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NF-kB inhibition by pyrrolidine dithiocarbamate attenuates experimental acute pancreatitis
incubated with cholecyslokiinn-octapeptide (CCK) Fallowing CCK treatment, Western immnoblotting (WB) and imnmnoprecipitation (1P) were periormed. Synthetic CCK-analogue caerulein (Co) at doses of 20 and 50btg&g was intraperitoneally injected to BALB/c mouse six times in every one hour to induce acute pancreatitis. Changes in serum amylase and his:ologic findings were examined The expressmn of RhoA, ROCK-2, E-cadherin, and Bca:coin were analyzed by WB [Results] 1) Pancreatic acinar model; CCK enhanced expression of RhoA, ROCK-2, E-cadherin and B-ca:cure from 3min and sustained up to 30 rain tollowing stimulation. IP demonstrated tbrmation ot :mmunocomplea (1C) of B-catenin with RhoA and E-cadherin. RhnA also formed IC with ROCK-2 by CCK-8 stimulation. 2) Pancreatitis model; In 20~tgjkg Cn it~[ection, slight interstitial edema and vacuolization of acinar cell were observed from 7 h following initial Cn injection. In 50 ~g/kg Cn injection, marked inters:toni edema and vacuolization were seen at 18 h. The level of serum amylase showed the peak at 12 h tollowing initial Cu injection. The expression of RhoA and ROCK-2 were detectable time-depeudemly. On the other hand, the expression of E-cadherin and B-catenin were detected fl'om 7 h tbllowing mtial rejection. Furthermore, specific ROCK-2 inhibitor, Y~27632 inhfhited the expression of not only ROCK-2 but also E-cadberin and B-catenin. IConclusion] Our resuhs suggested that RhoA-ROCK-2 signaling pathways interact with Ecadbenn and B-catenin in pancreatic acim and caerulein-induced pancreatitis.
the role of cyclooxygenase-1 (COX-l) and cyclooxygenase-2 (COX-2) in protective eftect of HGF administration in caerulein-induced pancreatitis (CIP). Methods: Acute pancreatitis was induced by s.c. infusion of caernlein (10 mcg/kg/h) for 5 h. HGF was administrated twice (30 min prior to caeruleni or saline infusion and 3 h later) at the dose 10 mcg/kg s.c. Activity of COX-1 and COX-2 were inhibited by resveratrul and mfecoxib, respectively (10 mg/kg, ig. 1 h before saline or caerulein infusion), hnmediately after cessation of caemlein or saline infusion the pancreatic blood flow (PBF), plasma lipase activity, plasma interleukin1 (IL-1) and mterleukin- l0 (IL-10) concentration, cell proliferation, and morphological signs of pancreatitis were examined. Expression of COXq and COX-2 mRNA transcripts was determined by RT-PCR Analysis of COX production was made by Western Blot. Resuhs: Administration of HGF or CIP alone, or their combination were without effect on COX-1 mRNA expression in pancreatic tissue. Expression of COX-2 mRNA was increased by HGF and CIP. Maximal increase in COX-2 mRNA expression was observed, when HGF administration was combined with C1P. The similar effect was observed, when we studied influence of HGF and CIP on pancreatic COX-2 production determined by Western Blot. Administration of HGF without induction of CIP increased plasma IL-10 and this effect was abolished by COX-2 inhibitor-rofecoxib. Treatment with HGF, during development of CIP, increased plasma IL-IO and attenuated the pancreatic damage, what was manifested by: (a) histological improvement of pancreatic integrity, (bY the partial reversion of the drop in DNA synthesis and PBF, (c) the reduction in pancreatitis evoked increase in plasma ]ipase, and IL-1. Administmtinn of resveratrol and rofecoxib alone was without eftect on development of CIP. Combination of rofecoxib with HGF reduced HGF-evoked increase in plasma IL-10 and abolished protective effect of HGF against pancreatic damage in CIP. Resveratrol did not aflect a protective effect of HGF administration. Conclusions: (1) HGF administration reduces COX-2 but not COX-1 expression; (2) COX-2 activity is necessary for protective effect of HGF in CIP.
I'1185 Protective Effects of Edaravone, a Novel Free Radical Scavenger, in CeruleinInduced Experimental Pancreatitis in Rats Sawako Kobayashi, Takuya Kitada, Hidck: Fuiii, Takao Yamada, Hiroki Sakaguchi, Shuichi Seki
Backgroundk~ims: Oxidative cellular injury induced by oxygen tree radicals have been imphcated in the pathogenesis of vanons human diseases including acute pancreatitis. The aim of this study was to examine whether edaravone (3-methyl-l-pbewl-2-pyrazoline-5one), a novel free radical scavenger, could attenuate pancreatic inju U in rats with ceruleininduced acute pancreatnis. Materials and Methods: Male Wister rats (200g) were divided into three groups. Group1 received 4 intrapentoneal injections of cerulein (50 mg/kg body weight) at lht interval. Group 2 received an intravenous injection of edarabone (3mg/kg body weight) 30 rain beibre each cerulein injection. Group 3 (control) received 4 intraperitoneal in[ecuons of saline at lhr interval as a control. Rats were sacrificed at 30 min after the last cerulein injection, and pancreatic injury was assessed by a histological examination and determination of serum amylase activW, Results: Cerulein administration induced pancreatic edema and increase of serum amylase acuvny in non4reated rats (Group i) as previously reported. In contrast, pretreatment of edaravone betore each cerulein injection (Group 2) resulted in a significant reduction in pancreatic edema evidenced by the measurement of the paocreas*%xly weight ratio and histological assessment. In addition, serum amylase activity was significantly reduced by pretreatment with edalavone. Conclusion: Although the mechanism remains to be investigated, these results suggest that edaravone have protective effects on cerulein-induced rat experimental pancreatitis.
