| BackgroundIn 1960, Jenings first described an event called reperfusion injury, which means the restoration of blood flow to an ischemic region leads to tissue injury at a greater rate than the original ischemic insult. The ischemia-reperfusion injury now has been observed in different tissues and organs including myocardium, cerebrum, liver, kidney, stomach, intestine, spinal cord, retina, limbs, and so on.Among the abdominal organs, the small intestine is probably the most sensitive to ischemia-reperfusion induced injury. It occurs frequently in a variety of clinical conditions, including trauma, hemorrhagic shock, septic shock, mesenteric arterial embolism or thrombosis, mesenteric venous thrombosis, intestinal strangulation, abdominal aneurysm surgery, as well as small intestinal transplantation, and is associated with high morbidity and mortality. Recently it's reported that high IAP(intra-abdominal pressure) with CO2 pneumoperitoneum induced oxidative stress injury in an experimental model. In another two studies, effects of intestinal ischemia-reperfusion injury on healing of intestinal and colonic anastomoses were demonstrated. Moreover, intestinal obstruction is a complication frequently occurs after abdominal surgery and is most commonly due to postoperative intraperitoneal adhesions. In a rat model, intestinal ischemia-reperfusion injury also aggravates adhesions formation. Intestinal local injury induced by mesenteric ischemia reperfusion event is characterized by intestinal dysfunction and failure, including digestion and ingestion dysfunction, motility derangement and disruption of mucosal integrity, which leads to bacterial translocation and endotoxin, release of intestine-derived inflammatory mediators, exaggerated inflammatory cascade, SIRS(systemic inflammatory response syndrome) induced, remote organ injury, and ultimately MODS and MOF development. Intestinal ischemia-reperfusion injury is a pivotal initiating event during the development of MOF. Therefore, it's valuable and significant to investigate the underlying mechanism and interventional strategy of intestinal ischemia reperfusion injury. A brief episode of sublethal ischemia protects tissue from injuries caused by subsequent ischemic insults and is termed"ischemic preconditioning"or IPC. This important phenomenon was first described in the heart by Murry et al. in 1986. A tissue that recovers from a reversible ischemic insult acquires an enhanced ability to tolerate a subsequent insult, and this ischemic preconditioning phenomenon has been observed in myocardium, kidney, liver, cerebrum, skeletal muscle and other organs sensitive to ischemia or hypoxia such as intestine, suggesting a possible ubiquitous survival mechanism endogenously built into various tissues. On the other hand, ischemic preconditioning has extensively been taken as a protective interventional strategy against ischemia-reperfusion injury in vitro and in vivo experimentally. Beneficial effects of ischemic preconditioning are confirmed in myocardial and cerebral tissues, and several small molecular agents have been shown playing a key role during cardiac preconditioning. The major mediators of ischemic preconditioning include adenosine, NO, bradykinin, opioids, and free oxygen radicals, although their precise roles and mechanisms are not clearly elucidated. However, protective effects of mesenteric ischemic preconditioning are not certified due to few relative studies performed previously.In recent years, many studies have shown that erythropoietin also acts as a tissue protecting factor, suggesting impressing new roles for erythropoietin in nonhaematopoietic tissues. It's traditionally believed that erythropoietin is a glycoprotein cytokine produced primarily by the kidney. Erythropoietin regulates the proliferation, differentiation, maturation targeting erythroid progenitor cells in bone marrow, releasing reticulocyte and increasing production of peripheral blood RBC. Recombinant human erythropoietin has been synthesized and currently used to correct renal anemia and anemia induced by chemotherapy or radiotherapy in tumor patients. Accumulating evidences have indicated alternative functions of erythropoietin that could be protective in an ischemia-reperfusion setting in such tissues as brain, kidney and heart. Although not fully understood, erythropoietin-mediated protective effects seem to involve its anti-oxidative, anti-inflammatory, anti-apoptotic