| Objective:Acute pancreatitis is a common acute abdomen with an increasing incidence.According to the Atlanta Classification,most of the cases of acute pancreatitis are mild,while there are about 15-20% of severe acute pancreatitis(SAP)with the mortality up to 10-30%.Respiratory distress syndrome(ARDS),caused by acute pancreatitis associated lung injury(APALI),is an early serious complication and is the leading cause of death in early stage of SAP.The occurrence and development of APALI are closely related to systemic inflammatory response syndrome(SIRS),in which a large number of inflammatory cells are activated and inflammatory mediators are released,causing "waterfall like reaction in the body.SIRS may lead to the damage of tissue cells,the increase of vascular endothelial barrier permeability,and the occurrence of multiple organ dysfunctions.Under the direct effect of inflammatory reaction and oxidative stress induced by SIRS,the lung tissue was damaged.Cell necrosis,hemorrhage,destruction of alveolar structure,fluid leakage and impaired ventilation function all appeared in lung tissues.In the later stage of SAP,APALI may also develop into pulmonary fibrosis,which seriously affects the prognosis and quality of life of patients.Currently,the treatments of APALI in clinic are mainly supportive treatments like liquid control and respiratory supports,but the effect is not ideal,and effective treatments are still lack.Therefore,the search for effective prevention and treatment of APALI has become an urgent problem in the clinical treatment of SAP.Pancreatitis associated ascitic fluid(PAAF),which contains a large number of pancreatic enzymes,inflammatory factors and other toxic substances,is a common complication in patients with moderate to severe pancreatitis.As the "storage room" of toxic substances,studies have shown that the abdominal ascites may aggravate the severity of acute pancreatitis through multiple mechanisms:(1)PAAF stimulates peritoneal macrophages to produce inflammatory mediators,which then enter the circulation via peritoneal resorption;(2)PAAF causes the decomposition of abdominal fat,resulting in increased free fatty acids in the local and systemic inflammation;(3)A large number of free hemoglobin in PAAF was absorbed into blood,which could aggravate the systemic inflammatory response.At the meantime,experimental results also show that,injection of a certain amount of PAAF into the abdominal cavity of animals can aggravate the disease from mild to severe pancreatitis,and significantly increase the degree of injury in lung tissue and other organs.However,PAAF are believed to be self absorption aseptic effusions in the past,and the use of positive puncture therapy is still controversial,meanwhile,none of the current guides for treatment of pancreatitis have mentioned this problem.In recent years,Based on these studies above and our clinical practices,we attempt to apply ultrasound guided abdominal paracentesis drainage(APD)in the treatment of SAP patients who have peritoneal effusion(?100ml)and a safe puncture drainage path was exist.Our retrospective study found that APD can reduce the mortality,decrease the organ failure rate,especially reduce the tracheal intubation rate,the average rate of ventilation time and the incidence of pneumonia in patients with APALI,shorten the recovery time,and do not increase the risk of infection(Liu WH,et al.Crit Care Med,2015;Liu L,et al.J Clin Gastroenterol,2015).APD can reduce the levels of inflammatory factors and trypsin both in systemic and local blood circulation to a certain extent,especially in the blood circulation around the local abdominal organs,which plays an important role in its treatment mechanism.However,the mechanisms through which APD reduce the severity of pancreatitis and protect the functions of different organs by reducing toxic substances in systemic and local circulation are not clear.The clinical application and promotion of APD were restricted by the unclear mechanisms;therefore,researches in the mechanism of APD have important significance.Oxidative stress induced by reactive oxygen species is one of the important mechanisms caused lung injury in acute pancreatitis.Xanthine oxidase(XOD)is an important source of oxygen free radicals.XOD can produce a large number of single electrons in the process of hypoxanthine catalytic and accelerate ROS generation by using oxygen molecules as electron