Tl188 COX-2 Gene Disruption Results in the Attenuation of NF-KB Activity Richard T. Ethridge, Michele I Slogoff, Dai H. Chung, B. Mark Evers Acute pancreatitis (AP) leads to severe inflammation and acinar ceil damage; the exact etiology of AP is not known. The inducible cyclooxygenase (COX) et~zyme, COX-2, appears to play a role in the inflammatory" response of certain tissues; nuclear factor-gB (NF-KB) is a stress activated transcription factor that can induce expression of COX-2. We have previously shown that the inhibition of COX-2 activity results in fire attenuation of AP severity. The purpose of this study*was to determine whether the alteration ot COX-2 expression, normally considered to be downstream of NF-gB, results in the feedback regulation of NF-KB acti~qty. METHODS. Swiss Webster mice or wild type (pigs2 ~j+) and knockout (ptgs2 ~) mice were injected Lp. with either saline (control) or caemlein (CAE; 50 mg/kg) hourly tbr 8 h and sacrificed at 0 h tollowing the completion of the injections. The COX-2 selective inhibitor (SC-58125; 10 p.g, i.p.) was injected in the Swiss Webster mice at the time of the first injection of CAE. Pancreata were harvested tbr protein and histology. Western blot was performed to determine the expression of p65, pSO, and IKB. Electmphoretic mobility shift assay (EMSA) was performed to assess NF4cB DNA binding activity. RESULTS. Induction of AP msuhed m increased NF-KB binding activity and IuB degradation in the Swiss Webster mice; in contrast, treatment with SC-58125 markedly decreased NF-KB induction and prevented IKB degradation. Similarly', AP resulted in induction of NFKB binding and IKB degradation in pigs2 +j+ mice; genetic deletion of pigs2 prevented the induction of NF-KB binding and decreased IKB expression. Additionally, as we have noted previously, treatment with a COX-2 selective inhibitor or genetic deletion of COX-2 resulted in decreased severity of AP. There was no change in p65 or p50 expression in any of the animals. CONCLUSIONS. The genetic deletion of the COX-2 gene and the chemical inhibition of COX-2 activity results in the decreased activity of NF-KB suggesting a role for COX-2 expression in feedback regulation of NF-KB activity. These data show that COX-2 activfiy/expression is necessary for the activity of NF-KB and suggest a potential role of COX-2 selective inhibitors for the inhibition of NF-KB mediated downstream activity
T1186 Expression and Regulation of JAK and STAT Proteins in Pancreatic Acinar Cells tiike Gallmeier, Irmgard Ploessl, Patricia btoubarak, Burkhard Goeke, Claus Schaefer, Andreas Wagner Purpose Pancreatitis development depends on acinar cell damage followed by an immune response which includes Th-1 cell activation and increase of pancreatic hrterferon (IFN)-y levels Acniar cells actively participate in the immune response through production of chemoand cytokmes Although the lanns kinase/signal transducer and activator of transcription(JAK/ 5TAT)-pathway represents the main effector of IFN-y and other c)r nothing is known about JARLSIAT proteins in acinar cells. Methods: Rat pancreatic acinar cells were prepared by collagenase digestion. Pancreatic lohules and acinar cells were treated with different concentrations of chotecystokinin (CCR), IFN-'y and AG-490 Expression of JAK/STAT proteins and phosphorylation on tyrosme-residues was measured by immunoprecipitatiord western blotting Alkaline phosphatase treatment was used to show specificity of antP pfiospho4TAT signals. Results: Western analysis showed expression of JAK1, JAR2, TYK2, as well as STATs 1, 2 3, 5, 6, but not STAT4 in Iobules and acinar cells. Prepared acinar cells showed in contrast to pancreatic lobules a strong upregnlation of STAT1 phosphorylation which rammed to baseline levels within 6 to 8h. IFN-y but not CCK also induced STAT1 pfiosphorylalion in acini and lobules :his efiect was dose dependently inhibited by the JAK2-inhibilor AG-490 w:th conrplete mhibitmn at llxM. Conchisinn: We showed for the tirst time the expression of severalJAK/STAT proteins and a functional JAK2/STAT1 pathway' in paucreatic acinar ceils interestingly, mechanical/metabolical stress during acinar cell ptvparatinn activated pancreatic STAT1 However, we tbund no crosstalk between CCKt~'ceptor stimulation and the JARTSTAT pathway. In contrast, acini and lobules sensitively responded to IFN-y with JAR2 med:ated STAT1 phosphorylation. We believe that these data support tim concept that acinar cells closely interact with immune cells during pancreatitis development Thus, acinar cells may not only" stimulate immune cells through production of ctmmo- and cytokines but also respond to increased pancreatic IFN-'/levels during pancreatitis
Tl189 NF-kB Inhibition by Pyrrolidine Dithiocarbamate Attenuates Experimental Acute Pancreatitis loannis T. Virlos, Emanuela Mazzon, lvana Serraino, Christoph Thiemernrann, Ajith K Siriwardena, Salvatore Cuzzocrea
Background: The nuclear lactor~kB (NF-kB) is a transcription lactor which plays a pivotal role in the induction of genes involved in dre response to injury" and inflammation. Dithiocarbamates are antioxidants which are potent inhibitors of NF-kB. Hypothes:s: This stud), tests the postulate that pyrrolidine dithiocarbamate (PDTC) attenuates experimental acute pancreatitis. Methods: Male wild type mice were used. All studies conformed to the 1991 American Physiology Association guidelines for the care of animals. Animals were allowed access to food and water ad libitum and were randomly allocated to one of three groups (n=nmnber of animals) as follows: Group i (n=28) intraperitoueal [i.p.] saline; Group II (n=29) i.p. cernlein 50 n:icrogms/kg and Group Iil (n=29) pre-treannent with PDTC 30 mg/kg prior to i.p. cernlein. Animals were sacrificed by intra-cardiac puncture at 6 hours and the following endpoints were evaluated: 1.Serum amylase and fipase 2.Histologic examination of pancreas and lung 3.lmmunohisrochemical localization of the adhesion molecule ICAM-I and nitrotyrosine in lung and pancreas 4Myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels in lung and pancreas Results: Intraperkoneal cemleiu resulted in severe acute pancreatitis characterized by edema, neutrophil infiltration, tissue hemorrhage and necrosis and elevated serum amylase and lipase. PDTC pre-treatment resulted in significant reduction in amylase and fipase (P<0.O01 vs cerulein; P = NS vs control). [mmunohistochemical examination demonstrated a marked increase in immunoreactiv~ty fi~rnitrotyrosine and ICAM-1 in the pancreas and lung of cemlein-treated mice while PDTC pre-treatmem abolished staining for both ICAMq and nitrotyrosine MPO and MDA in both lung and pancreas were elevated in ceruleinqnduced acute pancreatitis. PDTC pre-treatment reduced MPO activity in lung (P