potential. However, underlying mechanism of erythropoietin protective effects has not been verified concerning intestinal ischemia-reperfusion injury.Objective1. To investigate whether ischemic preconditioning plays a role in intestinal ischemia-reperfusion injury and its possible mechanisms using a rat mesenteric ischemic preconditioning model.2. To determine the possible tissue protective effects of recombinant human erythropoietin and the underlying related mechanisms using a rat mesenteric ischemia-reperfusion injury model.Methods1. Establishment of the rat mesenteric ischemia-reperfusion injury model and the mesenteric ischemic preconditioning model. Establishment of the rat mesenteric ischemia-reperfusion injury model is to performing a midline laparotomy under general anesthesia with SMA occlusion 30min subsequently reperfusion 60min using an atraumatic vascular micro clip. Establishment of the rat mesenteric ischemic preconditioning model is to performing a midline laparotomy under general anesthesia with SMA occlusion 5min subsequently reperfusion 5min for 2 cycles using an atraumatic vascular micro clip.2. MDA assay. Blood samples were gathered through ventricular puncturation, serum MDA contents were determined in a method of the thiobarbituric acid reaction, tetraethoxy propane as the primary standard, serum MDA content as an indicator reflecting lipid peroxidation.3. MPO activity. Intestinal tissue samples were taken from the distal ileum segments for the preparation of intestinal tissue homogenate, intestinal tissue MPO activities were measured by means of chromatometry, as an indicator of tissue neutrophil accumulation, in response to the local inflammation.4. Intestinal tissue specimen were taken from the distal ileum segments for the preparation of intestinal tissue section, stained with hematoxylin and eosin, examined under light microscopy and evaluated histopathologically using Chiu scoring system, detection of apoptosis by TUNEL method.5. Distal ileum segments were taken for the detection of intestinal tissues Bcl-2 protein expression using western blot method.Results1. In the rat mesenteric ischemia-reperfusion injury model, compared with the sham group, serum MDA content of rats in the ischemia-reperfusion group were found to have increased, as observed 8.42±0.08 vs 2.62±0.10nmol/ml, P<0.01, compared with the ischemia-reperfusion group, the EPO group serum MDA content of rats were found to have significant decreased, as observed 4.70±0.11 vs 8.42±0.08nmol/ml, P<0.01, compared with the sham group, ileal tissue MPO activity of rats in the ischemia-reperfusion group were found to have increased, as observed 2.04±0.04 vs 0.18±0.04U/g wet tissue, P<0.01, ileal tissue MPO activity of rats in the EPO group were found significant decreased compared with the ischemia-reperfusion group, 0.56±0.07 vs 2.04±0.04U/g wet tissue, P<0.01, histopathological features of normal ileal tissue and no evident mucosal injury were observed in the sham group, in the ischemia-reperfusion group, the villi were denuded to the level of the lamina propria and dilated capillaries, disintegration of lamina propria, hemorrhage, manifesting severe mucosal injury, in the EPO group, slight mucosal injury were observed, such as small subepithelial detachments, occasionally occurred subepithelial detachments exerted a slight amount of upward push on the mucosa epithelium, few denuded villus tips were observed, most villi are preserved intact, no disintegration of lamina propria, no hemorrhage or formation of ulceration, manifesting slight mucosal injury. Chiu scoring were low in the sham group, 0.33±0.21, compared with the sham group, Chiu scoring in the ischemia reperfusion group were higher, 3±0.26 vs 0.33±0.21, P=0.003, compared with the ischemia reperfusion group, Chiu scoring in the EPO group were found significant lower, 1.33±0.33 vs 3±0.26, P=0.006, a significant linear positive correlation exists between Chiu scoring and serum MDA content of rats, Y=0.294+7.532X, R2=0.837, r=0.915, P=0.010, a linear positive correlation also exists between Chiu scoring and ileal tissue MPO activity of rats, Y=1.599+0.148X, R2=0.817, r=0.904, P=0.013, TUNEL positive cell is rare in the sham group, compared with the sham group, the number of TUNEL positive cell in the ischemia-reperfusion group was found higher, 24.67±2.28 vs 3.67±0.62, P<0.01, compared with the ischemia reperfusion group, the number of TUNEL positive cell in the EPO group was found