Effect Of Prolease In The Lumen Of Rat Intestine On Inflammatory Reaction During Hemorrhagic Shock

When a variety of exogenous and endogenous factors caused cells and tissues of the body injuryed, the local or systemic body had a complex set of reactions that limited and eliminated the damage factors, absorbed the necrotic tissue, finally repaired the trauma.This complex defense reaction-based response was called inflammatory reaction. Any disease was accompanied by inflammatory reaction, such as infection, trauma, and other acute and chronic diseases. When the initial treatment of inflammatory reaction was not been timely and effectively, the body would produce a variety of inflammatory mediators which could form a "waterfall effect" that expanded the inflammatory reaction, even made it be uncontrolled.Then the body would undergo systemic inflammatory response syndrome(SIRS), even multiple organ dysfunction syndrome(MODS) or death. Therefore, it was important to treat the inflammatory reaction at early period that could control the progression of the disease and prevent its further deterioration.Decades of research, especially in the 1980s, the various steps of the inflammatory cascade had brought to light that leukocyte adhesion and leukocyte cytotoxic activity (e.g, oxygen free radical formation) were parts of the inflammatory cascade and may actually serve to injure innocent bystander cells and organs, such as obstruction of capillaries.This observation led to explorations designed to attenuate the injury side of the inflammatory cascade. Several of these ideas (e.g, oxygen free radical scavenging; leukocyte adhesion blockade) were tested in clinical trials, but emerged with only limited success as a protective measure in patients, even though preclinical studies gave indications to the contrary. This disappointment was a wakeup call in our thinking about inflammation and motivated us to approach the inflammatory cascade in an alternative fashion.In recent years, Geert W. Schmid-Schonbein, an American reseacher, proposed a new theory that fully activated digestive enzymes entering into the submucosal space and initiating self-digestion of an otherwise unprotected villus structure, a process that lead to complete destruction of their tissue matrix was the key factor to lead to SIRS or MODS under the condition of trauma and shock. The fully activated and powerful digestive enzymes in the lumen of the intestine, usually prevented from entrying into the mucosal barrier, would rapidly enter into the wall of the intestine under any condition that compromised this important barrier. They may be carried even into deeper muscle tissue layers of the intestinal wall. The self-digestion of intestine tissue could produce many activity factors that could enter the portal venous circulation, the intestinal lymphatics, and may aslo escape across the serous coat into the peritoneal fluid to active inflammatory cells and release inflammatory mediators. Through the intestinal perfusing serine protease inhibitor gabexater or nafamostat could significantly improve the survival rate of intestinal ischemia-reperfusion rats after reperfusion. The plasma activity level of experimental group rats were lower than control group rats. The plasma of experimental group rats aslo had the lower ability to active human neutrophils.Hemorrhagic shock was accompanied by inflammation reaction. It lead to death if the inflammation reaction could not be inhibited in time. Therefore, our experiment would investigate the effect of intestinal perfusion ulinastatin (UTI) on inflammatory reaction during hemorrhagic shock. This would offer new treatment for hemorrhagic shock.Materials and methods1. Animals and Experimental groups28 healthy wistar rats (weight,220-270g), both male and female, were divided into 4 groups randomly:no intestinal perfusion group,0.9% saline water intestinal perfusion group, UTI intestinal perfusion group and intravenous injection UTI group(Tab.1). The rats were normal diet 24 hours before the experiment. Tab.l Different interventions and ways of administration of different group2. The model of hemorrhagic shockThe rats were anesthetized by 3% sodium pentobarbital(50 mg/kg)intraperitoneal injection. The right femoral artery and vein were cannulated with a polyethylene tubing (Trocar,24G). The arterial line served to record the systolic, diastolic, and mean arterial pressure and the venous line served to administer salt solution containing UTI or not. Then the lumen of the proximal duodenum was cannulated with a PE-240 tubing. Another PE-240 tubing was inserted into the terminal ileum. The intestinal contents were gently flushed out through the two tubings.All above were done, except the no intestinal perfusion group, in the other three groups, a polypropylene tube and a peristaltic pump were connected to these catheters to set up an intestinal perfusion circuit with 50 mL of saline (37℃) at constant pressure and speed.Then every group exsanguinated(in 30min) to a mean arterial blood pressure of 40±5 mm Hg. Maintaining at this blood pressure for 10 min, UTI intestinal perfusion group was intestinal perfused UTI (50 000U/Kg)salt solution 10 ml and intravenous injected 0.9% saline solution 0.5ml.Intravenous injection UTI group was intravenous injected UTI (50 OOOU/Kg) saline solution 0.5ml and intestinal perfused 0.9% salt solution 10ml. 0.9% saline water intestinal perfusion group was intravenous injected 0.9% saline water 0.5ml and intestinal perfused 0.9% salt solution 10ml, and no intestinal perfusion group was only intravenous injected 0.9% saline water 0.5ml.3. Detection index3.1 The change of MAP duiring Hemorrhagic shockThrough blood pressure monitor, observed and recorded changes in MAP of rats duiring Hemorrhagic shock. The recording time inclouded basal blood pressure, before intestinal perfusion, lOmin after intestinal perfusion and intestinal perfusion over, aslo shock,30min after shock,60min after shock,90min after shock,120min after shock and 180min after shock.3.2 Survival timeRecord the survival time of rats. It was defined death when the heart of rat stopped beating without treatment in 5min.3.3 The expression of human PMN CD11b induced by