T1187 Inhibition of Cyclooxygenase-2 Reduces the Protective Effect of Hepatocyte Growth Factor Administration in Experimental Pancreatitis Zygmunt Warzecha, Artur Dembinski, Piotr Ceranowicz, Wieslaw W. Pawlik, Ryszard Sendur, Jaroslaw Biernat; Marcin Dembinski, Stanislaw J. Konturek, Romana Tomasaewska, Jerzy Stachura, Pete:" C Konturek, Detlef Shnppan, Toshikazu Nakamura
FIepatocyte growth factor (I-iGF) overexpression is observed in experimental and clinical acu*e pancreatitis. Moreover, previons studies has shown that administration of HGF reduces pancreatic damage in experimental pancreatins The aim of out studies was to determine
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AGA Abstracts
provide the first evidence that PDTC, a potent inhibitor of NF-kB activation atteuuates both pancreatic and lung miury and reduces nantmphil infihratirar, lipid peroxidation, arrd expression of ICAM-1 and nitrotymsiue in expenmental acute pancreatitis. Conclusion: The findings demonstrate that NF-kB inhibinon by PDTC ameliorates experimental mnrine acute pancreatins and provide important insight into the nrolecular biology of this disease,
218 amino acids, which was ahnost identical to human nineteen kD interacting protein-3 like (NIP3L) protein. The expression of rat NIP3L slighdy increased in the pancreas after induction of AP On the other hand, the expression of NIP3L m the lung showed a marked incl~ase by Northern and Western blot analyses. N1P3L immunoreactivity was found in alveolar and epithelial ceils of the control (sham) lung, and the ~mmunoreactivity in these cells elevated after induction of AP, Moreover, AP increased terminal deoxynucleotidyl transierase-mediated dUTP-biotin inck end labeling (TUNEL)-positive alveolar and bronchiolar ceils m the lung. Apoptosis indexes, defined as the ratio of TUNEbpositive cells, of both alveolar and bronchiole epithelial ceils on day 5 after induction of AP were significantly higher than those of control (sham). NIP3L protein levels in tbe Inng on day 2 and day 3, when the apoptosis indexes did not significantly increase, were significantly higher than those of control (sham). Conclusion: NIP3L might be involved in apoptosis of lung injury,, which is one of the major causes of death m severe AP
Tl190
Akt-NFkB Pathway Is Activated In Duct Ligation-lnduced Acute Pancreatitis In Rats Isaac Sarnud, Asgar Zaheer, Mark Yorek, Smita Zaheer, Rory Fisher Bile-pancreatic duct ligation in rats excludes bile-pancreatic juice from gut and induces acute pancreatitis. CCK-A and c:holinergic receptor-mediated exocr/ne pancreatic hyperstimulation secondat7 to bile-pancreatic juice exclusion ~ implicated in disease pathogenesis. Stimulation of these G protein coupled receptors can activate the Akt-NFkB pathway Nuclear Transcrip~ lion Factor kappa B (NFkB) is a downstream messenger of acinar cell signal transduction via the Akt pathway and is capable o/inr Nag adnar cell cytokina production. We hypothesize that the Akt-NFkB pathway is activated in figation-induced acute pancreatitis. METHODS: We studied rats w~th 1, 3, or 24 hra ot duct ligation or sham-operation (ducts dLssected but not ligated)(n= 5 rats/group). RESULTS: Compared to sham-controls, duct ligation induced acute pancreatitis as evidenced by hyperamylasemia and pancreatic morphologic changes. Electro-mobility shift assay showed a irma-dependent incraase in NFkB in pancreatic nuclear extracts after duct ligation (transcnption factor E-box was used as control; competing cold NFkB prone excluded non-specihc binding), immunobIot analysis of pancreatic homogehates showed mcreases in phospho-Akt and phospho-lkB that were also time-dependent and parallel with NFkB activation (actin immunobiots confirmed equal loadtirg of lanes) (lkB phosphorylation permits translocatinn of NFkB from the cytosol to the nucleus). CONCLUSIONS: 'Paere is a time-dependem and parallel increase in nuclear translocation of NFkB and acuvation of Akt and IkB after duct ligationdnduced acute pancreatitis in rats. These findings are consistent wuh the h)~otbesis that the Akt-NFkB pathway is activated in duct ligation induced acute pancreatitis. In this expemnentaI corollary of gallstone-reduced acute pancreatitts, bile-pancreatic juice exclusion-induced stimulation of acinar cell G protein coupled receptors could exacerbate acute pancreatitis by increased acinar cell production of pro-inflammatory cytokines via lbe Akt-NFkB pathway (Support: Carver Collaborative Pilot Grant; N1H #lK08DK062805)
Tl193
