The Role Of The Transcription Factor NF-kB Signaling Pathway In The Development Of Pancreatitis

Introduction:Pancreatitis is an inflammatory disease of the pancreas that causes severemorbidity and mortality. Specific and effective prevention and therapies for thisdisease are lacking largely because of a lack of understanding of the early cellularevents important in its pathophysiology. The transcription factor NF-kB is activatedearly in acinar cells during acute pancreatitis and increases transcription of multipleproinflammatory genes.With these complexities in the NF-kB signaling pathway, it isnot surprising that controversy has arisen in the literature concerning the role of thispathway in pancreatitis. Most early studies suggested that NF-kB was critical for theinitiation of pancreatitis. For example,pharmacological inhibition of NF-kB activationresulted in an amelioration of the disease However, because none of the inhibitorsutilized were highly specific, these data were not definitive. More definitive datacame from the observation that pancreatitis was induced by pancreatic expression ofp65by adenoviral-mediated gene transfer and transgenic expression active IKK2Thus, several studies supported that NF-kB increased pancreatitis severity as mightbe expected from its role in other inflammatory diseases.however, not all studies inthe pancreas supported NF-kB this concept.In contrast to the several other studies of its type, one early study involvingpharmacological inhibition of NF-kB during caerulein-induced pancreatitis suggestedthat NF-kB inhibition perpetuated acute pancreatitis. Also, in contrast to another studyby the same investigators, a different study on the effects of transgeneic expression ofactive IKK2reported that it had no ability to induce pancreatic damage. More recently,it has been reported that after genetic elimination of NF-kB signaling in pancreaticcells mice developed a more severe pancreatitis compared to those with wild-typeNF-kB. Thus, from these studies it has been suggested that NF-kB rather thancontributing to severity is in fact protective against pancreatitis. These apparentlyconflicting and contradictory outcomes have rendered the role of NF-kB inpancreatitis controversial.Objective:1.The establishment and utilization of the pancreas-specific NF-kB transgenic and knockout mice.To set up pancreatitis model and simulate the disease process2. In the process of pancreatitis, NF-kB gene mouse phenotype and preliminary studyof NF-kB regulation mechanism of pancreatitis process3. In-depth study of NF-κB signaling pathway and potential therapeutic targets forparticipation in the process pancreatitisMethods:1. Plasmid Construction1.1pGL3-P4Hα1Promoter Plasmid ConstructionA full-length cDNA encoding p65was excised from a pBluescript SK+plasmid as anXhoI/XbaI restriction fragment (from Dr. G. Nabel,). This was ligated behind aloxp-EGFP-loxp fragment and cloned into the EcoR I site of the pCAGGS vector(provided by Dr. Miyazaki). The pCAGGS vector contains a CMV promoter and achicken beta actin intron which has been proven to increase the level of expression16.The CMV-loxp-EGFP-loxpp65cassette was isolated by SpeI and Hind III digestionand submitted for pronuclear injection to generate floxed-p65mice. Transgenic micewith expression of constitutive active IKK2(floxed-IKK2) were developed using thesame strategy. Active IKK2construct was a gift of Dr. Michael Karin.TransgenicGFP expression in pups was visualized under a UV lamp.2. Animal ModelsTo build double transgenic triolp transgenic acinar cell specific pancreatitis micemodels.Intraperitoneal injection of caerulein50ug/kg or L-arginine4g/kgintraperitoneal injection to build a mouse acute pancreatitis model3. Immunohistochemical AnalysispSTAT3,pERK,F4/80,Caspase3,CD45,Ki-67,p65in atherosclerotic lesions wereidentified using appropriate primary antibodies.4. RT-PCRThe expression levels of IL-1b,TGF-b,Fibronectin,a-SMA,IL-6,TNF-a,RPS6mRNA were measured by RT-PCR after RNA extraction and reverse transcription. 5. EMSAFor the antibody-based supershift assay, antibodies against transcription factors wereadded to a mixture of biotin-labeled oligonucleotide probes and nuclear proteins.6. Western BlotAfter incubation with the primary and secondary antibodies and exposure, the proteinexpression levels of a-SMA,Amylase,pSTAT3,BCL2,LC3,Beclin1,ATG7,ATG12andGAPDH were determined7. Statistical AnalysisData were presented as mean±standard errors of the means and analyzed with SPSS13.0software and a p value<0.05was considered statistically significant.Results:1.Transgenic expression of p65in pancreatic acinar cells leads to compensatorychanges with no obvious phenotype.2.Transgenic expression of p65increased NF-kB activity and the severity ofcaerulein-induced3. Pancreatic specific expression of active IKK2induces pancreatitis.4Co-expression of IKK2and p65generates a severe form of pancreatitis withinflammation and autophagy.5. chloroquine administration significantly increased the severity of pancreatitis inthese mice6.IKK2-KO aggravated pancreatitis in different pancreatitis models7pSTAT3mediated expression of Bcl-2involved in IKK2-KO-induced acutepancreatitis8Inhibition of STAT3IKK2-KO mice will diminish the severity of pancreatitisConclusion:Take together, we found that in addition to the well established role of NF-kBcontrolling proinflammatory and anti-apoptotic genes expression, autophagy regulated by NF-kB components is also implicated in the development pancreatitis. Therefore,the NF-kB pathways directly impacts both pro-and anti-severity processes.Nonetheless, overall our data, and that in the literature, suggest that NF-kB activationincreases the severity of acute pancreatitis and can induce chronic pancreatitis. Thusinhibition of NF-kB activity would be appropriate and should be beneficial forpancreatitis prevention and treatment. pSTAT3mediated expression of Bcl-2involvedin IKK2-KO-induced acute pancreatitis.Inhibition of Stat3may provide a noveltherapeutic approach for pancreatitis.