T1831

Effect of the Selective Cyclooxygenase 2 (COX-2) Inhibitor On theExperimental Acute PancreatitisJae Hyuk Do, Seung Mun Jung, Chang Hwan Choi, Hyun Woong Lee, Hyoung-Chul Oh,Jeong Wook Kim, Jae G. Kim, Sae Kyung Chang

Background/Aims: Acute pancreatitis (AP) is an inflammatory disease. The patterns of acinarcell death, necrosis or apoptosis, is important to determinate the disease severity in AP.COX-2 plays an important role in inflammation. In AP, COX-2 inhibitor can amelioratesthe disease severity by decreased the inflammatory response. COX-2 inhibitor inducesapoptosis in the several cancers including pancreatic cancer. However it is not establishedhow COX-2 inhibitor affects to acinar cell death pattern in AP. The aims of this study is toinvestigate the effect of selective COX-2 inhibitor (NS-398) on the disease severity and thepatterns of acinar cell death on the experimental AP in male Swiss webster CD-1 mice.Methods: The mice were divided into four groups: saline injection group, cerulein injectiongroup, NS-398 with saline injection group and NS-398 with cerulein injection group. APwas induced by 8 hourly intraperitoneal injections (i.p.) of cerulein (50μg/kg). NS-398(10mg/kg) was given i.p. just before the cerulein or saline (0.5 mL) injection. The serumamylase was measured. The pancreas histology, necrosis, edema, inflammatory cell infiltra-tion, and vacuoles, was examined with H&E stain. The apoptosis of acinar cell was examinedwith Terminal deoxynucleotidyl transferase mediated X-dUTP nick-end labeling (TUNEL)staining. COX-2 protein expressions were examined with Western blot and immunohistoch-emical staining (IHC). Caspase-3 and Poly(ADP-ribose) polymerase-1 (PARP-1) proteinexpression were examined with Western blot. Results: The COX-2 protein expression wassignificantly reduced in NS-398 with cerulein injection group compared to cerulein injectiongroup in IHC and Western blot. The serum amylase level was not different between ceruleininjection group and NS-398 with cerulein injection group. Acinar cell necrosis, edema,inflammatory cell infiltration and vacuoles were significantly reduced in NS-398 with ceruleininjection group compared to cerulein injection group. Apoptosis is significantly increasedin NS-398 with cerulein injection group compared to cerulein group. Active caspase-3protein expression is increased in NS-398 with cerulein injection group compared to ceruleininjection group. PARP-1 protein expression was not different between cerulein injectiongroup and NS-398 with cerulein injection group. Conclusion: The selective COX-2 inhibitorameliorates the disease severity in cerulein induced experimental AP mediated by inductionof apoptosis.

T1832

Acute Biliary Pancreatitis Is Associated with Enhanced EnterohepaticCirculation of Bile Salts in a Rat ModelNiels G. Venneman, Jose J. ter Linde, L. Paul van Minnen, Andre Verheem, WillemRenooij, Louis M. Akkermans, Karel J. Van Erpecum

Acute pancreatitis leads to disturbed intestinal motility, small bowel bacterial overgrowthand bacterial translocation. Intestinal bile salts on the other hand enhance small intestinalmotility and prevent bacterial translocation, most likely by activation of intestinal bile saltresponsive nuclear receptor: Farnesoid X Receptor (FXR). Activation of FXR leads to inductionof intermediate SHP, and subsequently of intestinal barrier enforcing genes. Also, FXRactivation enhances secretion of the ileal hormone fibroblast growth factor-15 (FGF15),leading to a negative feedback signal of biliary and pancreatic secretion. However, effectsof acute pancreatitis on enterohepatic circulation of bile salts are unknown. Therefore,we explored enterohepatic bile salt circulation and expression of FXR-mediated bile salthomeostasis genes in liver and ileum, in a rat model of acute biliary pancreatitis. Acutepancreatitis was induced in male Sprague-Dawley rats by anterograde infusion of 0.5 mL15mM TDC (or PBS in control rats) into the bile duct (van Minnen, Pancreas 2006). Tocontrol for exogenous bile salts, control rats received 0.5 mL 15mM TDC intraduodenally,whereas pancreatitis rats received intraduodenal PBS. Bile salt hepatic secretion, intestinalcontent, fecal excretion, total pool size and serum level were determined 72 hrs after bileduct infusions. Also, mRNA expression of genes involved in uptake, synthesis, signalingand secretion of bile salts, in liver and ileum, was determined by qRT-PCR. Hepatic bilesalt secretion markedly increased in pancreatitis rats (449 ± 119 vs 90 ± 24 μmol/hr/kgbody weight in control rats, P=0.03), Muricholate was the major bile salt (68 ± 4 vs 46 ±4% in control rats, P=0.01). Intestinal bile salt content, serum level, and fecal secretion werenot significantly different. On the other hand, total bile salt pool size was much higher inpancreatitis rats (468 ± 23 vs 255 ± 81 μmol/kg body weight in control rats, P=0.04). IlealmRNA expression of ASBT, FXR, OSTα, OSTβ, MRP3, or FGF15 did not differ. Also, hepaticmRNA expression of NTCP, OATP1, OATP3, FXR, CYP7A, CYP27, BSEP and MRP2 wascomparable in pancreatitis and control rats. However, hepatic mRNA expression of MRP3was 1.5-fold higher in pancreatitis rats (P=0.04 vs control rats). Conclusions: In a rat modelof acute biliary pancreatitis, enterohepatic circulation of bile salts is enhanced.We hypothesizethat pro-inflammatory cytokines during acute pancreatitis interfere with FXR-mediated path-ways since the mRNA expression of bile salt homeostasis genes in liver and ileum areunaffected, which is inappropriate in case of enhanced bile salt circulation.

