Study On The Role And Mechanism Of CyPA In Severe Acute Pancreatitis

BackgroundAcute pancreatitis(AP) is a potential life-threatening illness,of which the clinical presentation ranges from a mild edematous, self-limiting disease with good prognosis to severe necrotizing inflammation.In patients with severe acute pancreatitis(SAP),multiple organ failure(MOF) is responsible for early disease mortality while sepsis supervenes later and remains the major cause of death. Accumulated evidences suggested that activated polymorphonuclear granulocyte(PMN) that released various pro-inflammatory cytokines, play a pivotal role in acute pancreatitis.Previous study have shown that tissue injury is PMN-dependent and protease and oxygen-radical-mediated in SAP.In particular,the severity of tissue injury can be mitigated by PMN depletion or by blocking PMN adhesion and infiltration with antibodies.Cyclophilin A(CyPA) is a ubiquitously distributed intracellular protein belonging to the cyclophilin family.However,CyPA plays an important role in the course of inflammatory responses.Indeed,elevated levels of CyPA have been reported in several different inflammatory diseases,including sepsis,acute lung injury,rheumatoid arthritis,and vascular smooth muscle cell disease.In the case of rheumatoid arthritis,a direct correlation between levels of CyPA and the number of PMN in the synovial fluid of rheumatoid arthritis patients was reported.In vitro studies have shown that extracellular CyPA is a potent chemoattractant for PMN.Furthermore,CyPA can induce a rapid inflammatory response, characterized by PMN influx,when injected in vivo.Previous studies have shown that CD 147,a typeⅠtransmembrane protein belonging to the immunophilin family,as the mam signaling receptor for extracellular CyPA and have shown that the presence of CD147 is essential for cyclophilin-mediated chemotaxis and signaling to occur in human leukocytes.Some findings demonstrated that CyPA-CD147 interactions do indeed contribute to the recruitment of PMN into tissues during an inflammatory response.Furthermore, blocking these interactions significantly inhibits PMN influx into inflamed tissues,providing a potentially novel therapeutic approach for the alleviation of inflammation-mediated diseases.No study has been found on the role of CyPA in SAP.ObjectiveIn our study,we expect to investigate the potential role and the underlying mechanisms of elevated serum CyPA involved in PMN infiltration in SAP.This article was composed of three parts:In part one, we aim to investigate the expression of serum CyPA in experimental SAP, and the impossible links between serum CyPA expression and the level of pancreas injury and PMN infiltration.In part two,we studied the expression of serum CyPA expression and investigated the value of evaluating the severity and prognosis of SAP patients.In part three,we demonstrated the phenomenon of the PMN delayed apoptosis and we further investigate the mechanism of CyPA involved in PMN delayed apoptosis in SAP.MethodsPart oneAdult male Sprague-Dawley(SD) rats were randomly divided into three groups:normal control group(NC group),mild acute pancreatitis group(MAP group) and severe acute pancreatitis group(SAP group). Animals in NC group received laparotomy only,rats in MAP group were induced by the retrograde injection of bilio-pancteatic duct with 0.5% sodium taurocholate(1.0mL/kg),rats in SAP group were induced by the retrograde injection of bilio-pancteatic duct with 5%sodium taurocholate (1.0mL/kg).Rats were sacrificed at 3h,6h,12h after induction,the blood serum,pancreas and lung tissue of rats were harvested.Serum amylase (AMS) activity was detected with biochemical analyzer;the serum level of CyPA was detected by ELISA method;myeloperoxidase activities (MPOs) of pancreas and lung tissue were enzymohistochemistrically detected by colorime try,using a BECKMAN DU640 model spectrophotometer.The pancreas and lung samples were removed for microscopic histological examination to score lesions.Part two69 patients with AP including 41 MAP and 28 SAP from December, 2006 to April,2008 were included in the study.Serum CyPA was detected by ELISA and peripheral PMN was isolated and CyPA mRNA and protein were detected by RT-PCR and Western Blot in 48h after