Study Of The Role Of NF-κB In Acute Necrotizing Pancreatitis Rat Model

Background and Objectives:Acute pancreatitis(AP) is a sudden inflammation of the pancreas which occurs suddenly and deteriorates quickly,severe case fatality rate is up to 50,however,it's pathogenesis has been obviously understood by now.Current study consider:in acute pancreatitis,a series of cascade reaction and "waterfull effection" caused by pro-inflammatory factor result in systemic inflammatory response syndrome(SIRS) and multiorgan functional disturbance symptom complex(MODS).Currently,it becomes focus research that how the abnormal expression of various kinds inflammatory factors or mediators effect acute pancreatitis's pathogenesis.Nuclear factor kappa B(NF-κB) is an important intracellular nuclear transcription factor,widely expressed in various cells, control the expression of many cytokines and play an important role in the regulation on cell proliferation,apoptosis and inflammation.Generous achievement show:proinflammatory factor,such as interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α), activate NF-κB and induce it's translocation from cytoplasm to nucleus in pancreatic acinar cell,thus NF-κB bind to situs in target gene and induce their transcription,then finally accelerate severity of acute pancreatitis.Pyrrolidine dithiocarbamate(PDTC),as special inhibitor of NF-κB,can block the expression of NF-κB and downstream cytokine after subcutaneous injection.Up to date,major research about AP's pathogenesis are based on rats' model.This topic is to explore the Effect of PDTC on expression of E-selectin and the activities of enzymes in oxidative stress in the pancreas of rats with ANP(acute necrotizing pancreatitis) through detecting the activity of NF-κB,MPO(myeloperoxidase),LPO(lipid peroxidase),NOS(nitric oxide synthase) and the changes of the E-selectin.Meathods.In first part of this experiment,rats model with ANP(including rats model with ANP +PDTC intervention)are induced by intraperitoneal injections of 20%L-arginine (totally 5 g/Kg,twice at interval of 1 h),and some items are detected to confirm these rats's model;In second part of this experiment,we show the effect of PDTC on expression of E-selectin and the enzymes about oxidative stress in rats' ANP.Thrity-six SD rats were assigned to 6 groups randomly and equally,that is,normal control,ANP,PDTC pretreatment group 1(1 mg/kg) and 2(100 mg/kg),and PDTC treatment group 1(1 mg/kg) and 2(100 mg/kg). PDTC pretreatment was preformed by subcutaneous injection of PDTC at 1 h before the first L-arginine's injection.For the rats in treatment groups,PDTC was given at 3 h after the first injection,the normal control are given intraperitoneal injection normal sodium instead of L-arginine.All rats were killed by taking suction of their blood at 24 h after the first injection of L-arginine.At the first part we observe below items:1)the morphous of rats' pancreas and the organs around pancreas were observed;2)the pancreatic tissue were dyed with haematoxylin-eosin and evaluated according to reformed Schmidt's standard;3)the serum amylase were detected by enzymic method.At the second part we observe below items: 1)Expressions of E-selectin were detected through immunohistochemical method;2) the activity of NF-κB-DNA binding were detected by means of the electrophoretic mobility shift assay(EMSA);3)the activity of MPO,LPO and NOS were detected by colorimetric method.Results.All rats were killed at 24 h.Then,in ANP groups,the rats can be observed such symptom after dissection as flatulence of gastrointestinal tube,part of intestinal necrosis,a large number of bloody ascites,swelling of the pancreas congestive obviously, visible and disseminated necrotic foci,there is peripancreatic ANPonif spot,the score of pathological specimens from pancreas were higher than the normal control(P＜0.05);and serum amylase values in ANP groups rise significantly vs the normal control(P＜0.05),these above items show the rats' ANP induced by L-arginine was successed.During 1 mg/kg PDTC intervention group,especially PDTC pretreatment group,the morphous of rats' pancreas and the peripancreatic organs form of a marked improvement,the pathological specimens' score and the serum amylase values decline significantly vs ANP groups.However,there is no statistical significance in the pathological specimens' score and the serum amylase values between 100 mg/kg PDTC intervention groups and ANP group(P＞0.05).In ANP groups and L-arginine plus PDTC intervention groups,1)The rat pancreatic expression of E-selectin,NF-κB,MPO,LPO,iNOS(inductible nitric oxide synthase) were higher than the normal control(P＜0.05),all above items were moderately down-regulated in 1 mg/kg PDTC intervention groups(especially in 1 mg/kg PDTC pretreatment group);2)the activity of cNOS(constitutive nitric oxide synthase) was lower than the normal control(P＜0.05),and 1 mg/kg PDTC intervention groups' cNOS activity near normal levels;3)100 mg/kg PDTC have little effect on all above items(P＞0.05).Conclusion:1)Rat model of ANP can be built by intraperitoneal injections of 20%L- arginine(totally 5 g/kg,twice at interval of 1 h);2)1 mg/kg PDTC intervention,especially 1 mg/kg PDTC pretreatment,can down-regulate the expression of E-selectin and depress the activity of the enzymes about oxidative stress by blocking the translocation of NF-κB,thus alleviate ANP' inflammation and show protection on ANP in rat.However,the 100 mg/kg PDTC show little protection on ANP.