Caspases and Poly(ADP-ribose) Polymemse (PARP) Regulate the Balance Between Apoptosis and Necrosis in Experimental Pancreatitis Ilya Gukovsky, Kynng J Nam, Jason H. Cbeng, Stephen J. PandoI, Anna S. Gukovskaya Background & Aims: Both apoptosis and necrosis occur m pancreatitis, and their relative contribution may determine pancreatitis seventy. However, the mechanisms of acinar cell death m panareatitis remain unknovm. Key mediators of apoptosis in other systems are specific cysteine proteases, the caspases; one important regulator of necrosis is PARP. PARP activity may be inhibited through cleavage by caspases. We sought to determine the roles of caspase and PARP activation in cell death responses of mouse and rat cerulein pancreatins. Methods: Pancreatitis was induced in mice by 7 injections of 50 b~g/kg cerulein, and in rats, by 6-h infusion of 5 }xg/kg/h cerulein. We measured apoptosis and necrosis in pancreas; pancreatic PARP activity by" protein polyADP-ribosylation; PARP cleavage, by Western blotting; and caspase activation, by fluorogenic assay. Results: The necrosis rate was -6-fold greater in mouse compared to rat cemlein pancreatitis; for apoptosls, this ratio was m',,ersed, The key" executioner caspase-3 was activated in both models of cerulein pancreatitis. Its activation was greater in the tat model, correlating with apoptotic rate. PARP was activated in both models, as well as m isolated pancreatic acini stimulated '.ruth a supramaximal dose (100 nM) of CCK-8 However, in contrast to caspases, PARP activity was greater in the mouse model. PARP activation was inhibited by 3-amtnobenaamide (3-AB; 30 mg/kg). 3AB decreased necrosis in the mouse model by 40%, but it did not affect necrosis in the rat model The reason for this could be diB):rent degree of PARP degradation in the two models. Indeed, we tbund significant PARP cleavage in the rat model but no PARP degradation in the mouse model. Thus, the more necrotic mouse cerulein pancreantis is associated "aith higher PARP and lower caspase activ'W, In contrast, ttre greater caspase activation in the rat model results in PARP degradation, limiting its activity and thus necrosis, The broadspectrum caspase inhibitor zVAD-fink (3 mg/kg) inhibited caspase activation and apoptosis Moreover, it also greatly inhibited PARP cleavage in the rat model and increased necrosks >2-fold, converting this model to a "necrotic" type. Conclusion: Caspases mediate apoptosis whereas PARP mediates necrosis in cerulein pancreatitis. Caspase activation protects from necrosis, at least in part, through PARP cleavage. The results suggest caspases and PARP as therapeutic targets to attenuate the severity of pancreatitis
Tl191
The Therapeutic Effect of Anti-Cdl4 Antibody Via Blocking Endotoxic Signal Transduction Pathway in Animal Model of Severe Acute Pancreatitis Xing-Peng Wang, Li-Ying Wu Ba&ground/Aims: To investigate the therapeutic effect of anti-CD14 antibody via blocking endotoxic signal ransduction pathway in animal model of severe acute pancreatins (SAP)Methods: Thirty-six BALB/c mice ,,',,ere randomly" dMded into SAP group and antiCD14 antibody treated group. SAP was induced by administration of seven intrapentoneal injections of cerulein (50mgj&g) at hourly intervals, hpopolysaccharide (LPS) was injected 6 hours after the first cerulein injection. Treatment with the anti-CD 14 antibody' was started 15 minutes bdore the LPS inlection. Serum levels of" amylase and lactate dehydrogenase (LDH) were measured at tha end of the experiments. The severity of pancreatitis was evaluated by histological scoring using a semi-quantitative analysis ot hematoxylin and eosin-stained sections of the pancreas. Semi-quantitative raverse transcription-polymerase clmin reaction (RT-PCR) was perfomrad to quantit), fire intmpancreatic TNF-alpha,ICAM-1 and E-selectin mRNA levds The expressions of unclear factor NF-kB and polyraorphonudear leukocyte (PMN)-spectfic antigen Mac-1 were investigated by the method of immunohistochemistry and western blotting 'The activity of PMN myeloperoxidase (MPO) was detennined}y/ zymohistochemist*T, Results: Seven times cerulein injection induced edematous pancreatitis, whereas when [PS was challenged, necrotizing pancreatitis developed. At 9, 12, 24 hours afier the first cerukin injection the blood and specimens of pancreas were harvested. Serum amylase and lipase levels progressively increased vAth a maxnnum reached at 12 hours. Treatment of anti-CD14 antibody signtflcantly decreased amyIase and LDH levels at 9 and 12 hours. Histologically, treatment of ann-CD14 antibody reduced the seventy of pancreatic injury including inflammatory cell mfihration, aud necrosis at 9 and 24 hours, intrapancreatic l-NF-alpha ICAM-I and E-selectin mRNA leveIs weee reduced in the anti-CD14 antibodytreated group at 912 hours. There was significant decrease of MPO actmty in anti-CDl4 antibody group at 12 and 24 hours Imraunohist~K'hemistry and western blotting also showed down-regulation, of nuclear tactor NF-kB and PMN-specific antigen Mac-], Conclusions: ~he anti-CD 14 antibody decreased the se',*en/y of expenmental pancreatitis, suggesting that the protective effect of the anti-CD14 antibody' was mediated, at least in part, through blocking endotoxin signal pathway.
T1194
Identification of Pancreatic Genes Expressed during the Initiation of Acute Pancreatitis: EGR-1 is a Key Regulator Chug D. Logsdon, Baoan Ji, Xue-Qing Chen, David E, Misek, Rork Kuick, Steve Ernst, Rebecca Najafian, Samir Hanash We hypothesized that genes expressed in pancreauc acinar cells during the initiation of acute pancreatins determine the severity of the disease Tberefi~re, we utilized microarrays to profile changes in gena expression and identit}/those genes commonly induced in rat pancreatic acinar cells within 1-4 hours in two in vivo models, caerulein and taurocholate administration, and one in vitro model, isolated acini treated with caerulein. This strategy yielded 72 genes common to these three models which represented a complex array of molecules, including those that are likely" to either reduce or increase the seventy" of the disease, Novel genes identified in the current study included: ATF3, BRF1, O~BPI3 and 8, CGRP, EGR-I, ephrmA1, villin2, ierredoxin, tbllistatin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, and thioredoxin reductase. To validate these microarray results, the role of EGR-1 was turther investigated in a rat model of secretagogue induced acute pancreatitis using QRT-PCR, western blotting, and immunocytocbemistry. EGR-1 expression occurred within acinar cells and correlated with the development of acute pancreatitis, To further investigate tire role oI EGR- 1 in acute pancreatitis EGWI deficient mice were treated with high concentrations of caerulein. Pancreatitis associated lung inflammation and pancreatic levels of the inflammation-related genes IL-6, MCP-I, PAI, TF and ICAM-1 were reduced in EGR-1 deficient mice compared to wild-type c(mtruls Thus, we identified EGR-1 and several other novel genes likdy to be important in tha development and seventy of acute pancreatitis
