| Abstract Objective:Acute pancreatitis (Acute pancreatitis, AP) is made of waterfall、multiple organ tissues pathology changes caused by the pancreas as initiating organ, the capillary permeability plays a very important role during the process as transmission and amplification. The study is apply the pancreatic bed capillary as window, chose ZO-1protein as the reaction index for capillary permeability, to explore the change of ZO-1protein expression in pancreatic bed capillary endothelial cells for rats with AP and the relationship with the change of capillary permeability, and apply the abdominal cavity drainage technology to rats model which is widely used clinically of the treatment of Severe Acute pancreatitis (Severe Acute pancreatitis, SAP). To locate the possible factors causing the change of ZO-1protein, and to explore the early abdominal cavity drainage for acute pancreatitis rats and its possible mechanism of intervention effects.Methods:Healthy SPF male Wistar rats were applied as experimental animals,body weight200-250g,120Wistar rats were randomly divided into3groups after adaptive feed for one week:sham operation group (Group A), SAP group (Group B), SAP drainage (Group C),Each group contains40rats. For each group, five time points were set at1h,3h,6h,12h and24h after operation, at each time point,8rats were assigned. Group A were laparotomized, only flip pancreas after the abdomen was closed, Group B were applied the retrograde cholangiopancreatography by micro-pump injection with5%sodium taurocholate (0.1ml/100g) to produce SAP model and the abdomen was closed after the identification of the pancreatic duct toward bleeding, edema and other signs, Group C were applied the uplink peritoneal drainage on the basis of Group B, namely put drainage tube into lower abdomen of the rat, connected to a drainage bag at the end of the drainage tube after layered abdomen closing. All rats were treated with basic fluid therapy, namely subcutaneous injection of40ml/kg of37℃saline on the back of rats, once every12h, significant Picchu was identified as a sign of success, preoperative and postoperative12hours were only with water but without food. Take rat peripheral venous blood, ascites and pancreatic tissue1h,3h,6h,12h and24h after surgery, inject intravenously of5%Evans blue (Evan’s Blue, EB)(lml/lkg)30minutes before sampling. Interleukin-8(IL-8), tumor necrosis factor-alpha (TNF-a), nuclear factor kappa B (NF-κB) and ZO-1changes in serum and ascites.were measured by ELISA. Amylase was assayed by Beck-man automatic biochemical analyzer, trypsin activity was measured using spectrophotometric detection.Pancreatic capillary permeability was applied spectrophotometric to detect Evan’s blue concentration around pancreatic tissue.Record amount of ascites.24h survival rate were observed. HE staining was applied for histological sections of pancreatic tissue, pathological changes were observed and pathology score were evaluated. The change of ZO-1protein expression of pancreatic bed capillaries was detected by Immunohistochemical method.Results:1, The general case of rats:no significant difference was shown for sham-operation group before or after surgery.The SAP rats appeared to be apathetic, less active, with poor appetite and small urine output; and the symptoms gradually worsened with time. Drainage group was similar to SAP group6hours after surgery,6h past the situations were better than that of SAP group, movement increased, urine output, and drainage time was extended for the drainage group, the symptoms gradually improved;2, The mortality at12h for SO group, SAP group and drainage group was0%,47.5%and12.5%.The total mortality at24h for SO group, SAP group and drainage group was0%,67.5%and22.5%3, Histopathological changes of pancreatic tissue:the pancreatic tissue for SO Group showed no abnormalities; the pancreatic tissue for SAP group showed early edema, punctate bleeding, scattered necrosis, with small amount of neutrophil infiltration, with time, acinar lobular structure damage, hemorrhage, necrosis, large number of neutrophils and mononuclear cell infiltration was observed by microscope; Drainage Group,3h after surgery, congestion and edema were observed at pancreas, but slightly lighter than that of SAP group, congestion and edema of the pancreas became obvious from6to12h with pancreatic spotty necrosis and small amount of interstitial infiltration of inflammatory cells, at24h pancreas congestive edema and necrosis were alleviated.4, The changing levels of the expression of TNF-alpha, NF-kappaB and IL-8in serum and ascites:TNF-α, NF-κB and IL-8level of SAP serum was significantly higher than that of SO group; the TNF-a, NF-κB and IL-8level of SAP rats ascites was significantly higher than that of drainage group (p<0.05).5, The hanging level of the expression of pancreatic amylase and trypsin of serum and ascites:serum pancreatic amylase activity of SAP Group and drainage group were higher than that of SO group (p<0.05), the ascites amylase of SAP group was significantly higher than that of drainage group (p <0.05), serum trypsin activity of SAP group was significantly higher than that of drainage group, and ascites trypsin activity at6h of SAP group was higher than that of drainage group, at12h the drainage group was higher than SAP group (p<0.05).6, The changing level of ZO-1expression:Serum ZO-1protein level of SAP group and drainage group were significantly higher than that of SO group (p <0.05); and followed steady decrease; ascites serum ZO-1protein level of SAP group were higher than that of drainage group (p<0.05), with time, ascites serum ZO-1protein level of SAP group gradually increased, ascites serum ZO-1protein level of drainage gradually decreased (p<0.05); ZO-1protein expression of pancreatic bed capillary wall of drainage group was significantly higher than that of SAP group (p<0.05); Serum ZO-1protein level was positively correlated with trypsin, amylase and TNF-a concentration, and the correlation coefficient between Serum ZO-1protein level and amylase was0.87, P<0.05, serum ZO-1protein level was positively correlated the damage of the pancreatic tissue (Pearson The correlation coefficient is0.96, P<0.05).7, The changes of pancreatic capillary barrier:the EB concentration of capillaries surrounding pancreatic tissue of SAP group was higher than that of drainage group (p<0.05), with times, the EB concentration of capillaries surrounding pancreatic tissue of SAP group increased gradually, that of drainage group reduced; the amount of ascites of drainage group were lower than that of SAP group at each time point.Conclusion:1, When rats were suffered with severe acute pancreatitis, the serum ZO-1protein concentration was significantly increased, and ZO-1protein expression in tissue was significantly decreased, the increase of trypsin and TNF-alpha appeared before the increase of ZO-1protein concentration possibly due to damage by high concentration trypsin and TNF-alpha and inhibition of ZO-1protein.2, With the reduced expression of ZO-1protein in tissues, the continuity and completeness of the capillary was damaged, capillary permeability increased and exudation increased. 3,Early drainage significantly reduced the general inflammatory response to reduce the concentration of ascites trypsin, TNF-a, NF-κB,IL-8, and other factors, and to also reduce the toxic factor in blood circulation, alleviate the capillary damage, protect the capillary barrier function, so as to reduce the mortality of rats. |
PDF Full Text Request