The Experimental Study On The Potential Role And Mechanism Of Cyclooxygenase-2 Involved In The Devolopment Of Severe Acute Pancreatitis And Associated Lung Injury In Rat

Background & AimsAcute pancreatitis(AP) is a clinical common acute abdomen. It's main character is pancreatic enzyme activation and local inflammation of pancreas, and may be or not associated with multiple organ dysfunction.It is frequently associated with systemic inflammatory response, about 15%～30% patients with acute pancreatitis develop Severe Acute Pancteatitis(SAP). It develops rapidly and effects the function of many important organs,for example lung,liver,kidney and heart,and develops systemic inflammatory response syndrome(SIRS), even multiple organ failure(MOF)with a mortality rate 30%. For the reason,severe acute pancreatitis(SAP) is still categoried as a common and severe disease vital to human health.Acute pancreatitis-associated acute lung injury(APALI), with high incidence in severe acute pancreatitis, is a systemic sequelae of vital importance, and adult respiratory distress syndrome(ARDS), a severe manifestion of pulmonary injury, might almost account for pancreatitis-associated mortality in the early stage of severe acute pancreatitis, and mortality was even up to 40% in these patients with acute lung injury or ARDS. Therefor, SAPALI was regarded as one of standards to evaluate severity degree of SAP in clinic. Thus, prevent and cure SAPALI or ARDS is very important to improve the therapeutic effect of SAP. Up to now,the pathophysiology of severe acute pancreatitis and SAPALI remained poorly understood, and also, the factors that determine the development and ultimate sequelae of pancreatitis were still not well established. Accumulated evidences suggested that various proinflammatory cytokines, such as TNF-α, interleukin-1(IL-1), and interleukin-6(IL-6), released form activated polymorphonuclear granulocytes(PMN) and monocytes/macrophages in pancreatic parenchyma, play a pivotal role in acute pancreatitis, proinflammatory cytokines not only contribute to the local inflammatory initiation and development of acute pancreatitis, but also the episode of systemic inflammatory response, overproduction of proinflammatory cytokines has been shown to deteriorate the severity of disease process. Conversely, the antiinflammatory cytokine, such as interleukin-4(IL-4), interleukin-10 (IL-10), has been documented to alleviate the severity of acute pancteatitis in experimental models.Recently, the role of prostaglandins and prostaglandin-derived mediators in mediating the systemic inflammatory response has been paid more attention in acute pancreatitis. Cyclooxygenase(COX) is the key enzyme for prostaglandin synthesis, and exists in two distinct isoforms: constitutive form(COX-1) and inducible form(COX-2). COX-1 is a house-keeping enzyme, and produces prostaglandin mediators for physiological reactions. Whereas COX-2 is an important proinflammatory, it is normally undetectable in most tissue, but overexpressed in inflammatory disease including acute pancreatitis, suggesting that COX-2 Might play an important role in the induction and progression of acute pancreatitis. Up to now, the precise role of COX-2 in SAP and SAPALI has not yet been well-established.ObjectiveIn our study, we expect to investigate the potential role and the underlying mechanisms of COX-2 involed in experimental severe acute pancreatitis and associated acute lung injury. This article was composed of two parts: In part one, we arm to investigate the diversity of the mediators of inflammatory, the expression of COX-2 in the pancreas tissue of SAP in rat, and the impossible link between COX-2 expression and the level of pancreas injury determined pathologically. In part two,we applied with a highly-select COX-2 inhibitor(Celecoxib) to pretreat SAP in rat, and we researched the histopathologic change of pancreas and lung tissue after pretreatment, in order to explore the impossible mechanism of Celecoxib in SAP and ALL MethodsAdult mail Sprague-Dawley rats were randomly divided into three groups: normal control group(group N), severe acute pancreatitis group(group SAP) and treatment group with Celecoxib(group T). Animals in group N received laparotomy only, animals in group SAP were induced by the retrograde injection of bilio-pancteatic duct with 5% sodium taurocholate(1.0ml/kg), animals in group T, Celecoxib was administrated affusedly down alimentary canal from mouth to stomach at 30mg/kg.d at 2 hours before the inducement of SAP. Rats were sacrificed at 3, 6, 12hours after induction, the blood serum, pancreas and lung tissue of rats were harvested. Serum amylase(AMS) activity was detected with biochemical analyzer; the serum levels of TNF-αand IL-6 were detected by ELISA method; serum