Tl192 Increased expression of nineteen kD interacting protein-3-1ike protein and the relationship to apoptosis in rat pancreatitis-associated lung injury Hayato Nakamura, Hidekazu Honda, Mitsuo Tashiru, Yasu)raki Kihara, Maknto Otsuki
Tl195 Leptin Plays An Anti-lnflammatory Role In Acute Pancreatitis Charalabos Pothoulakis, Manju Saluja, Pauline Amon, Amy Pan, Raiinder K, Dawra, Lalit C. Garg, Susan Biueber, Christos Mantzoros, Ashok K, Saluja
Background: Tha aim of the present study was to determine the underlying cellular mechamsms in the pancreas after acute pancreatitis (AP), and to stud), the pathogenesis of pancreatids-associated tung injury'. Method: We applied a difterantia[ display analysis to normal pancreas and pancreas of rats with AP, and examined the expression of the gena, identified by the analysis, in the hmg as wall as the panareas after AP Pancreatitis was induced by retrograde intradactal inclusion of 4% sodium taurocholate (100 ~xI/100 g body weight). Results: We cloned some expressed sequence tags, and identified one expressed sequence tag that was not registered in Gcnbank. Using the rapid amplification of cDNA endspolymerase chain reaction and searching the expressed sequence tags database, we obtained a transcript containing an open reading flame, The deduced protein was a polypeptidc of
AGA Abstracts
Background & Objectives: Leptin, an adipocyte-denved hormone is a product of the ob gena and functions to regulate body ,,;,eight and energy homeostasis. Leptin mediates its effect by binding to specific receptors (Ob-Rb) belonging to the cytokine super tamily, Recendy, it has shown that leptin and its receptor are also involved in the regulation of immune response m many' pathological states. Leptin receptors are known to be present in pancreas but their precise role in this organ is not well understood, in the present study, we have evaluated the role of leptin and its receptors (Ob-Rb) in acute pancreatitis, Methods:
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the role of cyclooxygenase-1 (COX-l) and cyclooxygenase-2 (COX-2) in protective eftect of HGF administration in caerulein-induced pancreatitis (CIP). Methods: Acute pancreatitis was induced by s.c. infusion of caernlein (10 mcg/kg/h) for 5 h. HGF was administrated twice (30 min prior to caeruleni or saline infusion and 3 h later) at the dose 10 mcg/kg s.c. Activity of COX-1 and COX-2 were inhibited by resveratrul and mfecoxib, respectively (10 mg/kg, ig. 1 h before saline or caerulein infusion), hnmediately after cessation of caemlein or saline infusion the pancreatic blood flow (PBF), plasma lipase activity, plasma interleukin1 (IL-1) and mterleukin- l0 (IL-10) concentration, cell proliferation, and morphological signs of pancreatitis were examined. Expression of COXq and COX-2 mRNA transcripts was determined by RT-PCR Analysis of COX production was made by Western Blot. Resuhs: Administration of HGF or CIP alone, or their combination were without effect on COX-1 mRNA expression in pancreatic tissue. Expression of COX-2 mRNA was increased by HGF and CIP. Maximal increase in COX-2 mRNA expression was observed, when HGF administration was combined with C1P. The similar effect was observed, when we studied influence of HGF and CIP on pancreatic COX-2 production determined by Western Blot. Administration of HGF without induction of CIP increased plasma IL-10 and this effect was abolished by COX-2 inhibitor-rofecoxib. Treatment with HGF, during development of CIP, increased plasma IL-IO and attenuated the pancreatic damage, what was manifested by: (a) histological improvement of pancreatic integrity, (bY the partial reversion of the drop in DNA synthesis and PBF, (c) the reduction in pancreatitis evoked increase in plasma ]ipase, and IL-1. Administmtinn of resveratrol and rofecoxib alone was without eftect on development of CIP. Combination of rofecoxib with HGF reduced HGF-evoked increase in plasma IL-10 and abolished protective effect of HGF against pancreatic damage in CIP. Resveratrol did not aflect a protective effect of HGF administration. Conclusions: (1) HGF administration reduces COX-2 but not COX-1 expression; (2) COX-2 activity is necessary for protective effect of HGF in CIP.
I'1185 Protective Effects of Edaravone, a Novel Free Radical Scavenger, in CeruleinInduced Experimental Pancreatitis in Rats Sawako Kobayashi, Takuya Kitada, Hidck: Fuiii, Takao Yamada, Hiroki Sakaguchi, Shuichi Seki
Backgroundk~ims: Oxidative cellular injury induced by oxygen tree radicals have been imphcated in the pathogenesis of vanons human diseases including acute pancreatitis. The aim of this study was to examine whether edaravone (3-methyl-l-pbewl-2-pyrazoline-5one), a novel free radical scavenger, could attenuate pancreatic inju U in rats with ceruleininduced acute pancreatnis. Materials and Methods: Male Wister rats (200g) were divided into three groups. Group1 received 4 intrapentoneal injections of cerulein (50 mg/kg body weight) at lht interval. Group 2 received an intravenous injection of edarabone (3mg/kg body weight) 30 rain beibre each cerulein injection. Group 3 (control) received 4 intraperitoneal in[ecuons of saline at lhr interval as a control. Rats were sacrificed at 30 min after the last cerulein injection, and pancreatic injury was assessed by a histological examination and determination of serum amylase activW, Results: Cerulein administration induced pancreatic edema and increase of serum amylase acuvny in non4reated rats (Group i) as previously reported. In contrast, pretreatment of edaravone betore each cerulein injection (Group 2) resulted in a significant reduction in pancreatic edema evidenced by the measurement of the paocreas*%xly weight ratio and histological assessment. In addition, serum amylase activity was significantly reduced by pretreatment with edalavone. Conclusion: Although the mechanism remains to be investigated, these results suggest that edaravone have protective effects on cerulein-induced rat experimental pancreatitis.