T1833

Chronic Pancreatitis Causes Up-Regulation of Brain-Derived NeurotrophicFactor (BDNF) in Sensory Neurons and Spinal CordMichael S. Hughes, Mohan Shenoy, Kshama R. Mehta, Pankaj J. Pasricha

Background/Aims: Pain in chronic pancreatitis, though common, remains difficult to treateffectively due to our lack of understanding of its underlying neurobiological mechanisms.We have previously shown that acute pancreatitis results in the up-regulation of severalneurotrophins including brain derived neurotrophic factor (BDNF) in sensory neurons.BDNF is a powerful modulator of short and long-term synaptic efficiency and is thoughtto exert its effect on TrkB receptors on second order neurons in the spinal cord. PancreaticBDNF expression has also been noted in specimens from patients with chronic pancreatitisand found to correlate with pain scores. The overall aim of this study is therefore to examine

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the role of BDNF in the pathogenesis of pain in an experimental model of chronic pancreatitis.Methods: Pancreatitis was induced by retrograde infusion of Trinitrobenzene Sulfonic Acid(TNBS) into the pancreatic duct of rats; control animals received an equal volume ofvehicle only. Twenty one days post-injection, pancreas specific pooled DRGs (T9-13) andcorresponding spinal cord segments were rapidly harvested from TNBS (n=5) or control(n=5) rats and snap frozen in liquid nitrogen. Cervical and lumbar DRGs that contain littleor no pancreatic afferents served as specificity controls. BDNF was quantified from theDRG and spinal cord extracts using an ELISA kit and was normalized to the total proteinconcentration and expressed as picograms of BDNF per microgram of total protein. Results:BDNF levels in DRGs (T9-13) were significantly higher in TNBS rats (0.031±.002) comparedto controls (0.022±.002, p<.006). Likewise, BDNF levels in corresponding thoracic spinalcord were significantly higher in TNBS rats (0.048±.007) compared to controls (0.029±.004,p<.05). In contrast BDNF levels in cervical and lumbar DRGs, as well as correspondinglumbar spinal cord segments were not significantly different between TNBS and controlrats (0.034±.007 vs 0.027±.002; 0.019±.004 vs 0.015±.001; 0.044±.006 vs 0.027±.003).Conclusion: BDNF is up-regulated in pancreas specific DRGs (T9-13) and spinal cord inTNBS rats in our rat model of chronic pancreatitis, supporting its role in mediating theneuronal sensitization seen in this condition. Studies are ongoing to assess the effects ofBDNF antagonism on the pain behavior in this model.

T1834

Role of XBP1 in the Protective Unfolded Protein Response to Limit ChronicEthanol-Induced Endoplasmic Reticulum Stress and Damage in the PancreasAurelia Lugea, Ilya Gukovsky, Samuel W. French, Fred S. Gorelick, Stephen J. Pandol

