The Therapeutic Effect And Mechanisms Of PS-341 For The Treatment Of Severe Acute Pancreatitis By Modulating The Ubiquitin-proteasome Pathway

Bankgroud and aims:severe acute pancreatitis(SAP)is a life threatening disease,which occurs in around 15%-25%of all patients with acute pancreatitis.Despite significant recent advances in supportive therapy and intensive care,the mortality rate still remains as high as 10%-30%.The exact mechanisms by which diverse etiological factors induce an attack are still unclear,but once the disease process is initiated,the severity of the disease is largely determined by a complex network of activated inflammatory mediators.Now it is believed that the progression of acute pancreatitis developed in three phases:local inflammation of the pancreas, a generalized inflammatory response,and finally sepsis with multiple organ damage.Recreuitment and activation of various inflammatory cells leads to acinar cell injury and causes an elaboration of various proinflammatory mediators such as tumour necrosis factor,IL-1,IL-2,IL-6 and other chemokines and anti-inflammatory factors such as IL-10 and IL-1 receptor antagonist.An imbalance in the pathway results in a sustained or exaggerated systemic inflammatory response syndrome, followed by widespread tissue damage and death from multiple organ dysfunction syndrome and septicaemia.Single cytokine antagonist therapy has not yes proven to be of clinical benefit in acute pancreatitis.Nuclear factor-κB(NF-κB)is a pluripotent transcription factor which plays an important role in regulating the host inflammatory,immune and anti-apoptotic responses.Recently,it has been demonstrated that NF-κB is rapidly activated after the induction of acute pancreatitis by cerulein. Under basal conditions,NF-κB is sequestered in an inactive form in the cytoplasm through an association with the inhibitory protein IκBα.When stimulated by inflammatory stress such as TNF-α,IL-1 or lipopolysaccharide(LPS),IκBαis phosphorylated,ubiquitylated,and subsequently degraded by the 26S proteasome.The degradation of IκBαallows NF-κB to translocate into the nucleus to direct the transcription of target genes encoding cytokines,chemokines,anti-apoptotic proteins and adhension molecules which are required for neutrophil adhension and migration.Therefore,inhibition of the ubiquitin-proteasome pathway may represent a novel therapeutic strategy for this severe disease.PS-341,also known as bortezomib,is a dipeptide boronic acid analogue that inhibits the activity of the 26S proteasome.It forms a covalent, reversible complex with the proteasome and is much more potent than its peptide aldehyde predecessors(such as MG-132 and calpain inhibitor-1). PS-341 has already been used in clinical trials for the treatment of multiple myeloma and other solid tumors due to its ability of blocking IκBαdegradation and subsequent NF-κB activation and the induction of cell apoptosis.In vivo drug distribution studies demonstrated that PS-341 accumulated in several gastrointestinal tissues,including the pancreas.This fact,combined with the recent implication of NF-κB activation in severe acute pancreatitis prompted us to investigate the effects of PS-341 on the development of severe acute pancreatitis.In the present study,using various experimental techniques,including small animal positron emission tomography(micro PET),we show that PS-341 has efficiently attenuated the severity of acute pancreatitis induced by cerulein and LPS in mice.This protective effect in vivo is associated with the inhibition of NF-κB activation,increased apoptosis of acinar cells and decreased neutrophil infiltration within the pancreas.Chapter 1 the therapeutic effect of PS-341 on severe acute pancreatitis Materials and methods:one hundred and forty four female ICR mice were used in this study altogether.Sixty four mice were used in the PS-341 treatment group,forty mice were used in the group of severe acute pancreatitis without PS-341treatment and forty were used in the normal group.Severe acute pancreatitis was induced by seven intraperitoneal injections of 50ug/kg cerulein at hourly intervals;lipopolysaccharide at a dose of 10mg/kg was administered inraperitoneally 5 hours after the first injection of cerulein.30 minutes before the administration of lipopolysaccharide,mice in the PS-341 treatment group were treated intraperitoneally with various doses of PS-341(0.1,0.2,0.5 and 1.0 mg/kg) in order to investigate the most effective therapeutic dose.The control groups were treated with an equal volume of vehicle.At different time point(8,12 or 24 hours after the first injection of cerulein),mice were anesthetized with 50mg/kg pentobarbital.Blood samples were collected from right jugular veins,allowed to clot and centrifuged at 3000 g for 5 minutes.The supernatant was collected and frozen at -20℃until assayed. Pancreas were quickly removed and either placed in formalin for histology or frozen in liquid nitrogen and stored at -80℃until use.Results:Injection of cerulein and lipopolysaccharide induced severe acute pancreatitis,the serum amylase,CRP,LDH,IL-1βand IL-6 concentrations and pancreatic MPO activity were significantly elevated,histological investigation revealed pancreatic interstitial edema,leukocyte infiltration and acinar cell damage.PS-341 treatment significantly inhibited NF-κB activation,while the acinar cell apoptosis was significantly enhanced, resulting in the improved parameters such as serum amylase,C-reactive protein,lactate dehydrogenase,interleukin-1β,interleukin-6 and pancreatic myeloperoxidase activity.Accordingly,the pancreatic damage was also markedly reduced.Conclusions:these observations demonstrate that PS-341 has significantly reduced the severity of acute pancreatitis induced by cerulein and lipopolysaccharide in mice.This protective effect is associated with the inhibition of NF-κB activation and increased acinar cell apoptosis within the pancreas.Chapter 2 the potential value of ¹⁸F-FDG-PET in the early diagnosis and evaluating the effects of interventions for severe acute pancreatitis Materials and methods:thirty-three female ICR mice weighing 20g±1g were used in this study.There are 11mice in each group.The induction of severe acute pancreatitis was in accordance with that reported in previous studies.30 minutes before the administration of lipopolysaccharide,mice in the PS-341 treatment group were treated intraperitoneally with 0.5mg/kg PS-341,while mice in the control groups were treated with an equal volume of vehicle.8 hours after the first cerulein injection,mice were anesthetized with 50mg/kg pentobarbital.3 mice were used for the ¹⁸F-FDG-PET examination and another 8 mice were used for the detection of pancreatic MPO activity.Results:the pancreatic appearance coulden't be seen in the PET image in normal group;while in those mice with severe acute pancreatitis,the ¹⁸F-FDG uptake was significantly increased,PS-341 treatment significantly reduced the uptake of ¹⁸F-fluorodeoxyglucose within the pancreas.MPO assay also demonstrated that induction of severe acute pancreatitis elevated the activity of MPO,PS-341 treatment significantly reduced MPO activiation.This result was in accordance with that of the Positron emission tomography.Conclusions:¹⁸F-fluorodeoxyglucose - Positron emission tomography could be a sensitive and promising means in evaluating the therapeutic effect and adjusting medical interventions in pancreatitis.