Tl188 COX-2 Gene Disruption Results in the Attenuation of NF-KB Activity Richard T. Ethridge, Michele I Slogoff, Dai H. Chung, B. Mark Evers Acute pancreatitis (AP) leads to severe inflammation and acinar ceil damage; the exact etiology of AP is not known. The inducible cyclooxygenase (COX) et~zyme, COX-2, appears to play a role in the inflammatory" response of certain tissues; nuclear factor-gB (NF-KB) is a stress activated transcription factor that can induce expression of COX-2. We have previously shown that the inhibition of COX-2 activity results in fire attenuation of AP severity. The purpose of this study*was to determine whether the alteration ot COX-2 expression, normally considered to be downstream of NF-gB, results in the feedback regulation of NF-KB acti~qty. METHODS. Swiss Webster mice or wild type (pigs2 ~j+) and knockout (ptgs2 ~) mice were injected Lp. with either saline (control) or caemlein (CAE; 50 mg/kg) hourly tbr 8 h and sacrificed at 0 h tollowing the completion of the injections. The COX-2 selective inhibitor (SC-58125; 10 p.g, i.p.) was injected in the Swiss Webster mice at the time of the first injection of CAE. Pancreata were harvested tbr protein and histology. Western blot was performed to determine the expression of p65, pSO, and IKB. Electmphoretic mobility shift assay (EMSA) was performed to assess NF4cB DNA binding activity. RESULTS. Induction of AP msuhed m increased NF-KB binding activity and IuB degradation in the Swiss Webster mice; in contrast, treatment with SC-58125 markedly decreased NF-KB induction and prevented IKB degradation. Similarly', AP resulted in induction of NFKB binding and IKB degradation in pigs2 +j+ mice; genetic deletion of pigs2 prevented the induction of NF-KB binding and decreased IKB expression. Additionally, as we have noted previously, treatment with a COX-2 selective inhibitor or genetic deletion of COX-2 resulted in decreased severity of AP. There was no change in p65 or p50 expression in any of the animals. CONCLUSIONS. The genetic deletion of the COX-2 gene and the chemical inhibition of COX-2 activity results in the decreased activity of NF-KB suggesting a role for COX-2 expression in feedback regulation of NF-KB activity. These data show that COX-2 activfiy/expression is necessary for the activity of NF-KB and suggest a potential role of COX-2 selective inhibitors for the inhibition of NF-KB mediated downstream activity
T1186 Expression and Regulation of JAK and STAT Proteins in Pancreatic Acinar Cells tiike Gallmeier, Irmgard Ploessl, Patricia btoubarak, Burkhard Goeke, Claus Schaefer, Andreas Wagner Purpose Pancreatitis development depends on acinar cell damage followed by an immune response which includes Th-1 cell activation and increase of pancreatic hrterferon (IFN)-y levels Acniar cells actively participate in the immune response through production of chemoand cytokmes Although the lanns kinase/signal transducer and activator of transcription(JAK/ 5TAT)-pathway represents the main effector of IFN-y and other c)r nothing is known about JARLSIAT proteins in acinar cells. Methods: Rat pancreatic acinar cells were prepared by collagenase digestion. Pancreatic lohules and acinar cells were treated with different concentrations of chotecystokinin (CCR), IFN-'y and AG-490 Expression of JAK/STAT proteins and phosphorylation on tyrosme-residues was measured by immunoprecipitatiord western blotting Alkaline phosphatase treatment was used to show specificity of antP pfiospho4TAT signals. Results: Western analysis showed expression of JAK1, JAR2, TYK2, as well as STATs 1, 2 3, 5, 6, but not STAT4 in Iobules and acinar cells. Prepared acinar cells showed in contrast to pancreatic lobules a strong upregnlation of STAT1 phosphorylation which rammed to baseline levels within 6 to 8h. IFN-y but not CCK also induced STAT1 pfiosphorylalion in acini and lobules :his efiect was dose dependently inhibited by the JAK2-inhibilor AG-490 w:th conrplete mhibitmn at llxM. Conchisinn: We showed for the tirst time the expression of severalJAK/STAT proteins and a functional JAK2/STAT1 pathway' in paucreatic acinar ceils interestingly, mechanical/metabolical stress during acinar cell ptvparatinn activated pancreatic STAT1 However, we tbund no crosstalk between CCKt~'ceptor stimulation and the JARTSTAT pathway. In contrast, acini and lobules sensitively responded to IFN-y with JAR2 med:ated STAT1 phosphorylation. We believe that these data support tim concept that acinar cells closely interact with immune cells during pancreatitis development Thus, acinar cells may not only" stimulate immune cells through production of ctmmo- and cytokines but also respond to increased pancreatic IFN-'/levels during pancreatitis
Tl189 NF-kB Inhibition by Pyrrolidine Dithiocarbamate Attenuates Experimental Acute Pancreatitis loannis T. Virlos, Emanuela Mazzon, lvana Serraino, Christoph Thiemernrann, Ajith K Siriwardena, Salvatore Cuzzocrea
Background: The nuclear lactor~kB (NF-kB) is a transcription lactor which plays a pivotal role in the induction of genes involved in dre response to injury" and inflammation. Dithiocarbamates are antioxidants which are potent inhibitors of NF-kB. Hypothes:s: This stud), tests the postulate that pyrrolidine dithiocarbamate (PDTC) attenuates experimental acute pancreatitis. Methods: Male wild type mice were used. All studies conformed to the 1991 American Physiology Association guidelines for the care of animals. Animals were allowed access to food and water ad libitum and were randomly allocated to one of three groups (n=nmnber of animals) as follows: Group i (n=28) intraperitoueal [i.p.] saline; Group II (n=29) i.p. cernlein 50 n:icrogms/kg and Group Iil (n=29) pre-treannent with PDTC 30 mg/kg prior to i.p. cernlein. Animals were sacrificed by intra-cardiac puncture at 6 hours and the following endpoints were evaluated: 1.Serum amylase and fipase 2.Histologic examination of pancreas and lung 3.lmmunohisrochemical localization of the adhesion molecule ICAM-I and nitrotyrosine in lung and pancreas 4Myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels in lung and pancreas Results: Intraperkoneal cemleiu resulted in severe acute pancreatitis characterized by edema, neutrophil infiltration, tissue hemorrhage and necrosis and elevated serum amylase and lipase. PDTC pre-treatment resulted in significant reduction in amylase and fipase (P<0.O01 vs cerulein; P = NS vs control). [mmunohistochemical examination demonstrated a marked increase in immunoreactiv~ty fi~rnitrotyrosine and ICAM-1 in the pancreas and lung of cemlein-treated mice while PDTC pre-treatmem abolished staining for both ICAMq and nitrotyrosine MPO and MDA in both lung and pancreas were elevated in ceruleinqnduced acute pancreatitis. PDTC pre-treatment reduced MPO activity in lung (P