Accumulation of unfolded proteins in the ER triggers the Unfolded Protein Response. UPRfacilitates ER related protein folding and degradation alleviating ER stress. The transcriptionfactor XBP1 is a key mediator of UPR. XBP1 target genes are chaperones, protein disulfideisomerases (PDIs) that assist disulfide bond formation during protein folding, and proteinsin ER degradation pathways. XBP1 is essential for ER development in the exocrine pancreas.We showed that ethanol feeding induces a protective UPR in rodent pancreas by upregulatingXBP1. To characterize the protective mechanisms of XBP1, we studied heterozygous nullXBP1+/- mice and wild type mice fed control or ethanol diets using the intragastric modelfor 4weeks.We analyzed pancreatic damage byH&E and TUNEL staining and EM; pancreaticmRNA levels of XBP1 target genes by real time RT-PCR; XBP1 mRNA splicing, as an indexof activation by RT-PCR, and PDI, XBP1 and the autophagosome marker LC3 by Westernblotting. XBP1+/- mice fed control diet were healthy and displayed normal pancreas. Acinarcells isolated from XBP1+/- mice were more susceptible to CCK induced ER stress than wildtype controls as indicated by persistent PERK and eiF2α phosphorylation and 20% decreasein amylase secretion suggesting defects in the secretory machinery of these cells. In thiscontext, mRNA levels of the XBP1 target genes ERdj4 and EDEM decreased 40% in XBP1+/-mice. Wild type mice fed ethanol displayed minor pancreatic damage. In these animals,ethanol induced a protective UPR with 10% increase in the chaperone BIP and 20% splicingof XBP1 mRNA. As a result, protein levels of XBP1 and PDI increased 50 and 30% respectivelyin wild type mice fed ethanol. In contrast, ethanol failed to elevate XBP1 and PDI content inXBP1+/- mice. Interestingly, Western blot analysis suggested that ethanol induced irreversibleoxidation of PDI, a state that compromises PDI ability to rearrange disulfide bonds duringprotein folding. Thus protein folding was impaired in XBP1+/- mice fed ethanol due tolower levels of functional PDI. Moreover, XBP1 deficiency and ethanol feeding togetherdecreased mRNA levels of the isomerases Grp58, the pancreas-specific PDIp, and ERdj4 by40-50 %. In XBP1+/- mice fed ethanol, defective UPR led to disorganized and dilated ER,loss of zymogen granules, decreased amylase content, accumulation of autophagic vacuolesand 5-10% acinar cell death (necrosis and apoptosis). In conclusion, ethanol-induced oxidat-ive changes of key ER proteins such as PDI may provide the trigger for pancreatic ER stresswhich, if not attenuated by a normal UPR and XBP1 response, will lead to pancreatic patho-logies.

T1835

Alcohol Withdrawal Promotes Regression of Pancreatic Fibrosis via Inductionof Pancreatic Stellate Cell (PSC) ApoptosisAlain Vonlaufen, Phoebe Phillips, Zhihong Xu, Xuguo Zhang, Lu Yang, Ron Pirola,Jeremy S. Wilson, Minoti V. Apte

Background: We have previously shown that alcohol withdrawal leads to regression ofpancreatic injury and disappearance of fibrosis in an animal model of alcoholic chronicpancreatitis. However, the mechanisms underlying these processes remain unknown. Wepostulate that one of the mechanisms responsible for resolution of pancreatic fibrosis is thedisappearance of activated PSCs via increased apoptosis. Aims: 1) To compare the effectsof alcohol withdrawal and alcohol continuation for 7 days on pancreatic fibrosis and PSCapoptosis. 2) To assess the effects of alcohol ± LPS on pancreatic stellate cell (PSC) apoptosisIn Vitro. Methods: 1) SD rats fed isocaloric Lieber-DeCarli diets ± alcohol for 10 weeks werechallenged with LPS (3 mg/kg; 1 IV injection / week x 3 weeks). Alcohol-fed rats were theneither switched to a non-alcohol (control) diet or continued on alcohol for 7 days. Thusthere were 4 groups in each experimental set (n=6 rats/group) - control diet + LPS (CL),alcohol diet + LPS (AL), alcohol diet + LPS continued on alcohol for 7 days (AL+A), alcoholdiet + LPS switched to control diet for 7 days (AL+C). Pancreatic sections were assessed fori) fibrosis / collagen deposition (morphometry of Sirius Red staining); ii) PSC apoptosis(TUNEL staining) and iii) presence of activated PSCs (α smooth muscle actin (αSMA)staining)). 2) Cultured rat PSCs were exposed to ethanol 10mM (E10) ± LPS 1μg/ml (L1)for 48h and effects on induced apoptosis assessed by Annexin V, Caspase-3 and TUNELstaining. Results: 1) Withdrawal of alcohol (AL+C group) led to resolution of pancreaticfibrosis and increased PSC apoptosis (see Table). Both alcohol and LPS significantly inhibitedPSC apoptosis In Vitro : Annexin V staining - % of control (mean±SEM); E10 70±10.2; L145.01±11.8; E10+L1 38.43±9.38; p<0.03 vs Control and E10+L1 vs E10; n=3 separate PSCpreparations. Similar results were obtained for caspase-3 and TUNEL. Conclusions: 1)Withdrawal of alcohol significantly inhibits disease (acinar necrosis, inflammation and fib-rosis) progression and reverses pancreatic fibrosis via increased PSC apoptosis In Vivo. 2)

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