithelial cells were dilated, microvilli were necrotic and denuded, mitochondrion were swollen with cracked cristae in I/R group rats. The injury of mucosal epithelia, the cell junction and mitochondrion of IPOST group and IPC group were began to recover. (3) Compared with Sham group, the mitochondrial membrane potential of intestinal mucosa significantly reduced in I/R group rats (P<0.05). However, ischemic postconditioning significantly improved the mitochondrial membrane potential compared with I/R group (P<0.05). There are no significance between IPOST group and IPC group (P>0.05).Conclusions 1. The ischemia-reperfusion injury in rat intestine resulted in injury ofintestinal mucosa and intestine dysfunction. Intestine tissue W/D, the intestinal bacteria translocation rate and viable bacterial counts, the endotoxin contents in portal and cava vein, the contents of TNF-αin cava venous serum, the hepatic functions, the contents of MDA, and the activity of MPO were obviously increased. However, the activity of SOD decreased. The Bcl-2 mRNA and protein expression decreased, while the Bax mRNA and protein expression were siginificantly increased. The mitochondrial respiratory function was significantly impaired, the MMP of intestinal mucosa significantly reduced. 2. Ischemic postconditioning played an important role in protection against intestine in rats with ischemia-reperfusion. Ischemic postconditioning could effectively reduce the intestinal bacteria/endotoxin translocation and decrease the release of TNF-αas well as protect the hepatic functions. 3. The antioxidative mechanism of ischemic postconditioning against ischemia-reperfusion of intestine in rats may be related to reducing the production of reactive oxygen species, maintaining the activities of antioxidant systems and suppressing neutrophils recruitment. 4. The anti-apoptosis mechanism of ischemic postconditioning against ischemia-reperfusion of intestine in rats was associated with upregulating the Bcl-2 mRNA and protein expression and downregulating the Bax mRNA and protein expression. 5. The mechanism of ischemic postconditioning against ischemia-reperfusion injury of intestine in rats was partly by maintaining the mitochondrial ultrastructural changes, respiratory function and the mitochondrial membrane potential. 6. The protective role and mechanism of ischemic postconditioning against ischemia-reperfusion injury in rat intestine were consistent with that of ischemic preconditioning. 7. This study may not only provide a novel approach for clinic use but also provide useful theoretical and practical evidences for reducing ischemia-reperfusion injury.