acceptors.As the lung tissue contains large amounts of oxygen,it may be damaged both directly and indirectly by oxygen free radicals easily.For example,leukocyte infiltration is a landmark event in lung injury and adhesion of leukocytes to endothelial cells is an important step in leukocyte infiltration from the blood vessels,which depends on a variety of molecules like P-selectin.Reactive oxygen molecules,at the same time,can promote the migration of leukocytes by up-regulation of P-selectin.Therefore,it is one of the effective measures to treat APALI by reducing the source of XOD to decrease the generation of oxygen free radicals.XOD is mainly exsit as xanthine dehydrogenase(XDH)in vascular endothelial cells of the digestive tract under physiological conditions.During acute pancreatitis,a large number of XDH dissociate from endothelial cells and transform into XOD by the function of trypsin in the blood.Meanwhile,the factors that promote the dissociation of XDH from endothelial cells in SAP are not clear yet.It is known that XDH is mainly linked to endothelial cells by sugar chains,and the amylase,which has a role in the dissociation of sugar chains,may play an important role in the dissociation of XDH.PAAF,which contains a large number of ?-amylase,may cause a second increase of the concentration of ?-amylase in the circulation around the intestinal tract by being re-absorption,and may promote the dissociation of XDH from the intestinal endothelial cells.In this case,early removal of ascites by APD can reduce the dissociation and transformation of XDH,and thus play a role in the protection of lung tissue and other organs.Therefore,the elucidation of this problem is of great significance for us to understand the mechanism of APD.In order to make this problem clear,we intend to investigate the effect of ?-amylase on the dissociation of XDH from intestinal endothelial cells by intraperitoneally injecting of PAAF to rats with mild acute pancreatitis.Then we made APD treated-severe acute pancreatitis rats model and to study the effect of APD on the dissociation and conversion of XDH.We also observe the changes of oxidative stress and expression of P-selectin and leukocyte infiltration in pulmonary tissue to verify the therapeutic effect of APD on APALI and its related mechanism.Methods and ResultsPart one: the protective effect of APD on lung tissue of SAP ratsMethods1.Effect of APD on the mortality of SAP ratsIn order to understand the effect of APD on the mortality of SAP rats,a total of 42 Male Sprague-Dawley rats were used.Animals were fasted overnight and randomly divided into three groups:(1)Sham group(n=14),in which the rats only underwent sham surgery;(2)SAP group(n=14),in which SAP was induced via a retrograde infusion of 5% sodium taurocholate(1ml/kg body weight)(Sigma)into the biliopancreatic duct over one minute;and(3)SAP+APD group(n=14),in which a modified surgical drainage tube(Baiduoan Company,Shandong,China)was used after the injection of sodium taurocholate.To allow for drainage,one end of the tube(Fr16,Branden,Shandong,China)was inserted into the right lower quadrant before the abdomen was closed,and the other end was connected to a negative-pressure ball device.To calculate the mortality rate,the numbers of surviving and dead rats in every group were determined 12 h after the surgery.2.Protective effect of APD on lung tissue of SAP ratsA total of 36 Male Sprague-Dawley rats were randomly divided into three groups:(1)Sham group(n=12),in which the rats only underwent sham surgery;(2)SAP group(n=12),in which SAP was induced via a retrograde infusion of 5% sodium taurocholate(1ml/kg body weight)(Sigma)into the biliopancreatic duct over one minute;and(3)SAP+APD group(n=12),in which a modified surgical drainage tube(Baiduoan Company,Shandong,China)was used after the injection of sodium taurocholate.Three hours after SAP induction or sham surgery,the rats in each group were anaesthetized,blood and PAAF were immediately collected,and tissues amples were quickly removed,frozen in liquid nitrogen and stored at-80 °C until use.The ascitic fluid in the APD device or in the abdominal cavity was collected,and its volume was calculated.Serum ?-amylase and trypsin activity;pathological change of lung tissue specimens under the microscope(HE staining)and pathological score of lung tissue wet weight(weighing method),Results1.The 12 h mortality rate of SAP rats was 57.1%,and the average amount of ascites was(10.1 ±1.3 ml).The 12 h mortality rate of rats in the SAP+APD group was 14.3%,and the average amount of ascites(7 ±0.9ml)were significantly lower than those in the SAP group.2.The ?