Experimental Observation Of Short-term Peritoneal Lavage Treatment With Protease Inhibitor In Severe Acute Pancreatitis

Objective:To establish the model of sodium taurocholate-induced severe acute pancreatitis and observe short-term peritoneal lavage treatment with ulinastatin in severe acute pancreatitis,and investigate its mechanism preliminarily.Method:A total of 80 Wistar rats were randomized into 5 groups,each 16 in every group: the sham operated control group(group A),SAP model group(group B),ulinastatin intravenous injection group(group C),normal saline peritoneal lavage group(group D),ulinastatin peritoneal lavage group(group E). The method of SAP animal model was according to Aho, way,anaesthesia with 2% sodium pentobarbital (0.3ml/100g) by intraperitoneal injection,entering the abdominal cavity through epigastric median incision,revealling pancreatic tissue,puncturing across the duodenal wall near duodenal papilla and retrograde inserting the cholangiopancreatic duct with the 4th scalp needle,and injection of 5% sodium taurocholate (0.1 ml/100 g),vision of pancreatic tissue hyperemia, edema, hemorrhage, necrosis about after 5-10min. Such changes showed the success of the preparation in rat model of SAP.Fasting but water was allowed after operation. Examine the level of IL-1β, TNF-α, amylase, trypase, NF-κB, TLR4 and pancreatic pathological change from blood, ascites, pancreatic tissue samples after operation made 6th,24th hour.Results:1. Pathological observation results: Group A: no significant pathological changes in the pancreas. Group B: obvious pancreatic hemorrhage and necrosis,saponifications,hemorrhagic ascites, gastrointestinal cavity flatulence. Large areas of necrosis,apparent red blood cell exudation and extensive infiltration of inflammatory cells can be watched under microscope. Group C: reductive pancreatic hemorrhage and necrosis,a small amount of saponifications, occasional hemorrhagic ascites and gastrointestinal cavity flatulence. The area of necrosis, red blood cell exudation and inflammatory cell infiltration is lower than group B significantly. Group D: the changes of general and under microscope were no significant compared with group B. Group E: the changes of general and under microscope were significant compared with group B,also with group C. Pathological changes in each group are progressive increased with time,that is, 24h was more serious than 6h. Pancreatic pathology score results: group E < group C < group B and there is no significant difference between group B and group D.2. Ascites amylase, trypsin test results: the difference between group B and group A was significant and that meaned the successful modeling. Group E was better than the other groups, followed by the group C, and there was no significant difference between group D and group B.3. Blood IL-1β, TNF-αtest results: the difference between group B and group A was significant and that meaned the successful modeling. There was no significant difference in the treatment between group D and group B. There was a significant difference compared group C,group E with group B. Also, significant differences had taken place between group C and group E, and group E was much better than group C.4. The expression results of NF-κB in pancreatic tissue: there was almost no NF-κB expression in pancreatic acinar cellular nucleus in group A and only a small amount of expression can be seen in acinar cellular cytoplasm. The expression of NF-κB was observed stronger in group B than the other groups in pancreatic acinar cellular nucleus and cytoplasm. The expression intensity in order: group B > group C > group E > group A, no significant difference between group D and B.5. The expression results of TLR4 in pancreatic tissue: expression happened in cells membrane and cytoplasm of main pancreatic duct epithelium and vascular endothelial cells in group A and there was a small amount of weak expression located in the cytoplasm of pancreatic acinar cells. The positive expression rate of group B was significantly higher than the other groups. The expression intensity in order: group B > group C > group E > group A, no significant difference between group D and B.6. The correlation analysis results between SAP rat NF-κB, TLR4 expression of the average optical density values and the corresponding rat pancreatic pathological score:the correlation coefficient between NF-κB and rat pancreatic pathological score is 0.9360, P = 0.000; the correlation coefficient between TLR4 and rat pancreatic pathological score is 0.9065, P = 0.000. That meaned the significant positive correlation between NF-κB, TLR4 expression of the average optical density values and the rat pancreatic pathological score. At the same time, after the score controlled, the correlation coefficient between NF-κB and TLR4 is 0.6009, P = 0.009, showing the certain degree of positive correlation between them.Conclusions:1. the expression of NF-κB and TLR4 in SAP rats was significantly increased, showing the consistency (r = 0.6009), and was closely related to the pancreatic pathology injury (rNF-κB = 0.9360, rTLR4 = 0.9065), showing the important pivotal roles in the pathogenesis of SAP.2. Ulinastatin peritoneal lavage can obviously inhibit amylase, trypsin activation ,reduce the expression of TLR4,NF-κB, reduce the level of TNF-α,IL-1βand relieve the injury of pancreatic tissue and the body compared with the other methods in this experiment.3. The way of peritoneal lavage treatment with protease inhibitor in severe acute pancreatitis is simple, effective,feasible.