disease onset,serum C-reactive protein(CRP) was measured at day 3 after disease onset.Other case data were collected including CT and APACHⅡscore and organ failure.The value of the CyPA in evaluating the disease severity and prognosis of patients with SAP was investigated.Part three30 patients with AP including 15 MAP and 15 SAP and 15 healthy volunteers from December,2006 to April,2008 were included in the study.Venous blood was collected in 48 hours after disease onset,CD 147 on PMN surface was detected by flow cytometry;to isolate peripheral PMN and detect its apoptosis by flow cytometry,using PT-PCR and Western Blot,XIAP expression was detected in PMN after coculture with human recombined CyPA or CD 147 mAb or CsA for 6 hs.ResultsPart one1.The serum AMS activities in SAP and MAP groups,were significantly increased in comparison with NC group after sodium taurocholate induction at each time point(3h,6h,12h),and the pathological scores of pancreas tissue injury(characteristic as edema, hemorrhage,necrosis and inflammation) were markedly increased (P＜0.001),the pathological scores of pancreas tissue injury(characteristic as edema,hemorrhage,necrosis and inflammation) in SAP group were markedly increased when compared with MAP group(P＜0.001). Therefore,in present study,rat models for SAP and MAP were well induced by standard retrograde infusion of bilio-pancreatic duct with 5% and 0.5%sodium taurocholate solution.2.Compared to NC and MAP groups,serum CyPA was increased in SAP group(P＜0.001) at each time point(3h,6h,12h);compared to MAP group,serum CyPA in SAP group was much higher(P＜0.001).There was high correlation between serum CyPA level and severity of pancreas damage.3.A great quantity PMN infiltrated pancreas and lung tissues in SAP group,MPOs activities of pancreas and lung were highly increased in SAP group compared to MAP and NC groups(P＜0.001).There were high correlations between serum CyPA and MPOs of pancreas and lung tissues. Part two1.Compared to NC group,serum CyPA and CRP_-72h were increased in MAP and SAP groups(P＜0.001);compared to MAP group,while serum CyPA in SAP group were much higher(P＜0.001).There was high correlation between serum CyPA and CRP_-72h(r=0.754,P＜0.001).2.Compared to no organ failure group,serum CyPA in organ failure group was elevated(P＜0.001);Compared to no pancreas necrosis group, serum CyPA in pancreas necrosis group was elevated(P＜0.001).3.Compared to CRP_-72h,serum CyPA can provide better information in evaluating the severity and prognosis of disease(P＜0.001).Part three1.The apoptosis of peripheral PMN from SAP group was delayed compared to NC group(P＜0.001) and MAP group(P＜0.001).2.CD 147 expression was detected on peripheral PMN from each group.Compared to NC group,PMN from MAP group and SAP group highly expressed CD 147(P＜0.001) and the expression CD 147 of SAP group was much higher than that of MAP group(P＜0.001).3.The expression of XIAP mRNA and protein in peripheral PMN from SAP group were much increased compared to NC group(P＜0.001) and MAP group(P＜0.001).4.The tendency of peripheral PMN apoptosis was bilateral after various concentration of CyPA(from 0ng/mL to 1000ng/mL) stimulation for 6hs.When the concentration was no more than 200ng/mL,CyPA inhibited the PMN apoptosis and the ability of inhibition was increased with the concentration increasing,while concentration was more than 200ng/mL,the ability of inhibition of CyPA on PMN was decreased.The XIAP expressions including mRNA and protein tendency were in accordance with apoptosis.5.The XIAP expressions of peripheral PMN including mRNA and protein from SAP group were markedly increased compared to NC group and MAP group after CyPA(200ng/mL) stimulation for 6 hs.6.CsA and CD147mAb can block the inhibition effect of CyPA (200ng/mL) on peripheral PMN.We presume that CyPA inhibits PMN apoptosis through interacting with CD 147 by increasing XIAP expression.ConclusionOur study demonstrated that serum CyPA was elevated in SAP, which may regulate PMN infiltrate,and can be an early marker for evaluating the severity of disease and prognosis.Furthermore,CyPA interacting with CD 147 on the surface of PMN,and inhibited PMN apoptosis through increasing XIAP expression and may play a vital role in PMN infiltration in SAP.