and lung tissue levels of TXB₂ and 6-keto-PGF_1α were detected by radioimmunoassay(RIA); myeloperoxi-das activity(MPO) of lung tissue was enzymohistochemistrically detected by colorime try, using a BECKMAN DU640 model spectrophotometer; the severity of pulmonary edema was determined by the ratio of the weight of wet lung tissue to that of dry lung tissue(w/d); The pancreas and lung samples were removed for microscopic histological examination to score lesions, expression of COX-2 in pancreas and lung tissue and expression of NF-κB in pancreas tissue were determined by immunohisto -chemistrical method, and expression of COX-2mRNA and COX-2 protein in lung tissue were determined by RT-PCR and Western-Blot.ResultsPart oneThe serum amylase activity, the serum levels of TNF-αand IL-6 ,TXB₂ and 6-keto-PGF_1α were significantly increased in comparison with the control in rat after sodium taurocholate induction at each time point(3, 6, 12hours), the pathological scores of pancreas tissue injury (characteristic as edema, hemorrhage, necrosis and inflammation) were markedly increased in group SAP(P<0.05), and the mortality in 24^th hr of rats in group SAP was 100%. Therefor, in present study, a rat model of SAP was well induced by standard retrograde infusion of bilio-pancreatic duct with 5% sodium taurocholate solution.In the study, expression of COX-2 was not detected in intact pancreas tissue, but overexpression of COX-2 could be observed in pancreas tissue in group SAP in rat from 3 hours to 12 hours, and the overexpression of COX-2 reached peak in 6 hours after sodium taurocholate. There was a considerable association of COX-2 protein with pathological scores of the level of pancreas injury at each time point(P<0.05).Part twoCelecoxib markedly down-regulated the level of serum AMS and TNF-α(P<0.05) , and bettered the character of ascites in rats in group SAP (P<0.05) ; the ratios TXB₂/6-keto-PGF_1α of lung tissue in grop T were significantly lower at 6, 12hours than those in group SAP. Pathologically, either under naked eye or light microscope, prophylactic administration of Celecoxib dramatically alleviated the severity of SAP and ALI, according to the pathological scores of pancreas and lung tissue injury, and resulted in a considerable decrease. in the ratio of w/d and MPO activity of lung tissue in rats with SAP (P<0.05) .In the study, little expression of COX-2 was observed in intact lung tissue in group N, but overexpression of COX-2 could be observed in lung tissue in group SAP in rat from 3 hours to 12 hours, and the overexpression of COX-2 reached peak in 6 hours after sodium taurocholate induction. Expression of NF-κB was not detected in intact pancreas tissue, but overexpression of NF-kB could be observed in pancreas tissue in group SAP in rat from 3 hours to 12 hours, and the overexpression of NF-kB reached peak in 3 hours after sodium taurocholate induction. Celecoxib dramatically inhibited the expression of COX-2, NF-kB protein in pancreas tissue and the expression of COX-2 protein in lung tissue in rats with SAP at each time piont (P<0.01) .RT-PCR and Western-Blot method, a little expression of COX-2mRNA and COX-2 protein were showed in lung tissue in group N. but expression of C0X-2mRNA and COX-2 protein were significantly up-regulated after sodium taurocholate challenge (P<0.01), and reached peak in 6 hours. Celecoxib dramatically inhibited the expression of COX-2mRNA and COX-2 protein in lung tissue in rats with SAP at eachtime piont (P<0.01) .ConclusionsOur research results clearly suggested that COX-2 was a primary response gene for severe acute pancreatitis, and plays a key role in the development of severe acute pancreatitis and associated acute lung injury. The over-expression of COX-2 was implicated to alter key components of inflammatory cascade, meanwhile product lots of prostaglandins and cause microcirculation disturbance in pancreas and lung tissue.Celecoxib could inhibit the expression of COX-2 gene, and it could have markedly protective effect to SAP and associated ALI of expremental rats.There were some impossible mechanisms: one mechanism could be infered that Celecoxib might inhibit TNF-αproduction, decrease stagnation of PMN and promote apoptosis of PMN in tissue to alliviate the injury of vascular endothelial cell and down-regulate the function of PMN; another could be infered that Celecoxib might decrease the level of TXA₂ and PGI₂ and correct the imbalance between TXA₂ and PGI₂ to improve microcirculation disturbance in pancreas and lung tissue; the other could be infered that Celecoxib might control considerable amplifying influence on the systemic inflammatory response by altering key components of the inflammatory cascade via suppression of NF-κB activation to inhibit over inflammatory response.