plasma of ratThe rat plasma that contained activity factors could active human PMN when they were mixed culture. Arterial blood (about 500μl at each time point) was collected before shock and 120 min,180min after shock.20μl was used to determine the circulating leukocyte counts. The remainder of the sample was centrifuged (500 g for 5 min) and the plasma was frozen (-80℃) until assayed.Blood from a healthy human volunteer was collected and was referred to as normal granulocytes. To assess the direct effects of the plasma specimens on CD11b expression,50μl Plasma was added to 200μl aliquots of whole blood from normal volunteers, which had been diluted 1:1 with 200μl of DMEM medium. After a 4-h incubation period,20μl of anti-human CD11b FITC-labeled monoclonal antibody (BD Pharmingen, San Diego CA) was added to the plasma-blood samples, after which the samples were gently and briefly vortexed, then placed on ice for 45 min in the dark. Subsequently, the red cells were lysed, the PMNs washed three times and then the level of CD11b expression was determined by flow cytometry as previously described.The data were expressed as mean fluorescence intensity. FITC-conjugated, isotype mouse IgGl antibody was used as an isotype control for nonspecific antibody binding.3.4 Leukocyte countThe leukocyte count was counted by manual method.2ml glacial acetic acid was added to 98ml distilled water which contained 10g/L methylene blue 3 drops.Then added 0.38ml the dilution to 20μl blood sample of rat, after which some was dropped on blood count board. The leukocyte count was counted by the light microscope 2-3 min later.3.5 Intestinal tissue biopsyWhen the rats were sacrificed and a 4-cm segment of the small intestine was excised, washed with 0.9% 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.4,Statistical analysisAll data were reported as the mean±standard deviation(x±s). Analysis of variance of repeated measure data was used to analyze the blood pressure data. The one-way ANOVA on ranks was used to assess significance between different groups at the same time point for surivial time, the expression of CD11b and leukocyte count. Spss 13.0 was used to analyze the data and p< 0.05 was considered to be significant.Result1. Before shock, the blood pressure is no statistic significance among different groups, F=0.904 (P=0.454,>0.05), and is aslo no statistic significance at different time within group, F=1.174 (P=0.322,>0.05). But it is significant difference after shock, F=5.814 (P=0.006,<0.05). The blood pressure of UTI intestinal perfusion group is higher than 0.9% saline water intestinal perfusion group (P=0.004,<0.05). Compared the blood pressure of intravenous injection UTI group with 0.9% saline water intestinal perfusion group, the former is higher at the same time (P=0.049, <0.05). But there were no significant differences between no intestinal perfusion group and 0.9% saline water intestinal perfusion group (P=0.544,>0.05), aslo between UTI intestinal perfusion group and intravenous injection UTI group (P=0.224,>0.05).2. The surivial time of UTI intestinal perfusion group is longer than no intestinal perfusion group (P=0.008,< 0.05), aslo longer than 0.9%saline water intestinal perfusion group (P=0.013,< 0.05) and intravenous injection UTI group (P=0.039, < 0.05). But the surivial time of intravenous injection UTI group is no significant differences compared with 0.9% saline water intestinal perfusion group or no intestinal perfusion group (P>0.05).3. While the activity of rat plasma increases during shock, the expression of PMN CD11b raises. The plasma activity of different groups is no statistic significance before shock, F=0.147 (P=0.930,>0.05).The expression of PMN CD11b induced by UTI intestinal perfusion group rats is lower than no intestinal perfusion group rats (P=0.001,<0.05) at 120min after shock, aslo lower than 0.9%saline water intestinal perfusion group rats (P=0.004,<0.05) and intravenous injection UTI group rats (P=0.004,<0.05). The plasma activity of intravenous injection UTI group is no statistic significance with 0.9%saline water intestinal perfusion group (P= 1.000,>0.05).The expression of PMN CD11b induced by UTI intestinal perfusion group rats is lower than no intestinal perfusion group rats (P=0.040,<0.05) at 180min after shock, aslo lower than 0.9%saline water intestinal perfusion group rats (P=0.048.<0.05) and intravenous injection UTI group rats (P=0.033,<0.05). The plasma activity of intravenous injection UTI group is no statistic significance with 0.9%saline water intestinal perfusion group (P=0.962,>0.05).4. The leukocyte count of different groups is no statistic significance before shock, F=0.508 (P=0.681,>0.05). But the leukocyte count of UTI intestinal perfusion group is higher than no intestinal perfusion group rats (P<0.001)at 120min after shock, aslo higher than 0.9%saline water intestinal perfusion group rats (P<0.001) and intravenous injection UTI group rats (P<0.001). The leukocyte count of intravenous injection UTI group is no statistic significance with 0.9%saline water intestinal perfusion group (P=0.767,>0.05).The leukocyte count of UTI intestinal perfusion group is higher than no intestinal perfusion group rats(P<0.001)at 180min after shock, aslo higher than 0.9% saline water intestinal perfusion group rats(P=0.001,<0.05)and intravenous injection UTI group rats (P=0.001,<0.05). The leukocyte count of intravenous injection UTI group is no statistic significance with 0,9%saline water intestinal perfusion group (P=0.787,>0.05)5. Histological sections of the small intestine from no intestinal perfusion group rats are a loss of the mucosal barrier and villi with a high amount of leukocytes infiltrating the whole intestine. Intra-intestinal pancreas protease inhibition further reduced gut damage.Conclusion1. Intestinal perfusion UTI can postponable the decline of blood pressure and prolong the survival time of rats duiring hemorrhagic shock.2. As pancreatic proteases inhibition, intestinal perfusion UTI can reduce the level of inflammatory reaction, improve the count decrease of leukocyte.3. Intestinal perfusion UTI can protect intestinal mucosa, reduce the damage of intestinal mucosa duiring hemorrhagic shock.