decreased, as measured 11.0±1.07 vs 24.67±2.28, P<0.01, a significant spearman nonparametric positive correlation exists between Chiu scoring and the number of TUNEL positive cell, rs=0.845, P<0.05.2. In the rat mesenteric ischemic preconditioning model, compared with the ischemia-reperfusion group, serum MDA content of rats in the IPC group were found to have significant decreased, as observed 5.50±0.14 vs 8.42±0.08nmol/ml, P<0.01, compared with the sham group, the glibenclamide group serum MDA content of rats were found to have significant increased, as observed 8.22±0.10 vs 2.62±0.10nmol/ml, P< 0.01, compared with the ischemia-reperfusion group, serum MDA content of rats in the glibenclamide group were found no siginificant difference, 8.22±0.10 vs 8.42±0.08nmol/ml, P>0.05, compared with the ischemia-reperfusion group, ileal tissue MPO activity of rats in the IPC group were found to have decreased, as observed 0.73±0.06 vs 2.04±0.04U/g wet tissue, P<0.01, ileal tissue MPO activity of rats in the glibenclamide group were found significant increased compared with the sham group, 1.97±0.06 vs 0.18±0.04 U/g wet tissue, P<0.01, compared with the ischemia-reperfusion group, ileal tissue MPO activity of rats in the glibenclamide group were found no significant difference, 1.97±0.06 vs 2.04±0.04 U/g wet tissue, P>0.05, histopathological features of slight mucosal injury were observed in the IPC group, subepithelial detachments exerted a slight extent of upward push on the mucosa epithelium, few villous denuded found, the mucosal integrity is proximately preserved, manifestation like that in the EPO group, in the ischemia-reperfusion group, the villi were denuded to the level of the lamina propria and dilated capillaries, disintegration of lamina propria, hemorrhage, manifesting severe mucosal injury, in the glibenclamide group, mucosal injury were observed significantly heavier than that in the IPC group, such as subepithelial detachments development into the deeper tissue, exerted a massive amount of upward push on the mucosa epithelium, quite a few villi tips were denuded and even to the level of propria, disintegration of lamina propria could be found, large subepithelial detachments along the villi were frequently occurred, manifestation approximate that in the ischemia-reperfusion group. Chiu scoring were assessed among groups, compared with the ischemia-reperfusion group, Chiu scoring in the IPC group were found to have significant decreased, as observed 1.50±0.34 vs 3±0.26, P=0.014, compared with the ischemia-reperfusion group, Chiu scoring in the glibenclamide group were found no significant difference, 2.67±0.33 vs 3±0.26, P=0.38, on the other hand, while compared with the sham group, Chiu scoring in the glibenclamide were found to have significant increased, as observed 2.67±0.33 vs 0.33±0.21, P=0.003, TUNEL positive cell is rare in the sham group, which is occasionally found in the tip of villi, compared with the ischemia-reperfusion group, the number of TUNEL positive cell in the IPC group was found to have significant decreased, as observed 12.0±1.16 vs 24.67±2.28, P<0.01, compared with the ischemia reperfusion group, the number of TUNEL positive cell in the IPC group was found to have no significant difference, as measured 22.83±1.35 vs 24.67±2.28,P>0.05, while compared with the sham group, the number of TUNEL positive cell in rat ileal mucosal in the glibenclamide group were observed to have significant increased, as measured 22.83±1.35 vs 3.67±0.62, P<0.01, in the sham group, Bcl-2 protein expression of rat ileal tissue samples were weak, while in the ischemia-reperfusion group, in the glibenclamide group and in the IPC group, ileal tissue samples Bcl-2 protein expression were found to be substantially stronger when compared with that observed in the sham group, ileal tissue samples Bcl-2 protein expression of rats among groups the IPC group seems to be the strongest.Conclusions1. Ischemic preconditioning ameliorate rat mesenteric ischemia-reperfusion injury, glibenclamide, an ATP sensitive potassium channel blocker abolishes the favorable effects induced by ischemic preconditioning, suggesting KATP channel maybe involved in the mechanisms of IPC.2. rHuEPO protects the intestine of rats against mesenteric ischemia-reperfusion injury mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic activities, EPO mimics the protective effects induced by IPC. |
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