T1187 Inhibition of Cyclooxygenase-2 Reduces the Protective Effect of Hepatocyte Growth Factor Administration in Experimental Pancreatitis Zygmunt Warzecha, Artur Dembinski, Piotr Ceranowicz, Wieslaw W. Pawlik, Ryszard Sendur, Jaroslaw Biernat; Marcin Dembinski, Stanislaw J. Konturek, Romana Tomasaewska, Jerzy Stachura, Pete:" C Konturek, Detlef Shnppan, Toshikazu Nakamura
FIepatocyte growth factor (I-iGF) overexpression is observed in experimental and clinical acu*e pancreatitis. Moreover, previons studies has shown that administration of HGF reduces pancreatic damage in experimental pancreatins The aim of out studies was to determine
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AGA Abstracts
provide the first evidence that PDTC, a potent inhibitor of NF-kB activation atteuuates both pancreatic and lung miury and reduces nantmphil infihratirar, lipid peroxidation, arrd expression of ICAM-1 and nitrotymsiue in expenmental acute pancreatitis. Conclusion: The findings demonstrate that NF-kB inhibinon by PDTC ameliorates experimental mnrine acute pancreatins and provide important insight into the nrolecular biology of this disease,
218 amino acids, which was ahnost identical to human nineteen kD interacting protein-3 like (NIP3L) protein. The expression of rat NIP3L slighdy increased in the pancreas after induction of AP On the other hand, the expression of NIP3L m the lung showed a marked incl~ase by Northern and Western blot analyses. N1P3L immunoreactivity was found in alveolar and epithelial ceils of the control (sham) lung, and the ~mmunoreactivity in these cells elevated after induction of AP, Moreover, AP increased terminal deoxynucleotidyl transierase-mediated dUTP-biotin inck end labeling (TUNEL)-positive alveolar and bronchiolar ceils m the lung. Apoptosis indexes, defined as the ratio of TUNEbpositive cells, of both alveolar and bronchiole epithelial ceils on day 5 after induction of AP were significantly higher than those of control (sham). NIP3L protein levels in tbe Inng on day 2 and day 3, when the apoptosis indexes did not significantly increase, were significantly higher than those of control (sham). Conclusion: NIP3L might be involved in apoptosis of lung injury,, which is one of the major causes of death m severe AP
Tl190
Akt-NFkB Pathway Is Activated In Duct Ligation-lnduced Acute Pancreatitis In Rats Isaac Sarnud, Asgar Zaheer, Mark Yorek, Smita Zaheer, Rory Fisher Bile-pancreatic duct ligation in rats excludes bile-pancreatic juice from gut and induces acute pancreatitis. CCK-A and c:holinergic receptor-mediated exocr/ne pancreatic hyperstimulation secondat7 to bile-pancreatic juice exclusion ~ implicated in disease pathogenesis. Stimulation of these G protein coupled receptors can activate the Akt-NFkB pathway Nuclear Transcrip~ lion Factor kappa B (NFkB) is a downstream messenger of acinar cell signal transduction via the Akt pathway and is capable o/inr Nag adnar cell cytokina production. We hypothesize that the Akt-NFkB pathway is activated in figation-induced acute pancreatitis. METHODS: We studied rats w~th 1, 3, or 24 hra ot duct ligation or sham-operation (ducts dLssected but not ligated)(n= 5 rats/group). RESULTS: Compared to sham-controls, duct ligation induced acute pancreatitis as evidenced by hyperamylasemia and pancreatic morphologic changes. Electro-mobility shift assay showed a irma-dependent incraase in NFkB in pancreatic nuclear extracts after duct ligation (transcnption factor E-box was used as control; competing cold NFkB prone excluded non-specihc binding), immunobIot analysis of pancreatic homogehates showed mcreases in phospho-Akt and phospho-lkB that were also time-dependent and parallel with NFkB activation (actin immunobiots confirmed equal loadtirg of lanes) (lkB phosphorylation permits translocatinn of NFkB from the cytosol to the nucleus). CONCLUSIONS: 'Paere is a time-dependem and parallel increase in nuclear translocation of NFkB and acuvation of Akt and IkB after duct ligationdnduced acute pancreatitis in rats. These findings are consistent wuh the h)~otbesis that the Akt-NFkB pathway is activated in duct ligation induced acute pancreatitis. In this expemnentaI corollary of gallstone-reduced acute pancreatitts, bile-pancreatic juice exclusion-induced stimulation of acinar cell G protein coupled receptors could exacerbate acute pancreatitis by increased acinar cell production of pro-inflammatory cytokines via lbe Akt-NFkB pathway (Support: Carver Collaborative Pilot Grant; N1H #lK08DK062805)
Tl193
Caspases and Poly(ADP-ribose) Polymemse (PARP) Regulate the Balance Between Apoptosis and Necrosis in Experimental Pancreatitis Ilya Gukovsky, Kynng J Nam, Jason H. Cbeng, Stephen J. PandoI, Anna S. Gukovskaya Background & Aims: Both apoptosis and necrosis occur m pancreatitis, and their relative contribution may determine pancreatitis seventy. However, the mechanisms of acinar cell death m panareatitis remain unknovm. Key mediators of apoptosis in other systems are specific cysteine proteases, the caspases; one important regulator of necrosis is PARP. PARP activity may be inhibited through cleavage by caspases. We sought to determine the roles of caspase and PARP activation in cell death responses of mouse and rat cerulein pancreatins. Methods: Pancreatitis was induced in mice by 7 injections of 50 b~g/kg cerulein, and in rats, by 6-h infusion of 5 }xg/kg/h cerulein. We measured apoptosis and necrosis in pancreas; pancreatic PARP activity by" protein polyADP-ribosylation; PARP cleavage, by Western blotting; and caspase activation, by fluorogenic assay. Results: The necrosis rate was -6-fold greater in mouse compared to rat cemlein pancreatitis; for apoptosls, this ratio was m',,ersed, The key" executioner caspase-3 was activated in both models of cerulein pancreatitis. Its activation was greater in the tat model, correlating with apoptotic rate. PARP was activated in both models, as well as m isolated pancreatic acini stimulated '.ruth a supramaximal dose (100 nM) of CCK-8 However, in contrast to caspases, PARP activity was greater in the mouse model. PARP activation was inhibited by 3-amtnobenaamide (3-AB; 30 mg/kg). 3AB decreased necrosis in the mouse model by 40%, but it did not affect necrosis in the rat model The reason for this could be diB):rent degree of PARP degradation in the two models. Indeed, we tbund significant PARP cleavage in the rat model but no PARP degradation in the mouse model. Thus, the more necrotic mouse cerulein pancreantis is associated "aith higher PARP and lower caspase activ'W, In contrast, ttre greater caspase activation in the rat model results in PARP degradation, limiting its activity and thus necrosis, The broadspectrum caspase inhibitor zVAD-fink (3 mg/kg) inhibited caspase activation and apoptosis Moreover, it also greatly inhibited PARP cleavage in the rat model and increased necrosks >2-fold, converting this model to a "necrotic" type. Conclusion: Caspases mediate apoptosis whereas PARP mediates necrosis in cerulein pancreatitis. Caspase activation protects from necrosis, at least in part, through PARP cleavage. The results suggest caspases and PARP as therapeutic targets to attenuate the severity of pancreatitis
