The Protective Effect Of PS-341 In Severe Acute Pancreatitis-associated Lung Injury By Inhibiting Neutrophil Recruitment

Severe acute pancreatitis(SAP) is a serious disease with highmorbidity in clinic. For the sudden occurring and rapid progressing, italways leads to systemic inflammatory response syndrome and distant organinjury. Acute pancreatitis associated lung injury(APAL) is the most frequentand potentially the most serious. Acute Pancreatitis-associated lung injurythat manifests itself clinically as acute respiratory distress syndrome ispresent in up to75% of patients with severe acute pancreatitis, and 30% -50% patients with this complication will die. Finding a more effective drugto reduce the high mortality of SAP is a serious task for us.The development of SAP involves a complex cascade of events. Thecurrent widely accepted theory is that activated pancreatic enzymes initiateautodigestion and pancreatic injury, the activated NF-kB is important for theactivation of pancreatic enzymes. The strong local inflammatory responsemay lead to a systemic inflammatory response syndrome. Excessivesystemic inflammatory response syndrome may lead to distant organ damageand multiple organ dysfunction syndrome.The lung is the first and the mostcommon involved organ. It has been proved that neutrophil and adhensionmolecules are the important mediators for acute pancreatitis-associated lunginjury. So reduced neutrophl infiltration and adhension molecules may relieve pancreatitis-associated lung injury.PS-341, also known bortezomib, is the only proteasome inhibitorlicensed to be applied in the clinic. PS-341 can inhibit the activation ofNF-kB, so PS-341 can inhibit the release of cytokins and adhensivemolecules. Activated NF-kB is the main pathogenisis inpancreatitis-associated lung injury. So PS-341 may be a promising drug toameliorate pancreatitis-associated lung injury.In this study, SAP was induced by intraperitoneal injection of celulin(50ug/kg) seven times, at 1 h intervals, and a single i.p. injection oflipopolysaccharide (10mg/kg) 5 hour after the initial cerulin injection.PS-341(0.5mg/kg) or DMSO (50%) was injected i.p. 1/2 hour before thesixth injection of cerulein. Two hour after the last injection of cerulin, ratswere anesthetized with 50mg/kg pentobarbital. We harvested serum foramylase and CRP and LDH test. The pancreas and lungs were also harvestedfor pathological examination and the level of MPO and adhension molecules(ICAM-1, E-selectin, P-selectin). Their differences between groups wereestimated to find whether PS-341 can protect pancreatitis-associated lunginjury in mice.Materials and methodsAnimals: Young female ICR mice, provided by the animal carecommittee of Zhejiang university, weighing 19～21g were used for this study.Groups: The rats were randomly divided into control group (n=20), SAP+DMSO group(n=20), SAP+PS341 group(n=20).Animal model: SAP was induced by seven intraperitoneal injections of50ug/kg celulein at hourly intervals, 10mg/kg LPS was administeredintraperitoneally 5 hours after the first injection of cerulein. 30 minutesbefore the administration of LPS, mice were treated intraperitoneally eitherwith 0.5mg/kg PS-341(dissolved in 50% DMSO) or with an equal volume of50% DMSO (0.2ml, diluted by saline). Control mice received 50% DMSOand physiological saline instead of cerulein and LPS in the same manner.The animals were allowed to drink freely but no foods.Preparation of samples: 2 hours after the last cerulein injection, themice were anesthetized with 50mg/kg pentobarbital. Blood samples werecollected from right jugular veins, the supernatant was collected and frozenat -20℃until assayed. Pancreas and lungs were quickly removed and eitherplaced in formalin for histology or frozen in liquid nitrogen and strored -80℃until use.Biochemical measurements: The serum levels of amylase, C-reactiveprotein(CRP) and lactate dehydrogenase(LDH) were dectected using amedical automatic chemical analyzer. Lung myeloperoxidase (MPO) activity,as a marker of tissue neutrophil infiltration, was measured from the opticaldensity (at 460 nm) changes resulting from decomposition of H2O2 in thepresence of o-dianisdine.Histological examination: sections of the specimens of pancreat and lung were stained with HE, and then observed under a light microscopeRT-PCR for the detection of adhension molecules (ICAM-1,E-selectin,P-selectin) : total RNA from lung was extracted , polymerasechain reaction products were analyzed.ResultsLevels of amylase and CRP and LDH:Amylase and CRP in serum increased significantly in AP group as comparedwith normal controls (p<0.05)Amylase and CRP in serum decreased significantly in AP+ps-341 group ascompared with AP group (p<0.05)Light microscopic examination:Pancreas:AP+ps341 group: mucosal edema moderately, focal necrosis andinflammatory cell infiltration damage diminished remarkably compared withAP group: mucosal edema obviously, massive necrosis, epithelialdegeneration, hemorrhage and inflammatory cell infiltration were revealedin mucosal or submucosaNormal control: normal or a mild degree of interstitial edema, pancreaticacinus remain complete, no hemorrhage or necrosis, no inflammatory cellinfiltrationLung:AP+ps341 group: interstitial edema mediately, inflammatory cell infiltration decreased compared with AP group, no alveolar hemorrhage and hyalinemembranesAP group: interstitial edema obviously, alveolar hemorrhage andinflammatory cell infiltration, hyaline membranes were presentNormal control: no interstitial edema, no hemorrhage, few inflammatory cellinfiltration, no hyaline membranesLevels of MPO in tissues:MPO in tissues increased significantly in AP group as compared with normalcontrols (p<0.05)MPO in tissues decreased significantly in AP+ps-341 group as comparedwith AP group (p<0.05)Levels of adhension molecules(ICAM-1,E-selectin,P-selectin) :adhension molecules increased significantly in AP group as compared withnormal controls (p<0.05)adhension molecules decreased significantly in AP+ps-341 group ascompared with AP group (p<0.05)Conclusions1. PS-341 inhibited the increases in serum amylase and CRP and LDH;2. PS-341 treatment improved pancreatic and lung histology;3. PS-341 reduced the level of MPO; 4. PS-341 reduced the level of ICAM-1,E-selectin,P-selectin5. PS-341 succeed to ameliorate severe acute pancreatitis -associated lunginjury in this rat model...