| Intestinal ischemia-reperfusion is a common clinical even with high rates of complication and death,which was highly concerned in the1960’s.Experimental observations indicate that Intestinal ischemia-reperfusion (I/R) is a localized pathology that often leads to multi-organ dysfunction and shock, which is accompanied by a strong inflammatory reaction. But the source of the inflammatory mediators in shock has remained uncertain. In the past, many researchers believed that the translocation of bacteria and endotoxin goes via the circulation as well as possibly the lymphatics from the lumen of the intestine may involve in the source of inflammation and shock. Recently, some researchers proposed a new theory named auto-digestion, they suggested that pancreatic digestive enzymes play a central role in the production of inflammatory mediators. Pancreatic digestive enzymes, compartmentalized in the lumen of the intestine in normal condition, may pass across the mucosal barrier of the ischemic intestine into the extracellular submucosal space and initiate the production of inflammatory mediators.Accordingly, pancreatic protease inhibitors in the ischemic intestine may help to protect against inflammation and shock.Urinary trypsin inhibitor (UTI), one of the Kunitz-type pancreatic protease inhibitors found in urine, is synthesized from inter-a-trypsin inhibitor through proteolytic cleavage by neutrophil elastase at the site of inflammation. Various serine proteases such as trypsin, chymotrypsin, neutrophil elastase, and plasmin are inhibited by UTI. Intra-intravenously administered of UTI has been prove to ameliorates inflammation in several inflammatory models such as ischemia-reperfusion injury, septic shock, and hemorrhagic shock in vivo. However, working mainly as a pancreatic protease inhibitor, UTI has not been tested intra-intestinally. Our study aims to confirm the auto-digestive theory and try to explain the mechanisms involved in the anti-inflammation role of UTI.1、Materials and methods1.1Animals and Experimental groupsThe animals used in this experiment were from the Laboratory Animal Center of Southern Medical University of China. The research protocols were approved by the Animal Care Committee of Southern Medical University of China and performed in concordance with National Institute of Health guidelines.48healthy Wistar rats, weighing200-250g, fasted8hours before the experiments, were divided into4groups randomly:sham group, SMAO group, SMAO+NS group and SMAS+UTI groups.1.2Experimental procedureAfter general anesthesia (50mg of pentobarbital sodium per kilogram of body weight, given intraperitoneally, the animals were placed on a heating pad(37℃-38℃). The left femoral artery and vein were cannulated with a polyethylene tubing (PE-50). The arterial line served to record the mean arterial pressure and the venous line served to administer fluid. The superior mesenteric artery were isolated through an abdominal midline incision and were looped with3-0silk thread at their aortic origin. The threads were guided through the tubing out of the body. The arteries were occluded by application of tension on these threads. The lumen of the proximal duodenum and the terminal ileum were cannulated with tubes. Intestinal ischemia was produced by occlusion of the superior mesenteric artery for100min and was followed by reperfusion of the vessel for80min immediately in the SMAO group. Animals in the SMAO+NS group and SMAO+UTI group were subjected to superior mesenteric artery occlusion (SMAO) shock after intestinal perfusion of saline (40ml,37℃,10ml/min) with or without UTI(100U/ml). Sham group (n=6) underwent surgery without intestinal lavage or ischemia. Intestinal perfusion was repeated20min and60min after reperfusion by30ml of saline with or without UTI. Six Rats in each groups without blood drawing were observed until cardiac arrest without treatment in5min. After80min of reperfusion, catheters were removed and skin wounds were sutured. Another six rats were taken sample for testing. After the first lavage, intestinal fluid was collected and centrifuged at500g for5minutes. Aliquots of the supernatant were stored at-70℃until later measurements of protease activity.2ml of heparinized blood (10U/mL) was drawn80min after reperfusion,1ml of which was used to measure the expression of PMN CD11b by flow cytometric detection. The remainder of the sample was centrifuged (500g for5minutes), and plasma was frozen (at-70℃) for later measurements of TNF-α and IL-1. After euthanasia, a5-cm segment of the small intestine was excised, rinsed with lOmM phosphate-buffered saline (PBS), and fixed in formalin (10%) for24hours.1.3. Detection index1.3.1The change of MAP duiring Intestinal ischemia-reperfusionThrough blood pressure monitor, observed and recorded changes in MAP of rats duiring Intestinal ischemia-reperfusion.1.3.2Survival timeRecord the survival time of rats. It was defined death when the heart of rat stopped beating without treatment in5min.1.3.3The expression of PMN CD11b10μl of anti-rat CD11b FITC-labeled monoclonal antibody was added to the100μl blood samples,after which the samples were gently and briefly vortexed,then placed on ice for15min in dark. Subsequently,the red cells were lysed,the PMNs washed three times and then the level of CD11b expression was determined by flow cytometry. The data were expressed as mean fluorescence intensity. FITC-conjugated,isotype mouse IgGl antibody was used as an isotype control for nonspecific antibody binding.1.3.4TNF-a and IL-1Levels TNF-a and IL-1Levels were measured by enzyme-linked immunosorbent assay.1.3.5Protease Activity Measurements Protease activity in intestinal fluid was measured with a kit (Enz-Chek Protease Assay Kits).1.3.6Histological examination of the small intestineWhen the rats were sacrificed and a4-cm segment of the small intestine was excised, washed with0.9%Salt solution forth and fixed in formalin. One day later, the intestinal segment was embedded in resin, stained with toluidine blue, and histological sections were investigated under a microscope to assess tissue damage.1.4Statistical analysisData are presented as mean±SD. Analysis of variance of repeated measure data was used to analyze the blood pressure data. To assess differences in the expression of PMN CDl lb,leukocyte count and TNF-a,IL-1,IL-6levels between allgroups, the1-way analysis of variance was used. Spss13.0was used to analyze the data and p<0.05was considered to be significant.2. Result2.1Survival timeThe survival time of sham group is longer than24hours. The average survival time of MSMAO is0.48, which is significant shorter than both SMAO+NS group (2.1hours)and SMAO+UTI group(6.4hours)(P<0.05).The animals in SMAO+UTI group can live significant longer than SMAO+NS group (P<0.05).2.2Arterial blood pressureSMAO leads to a significant increase in mean arterial pressure, while reperfusion causes a rapid decrease in blood pressure after reperfusion of arteries in SMAO group. In contrast, flushing of the intestinal lumen using saline with or without UTI both help to maintain the mean arterial pressure. After30min of ischemia, mean arterial pressure in SMAO+UTI group was significant higher than SMAO+NS group.2.3The expression of PMN CD11bAfter80minutes of reperfusion, SMAO caused an increase in the expression of PMN CD11b. The plasma from the SMAO+NS group expression significant higher level of PMN CD11b (76.43%±3.80%), compared with the SMAO+UTI group (57.94%±8.95%) and SHAM group (50.87%±11.16%).2.4TNF-α and IL-1LevelsDuring ischemia-reperfusion injury, SMAO caused dramatically increase of inflammatory factors. The SMAO+NS group had significantly increased levels of TNF-a and IL-1compared with sham group and SMAO+UTI group(Table1)2.5Intestinal protease activityAfter80minutes of reperfusion, the intestinal proteaseactivity was significantly reduced in SMAO+UTI group(168.09Π16.22μug/mL) compared with SMAO+NS group(282.85±13.90μg/mL)(P<0.05).2.6Histological examination of the small intestineHistological sections of the intestine revealed ischemia and reperfusion-induced injury in the form of a complete loss of villi, ulcerations, and a high number of infiltrating leukocytes into the mucosa and submucosa in the SMAO+NS group. The intestinal injury was improved in the SMAO+UTI group as seen in the form of intact villi and no ulcerations. However, there were several leukocytes infiltrating the mucosa and the submucosa.3. Conclusion1. Intestinal perfusion UTI can postpone the decline of blood pressure and prolong the survival time of rats during Intestinal ischemia-reperfusion。2. As pancreatic proteases inhibition,intestinal perfusion UTI can reduce the level of inflammatory reaction. 3. Intestinal perfusion UTI can protect intestinal mucosa, reduce the damage of intestinal mucosa during hemorrhagic Intestinal ischemia-reperfusion. |
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