-amylase and trypsin activities in the serum of rats of the SAP group increased significantly than those in the rats of sham operation group.The serum ?-amylase and trypsin activities in the rats of SAP+ADP group were significantly decreased than those in the SAP group.3.The pathological observation of lung tissues of rats in the three groups shows that: three hours later,there are hemorrhage,necrosis and inflammatory cells infiltration the lung tissue of rats in SAP group.Compared with rats in the sham operation group,the pathological damage score and dry-wet ratio of lung tissue of rats in the SAP group significantly increased.The pathological damage score and dry-wet ratio of lung tissue of rats in SAP+APD group rats decreased significantly than lung tissue of rats in SAP group.ConclusionsThese results suggest that APD has therapeutic effect on SAP rats,especially on lung tissue,which can reduce the lung parenchyma injury and inflammatory cell infiltration.Part two: The role of XDH/XOD in the effects of APD on APALIMethods1.Effects of APD on intestinal XDH dissociation and transformationA total of 36 Male Sprague-Dawley rats were randomly divided into three groups:(1)the sham group(n=12),in which the rats only underwent sham surgery;(2)the SAP group(n=12),in which SAP was induced via a retrograde infusion of 5% sodium taurocholate(1ml/kg body weight)(Sigma)into the biliopancreatic duct over one minute;and(3)the SAP+APD group(n=12),in which a modified surgical drainage tube(Baiduoan Company,Shandong,China)was used after the injection of sodium taurocholate.Three hours after SAP induction or sham surgery,serum,intestinal and lung tissues were retrieved for the following tests: XDH and XOD activity(ELISA),serum XDH and XOD activity(ELISA),lung SOD activity and MDA level(spectrophotometry);lung XOD and XDH activity(ELISA).2.Effects of ?-amylase in PAAF on intestinal XDH dissociation and transformationTo measure the effect of PAAF on the mobilization of XDH/XOD from the intestine,another 24 rats with mild pancreatitis were used.Mild pancreatitis was experimentally induced by intraperitoneal injection s of two doses of caerulein at 20 ?g/kg over three consecutive days,as previously described Three days after the induction of pancreatitis,24 rats were randomly divided into the following three groups:(1)Controls(n=8): Rats with mild pancreatitis were intraperitoneally injected with 5ml of asaline solution.(2)PAAF injection(PI)(n=8): Fresh PAAF obtained from the abdominal cavity of rats with SAP was centrifuged,and 5ml of sterile supernatant was intra-peritoneally injected into rats with mild pancreatitis.(3)PAAF+?-amylase inhibitor injection(DPI)(n=8): Rats with mild pancreatitis were intraperitoneally injected with 5ml of PAAF and 0.5mg of an ?-amylase inhibitor(Sigma,St.Louis,MO,USA).PAAF and the ?-amylase inhibitor were previously incubated at 25 ?for 10 min.Three hours after the injections,rats were anaesthetized and blood and intestinal tissues were collected and immediately frozen and stored at-80? until use.Three hours after SAP induction or sham surgery,serum,intestinal and lung tissues were retrieved for the following tests: XDH and XOD activity(ELISA),serum XDH and XOD activity(ELISA).Results1.The activity of XDH and XOD in lung tissue of rats Compared with sham operation group,with severe acute pancreatitis was significantly increased,while the activity of XDH and XOD in lung tissue of rats after intraperitoneal drainage was lower than that of untreated rats.2.Compared with the sham group,The MDA levels in lung tissues of rats with SAP increased and the activity of SOD decreased.After the treatment of abdominal drainage,the MDA levels in rats of SAP+APD group decreased,while the SOD activity significantly increased compared to the rats in the SAP group.3.Immunohistochemistry and Elisa assay show that the expression of XDH in intestinal vascular endothelial cells in rats with SAP was decreased compared with that in the sham group.The expression of XOD in rats in SAP group was slightly increased.The expression of XDH and XOD in the serum and lung tissue increased compared with those in the sham operation group.After the treatment of abdominal drainage,the expression of XDH in intestinal endothelial cells of rats was higher thanth that in the rats of SAP group.The expression of XOD in intestinal endothelial cells and the expression of XDH and XOD in serum and lung tissue of rats in SAP group decreased slightly compared with those in the rats of SAP group.4.The expressions of serum XDH and XOD increased significantly,while the expression of intestinal XDH significantly decreased in rats with mild pancreatitis receiving intra-peritoneal injection of PAAF than those in rats with mild pancreatitis receiving intra-peritoneal injection of PAAF+?