Tl191
The Therapeutic Effect of Anti-Cdl4 Antibody Via Blocking Endotoxic Signal Transduction Pathway in Animal Model of Severe Acute Pancreatitis Xing-Peng Wang, Li-Ying Wu Ba&ground/Aims: To investigate the therapeutic effect of anti-CD14 antibody via blocking endotoxic signal ransduction pathway in animal model of severe acute pancreatins (SAP)Methods: Thirty-six BALB/c mice ,,',,ere randomly" dMded into SAP group and antiCD14 antibody treated group. SAP was induced by administration of seven intrapentoneal injections of cerulein (50mgj&g) at hourly intervals, hpopolysaccharide (LPS) was injected 6 hours after the first cerulein injection. Treatment with the anti-CD 14 antibody' was started 15 minutes bdore the LPS inlection. Serum levels of" amylase and lactate dehydrogenase (LDH) were measured at tha end of the experiments. The severity of pancreatitis was evaluated by histological scoring using a semi-quantitative analysis ot hematoxylin and eosin-stained sections of the pancreas. Semi-quantitative raverse transcription-polymerase clmin reaction (RT-PCR) was perfomrad to quantit), fire intmpancreatic TNF-alpha,ICAM-1 and E-selectin mRNA levds The expressions of unclear factor NF-kB and polyraorphonudear leukocyte (PMN)-spectfic antigen Mac-1 were investigated by the method of immunohistochemistry and western blotting 'The activity of PMN myeloperoxidase (MPO) was detennined}y/ zymohistochemist*T, Results: Seven times cerulein injection induced edematous pancreatitis, whereas when [PS was challenged, necrotizing pancreatitis developed. At 9, 12, 24 hours afier the first cerukin injection the blood and specimens of pancreas were harvested. Serum amylase and lipase levels progressively increased vAth a maxnnum reached at 12 hours. Treatment of anti-CD14 antibody signtflcantly decreased amyIase and LDH levels at 9 and 12 hours. Histologically, treatment of ann-CD14 antibody reduced the seventy of pancreatic injury including inflammatory cell mfihration, aud necrosis at 9 and 24 hours, intrapancreatic l-NF-alpha ICAM-I and E-selectin mRNA leveIs weee reduced in the anti-CD14 antibodytreated group at 912 hours. There was significant decrease of MPO actmty in anti-CDl4 antibody group at 12 and 24 hours Imraunohist~K'hemistry and western blotting also showed down-regulation, of nuclear tactor NF-kB and PMN-specific antigen Mac-], Conclusions: ~he anti-CD 14 antibody decreased the se',*en/y of expenmental pancreatitis, suggesting that the protective effect of the anti-CD14 antibody' was mediated, at least in part, through blocking endotoxin signal pathway.
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Identification of Pancreatic Genes Expressed during the Initiation of Acute Pancreatitis: EGR-1 is a Key Regulator Chug D. Logsdon, Baoan Ji, Xue-Qing Chen, David E, Misek, Rork Kuick, Steve Ernst, Rebecca Najafian, Samir Hanash We hypothesized that genes expressed in pancreauc acinar cells during the initiation of acute pancreatins determine the severity of the disease Tberefi~re, we utilized microarrays to profile changes in gena expression and identit}/those genes commonly induced in rat pancreatic acinar cells within 1-4 hours in two in vivo models, caerulein and taurocholate administration, and one in vitro model, isolated acini treated with caerulein. This strategy yielded 72 genes common to these three models which represented a complex array of molecules, including those that are likely" to either reduce or increase the seventy" of the disease, Novel genes identified in the current study included: ATF3, BRF1, O~BPI3 and 8, CGRP, EGR-I, ephrmA1, villin2, ierredoxin, tbllistatin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, and thioredoxin reductase. To validate these microarray results, the role of EGR-1 was turther investigated in a rat model of secretagogue induced acute pancreatitis using QRT-PCR, western blotting, and immunocytocbemistry. EGR-1 expression occurred within acinar cells and correlated with the development of acute pancreatitis, To further investigate tire role oI EGR- 1 in acute pancreatitis EGWI deficient mice were treated with high concentrations of caerulein. Pancreatitis associated lung inflammation and pancreatic levels of the inflammation-related genes IL-6, MCP-I, PAI, TF and ICAM-1 were reduced in EGR-1 deficient mice compared to wild-type c(mtruls Thus, we identified EGR-1 and several other novel genes likdy to be important in tha development and seventy of acute pancreatitis
Tl192 Increased expression of nineteen kD interacting protein-3-1ike protein and the relationship to apoptosis in rat pancreatitis-associated lung injury Hayato Nakamura, Hidekazu Honda, Mitsuo Tashiru, Yasu)raki Kihara, Maknto Otsuki
Tl195 Leptin Plays An Anti-lnflammatory Role In Acute Pancreatitis Charalabos Pothoulakis, Manju Saluja, Pauline Amon, Amy Pan, Raiinder K, Dawra, Lalit C. Garg, Susan Biueber, Christos Mantzoros, Ashok K, Saluja
Background: Tha aim of the present study was to determine the underlying cellular mechamsms in the pancreas after acute pancreatitis (AP), and to stud), the pathogenesis of pancreatids-associated tung injury'. Method: We applied a difterantia[ display analysis to normal pancreas and pancreas of rats with AP, and examined the expression of the gena, identified by the analysis, in the hmg as wall as the panareas after AP Pancreatitis was induced by retrograde intradactal inclusion of 4% sodium taurocholate (100 ~xI/100 g body weight). Results: We cloned some expressed sequence tags, and identified one expressed sequence tag that was not registered in Gcnbank. Using the rapid amplification of cDNA endspolymerase chain reaction and searching the expressed sequence tags database, we obtained a transcript containing an open reading flame, The deduced protein was a polypeptidc of
AGA Abstracts
Background & Objectives: Leptin, an adipocyte-denved hormone is a product of the ob gena and functions to regulate body ,,;,eight and energy homeostasis. Leptin mediates its effect by binding to specific receptors (Ob-Rb) belonging to the cytokine super tamily, Recendy, it has shown that leptin and its receptor are also involved in the regulation of immune response m many' pathological states. Leptin receptors are known to be present in pancreas but their precise role in this organ is not well understood, in the present study, we have evaluated the role of leptin and its receptors (Ob-Rb) in acute pancreatitis, Methods:
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