-amylase inhibitor.The expression of XOD increased slightly in rats with mild pancreatitis receiving intra-peritoneal injection of PAAF than those in rats with mild pancreatitis receiving intra-peritoneal injection of PAAF+?-amylase inhibitor.ConclusionsThe above results indicate that re-absorption of ?-amylase in ascites could mobilize large number of intestinal XDH dissociation from endothelial cells.The increased protease activity during pancreatitis may also play an important role in the transformation of XDH to XOD.Part three:The role of P-selectin in the effect of APD on APALIMethodsA total of 36 Male Sprague-Dawley rats were randomly divided into three groups:(1)the sham group(n=12),in which the rats only underwent sham surgery;(2)the SAP group(n=12),in which SAP was induced via a retrograde infusion of 5% sodium taurocholate(1ml/kg body weight)(Sigma)into the biliopancreatic duct over one minute;and(3)the SAP+APD group(n=12),in which a modified surgical drainage tube(Baiduoan Company,Shandong,China)was used after the injection of sodium taurocholate.Three hours after SAP induction or sham surgery,rats were sacrificed and lung tissues were obtained for the following detection: The expression of P-selectin levels(real-timePCR and Western blot)in lung;Lung tissue MPO(ELISA),the number of neutrophils in bronchoalveolar lavage fluid were all tested.Results1.The expression of P-selectin in lung tissue of rats with SAP was significantly higher compared with that in sham operation group.The expression of P-selectin in lung tissue of rats in SAP+APD group decreased compared with that in the SAP group.2.The number of neutrophils in bronchoalveolar lavage fluid and MPO activity of rats in SAP group were significantly higher compared with those in the rats of sham operation group and SAP+APD group.ConclusionsThese results indicate that APD can reduce the level of oxidative metabolism in lung tissue and reduce the expression of P-selectin in lung tissue which is closely related to leukocyte adhesion.Thereby APD can reduce the degree of leukocyte infiltration in lung tissue.Statistical analysisStatistical analyses were performed using SPSS version 19.0(IBM Corporation,Somers,NY,USA).Normally distributed data are expressed as the mean±standard deviation(SD)and were compared using a one-way analysis of variance(ANOVA)with Dunnett's multiple comparison tests.Data with an asymmetrical distribution are presented as median values and the range,and Kruskal-Wallis H tests followed by Mann-Whitney U tests with Bonferroni corrections were performed.Values of p<0.05 were considered to be statistically significant.SummaryThe injection of PAAF retrograde intraperitoneal into rats with mild pancreatitis,can cause dissociation of XDH from intestinal endothelial cells significantly increased,meanwhile,XDH and XOD activity in circulation and lung tissue increased.However,use of ?-amylase inhibitor can antagonize those effects,indicating that ?-amylase in PAAF can promote the dissociation of XDH from intestinal endothelial cells by been resorption.APD can reduce the dissociation from endothelial cells and conversion to XOD of XDH,and reduce the content of XDH and XOD in serum and lung tissue in rats with SAP through the removal of PAAF.Thereby APD can reduce the generation of oxygen free radicals and oxidative stress in the lung tissue.At the same time,the expression of P-selectin induced by oxygen free radical was decreased,and the infiltration of white blood cells in lung tissue was reduced after APD.In summary,our results show that APD inhibits XDH dissociation from intestinal endothelial cells and transformation to XOD by reducing re-absorption of ?-amylase.Thus APD could reduce the production of oxygen free radicals in lung,protect lung tissue in SAP rats(Fig 1). |
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