Mouse model of PRSS1 p.R122H-related hereditary pancreatitis highlights context-dependent effect of autolysis-site mutation

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.     

Effects of Octreotide Pretreatment in Experimental Acute Pancreatitis

Background: Severity of systemic lesions and mortality of experimental acute pancreatitis (AP) are reduced after pancreatic enzyme content reduction induced by cerulein administration. Octreotide has been used both pro- phylatically and therapeutically in AP. The possible effects of octreotide on pancreatic enzyme content and its influence on pulmonary lesions of experimental AP were assessed in this study. Methods: Wistar male rats were divided in two branches: Branch I - Animals divided into three groups: Group Sa (n = 10) intravenous saline infusion; Group Ce (n = 10) intravenous cerulein infusion, (0.133 ig/kg ^-1· h ^-1 ) and Group Oc (n = 10) SC octreotide (10 ig/kg ^-1 ). Trypsin, elastase and amylase pancreatic contents as well as serum amylase were determined thereafter in all three groups; Branch II - Rats treated as in branch I, were submitted to sodium taurocholate AP (Groups Sa+AP, Ce+AP and Oc+AP). Two hours thereafter amylase and TAP assays were performed in serum, ascites and pancreatic tissue in eight animals of each group. Pulmonary histology was studied by morphometry 24 h after AP in the remaining animals.Results: Increased serum amylase and pancreatic enzyme contents were observed in octreotide-treated animals when compared to animals receiving saline or cerulein. After AP increases of serum and ascitic fluid amylase and of pancreatic TAP were observed in octreotide pre-treated animals when compared to saline and cerulein groups. Pulmonary interstitial and alveolar edema after AP was significantly increased in rats receiving octreotide as compared to the cerulein group.Conclusion: Octreotide administration acutely increases the enzymatic content of the pancreas and thus may have a potential deleterious influence in the evolution of AP.     

Mouse model suggests limited role for human mesotrypsin in pancreatitis

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.     

Increased serum trypsinogen 2 and trypsin 2-α1 antitrypsin complex values identify endoscopic retrograde cholangiopancreatography induced pancreatitis with high accuracy

Aims—To evaluate the clinical utility of two newtests for serum trypsinogen 2 and trypsin 2-α₁antitrypsin complex (trypsin 2-AAT) in diagnosing and assessing theseverity of acute pancreatitis (AP) induced by endoscopic retrogradecholangiopancreatography (ERCP).
Patients—Three hundred and eight consecutivepatients undergoing ERCP at Helsinki University Central Hospital in1994 and 1995.
Methods—Patients were followed prospectively forpancreatitis and clinical outcome. They were tested for serumtrypsinogen 2, trypsin 2-AAT, and amylase in samples obtained beforeand one, six, and 24 hours after ERCP.
Results—Pancreatitis developed in 31 patients(10%). Their median serum trypsinogen 2 increased 26-fold to1401 µg/l at six hours after the procedure and trypsin 2-AAT showedan 11-fold increase to 88 µg/l at 24 hours. The increase in bothmarkers was stronger in severe than in mild pancreatitis, and inpatients without pancreatitis there was no significant increase.Baseline trypsinogen 2 and trypsin 2-AAT concentrations were elevatedin 29% and 32% of patients, respectively. The diagnostic accuracy ofa threefold elevation over the baseline value was therefore analysed.The sensitivity and specificity of these parameters in the diagnosis ofpost-ERCP pancreatitis was 93% and 91%, respectively, for serumtrypsinogen 2 at six hours after the examination, and 93% and 90%,for trypsin 2-AAT at 24 hours.
Conclusions—Serum trypsinogen 2 and trypsin 2-AATreflect pancreatic injury after ERCP. High concentrations areassociated with severe pancreatic damage. The delayed increase intrypsin 2-AAT compared with trypsinogen 2 appears to reflect thepathophysiology of AP. A greater than threefold increase in trypsinogen2 six hours after ERCP is an accurate indicator of pancreatitis.

Keywords:trypsinogen 2; trypsin 2-α₁antitrypsin complex; ERCP; pancreatitis

     

Trypsin activity

A normal serum amylase level is found in up to 32% of patients with acute alcoholic pancreatitis. This underlines the need for more sensitive diagnostic tests in this frequent cause of pancreatitis. Animal and human studies have shown that chronic alcohol consumption leads to important modifications in trypsinogen metabolism. The present work has prospectively analyzed admission serum trypsin activity with a new biochemical test and usual markers such as amylase, lipase, and immunoreactive trypsin in 32 attacks of acute pancreatitis. Seventeen were due to alcohol and 15 to other causes, including 11 with gallstone pancreatitis. High trypsin activity (median: 235 units/liter; range: 165–853) was found in all patients with acute alcoholic pancreatitis even when the amylase level was normal on admission (3/17: 18%). Trypsin activity did not differ between nonalcoholic pancreatitis (N=15): 84 units/liter (42–98), alcoholic controls (N=15): 77 units/liter (40–122), and healthy controls (N=62): 81 units/liter (15–143). The difference was not related to the severity of disease or circulating α₂-macroglobulin, α₁-protease inhibitor, or immunoreactive trypsinogen levels. Lipase/amylase ratio was less discriminant than trypsin activity between alcoholic and nonalcoholic diseases. We conclude that serum trypsin activity seems specific to acute alcoholic pancreatitis and should be included in new prospective studies assessing biochemical testing of alcohol-related pancreatic diseases.     

Chronic progression of cerulein-induced acute pancreatitis in trypsinogen mutant mice

T7K24R mice carry mutation p.K24R in mouse cationic trypsinogen (isoform T7), which is analogous to the human hereditary pancreatitis-associated mutation p.K23R. The mutation renders trypsinogen more prone to autoactivation. We recently reported that T7K24R mice exhibit increased severity of acute pancreatitis induced by repeated cerulein injections. The objective of the present study was to test whether trypsinogen mutant mice are prone to develop chronic pancreatitis, as observed in patients. We characterized the natural course of cerulein-induced pancreatitis in T7K24R mice and the C57BL/6N parent strain from the acute episode to 3 months post-attack. As expected, an acute episode of pancreatitis in C57BL/6N mice was followed by rapid recovery and histological restitution. In stark contrast, T7K24R mice developed progressive chronic pancreatitis with acinar cell atrophy, persistent macrophage infiltration, and diffuse fibrosis. The nadir of pancreas damage occurred on days 5–6 after the acute episode and was accompanied by digestive dysfunction. Remarkably, histological recovery was markedly delayed and permanent, chronic changes were still detectable 1–3 months after the acute pancreatitis episode. We conclude that during cerulein-induced acute pancreatitis in T7K24R mice, trypsin triggers an autonomous inflammatory program resulting in chronic disease progression, even after the cessation of cerulein-mediated injury. We propose that this uniquely trypsin-dependent mechanism explains the development of hereditary chronic pancreatitis in humans. Trypsin inhibition during acute attacks should prevent or delay progression to chronic disease.     

Korean red ginseng ameliorated experimental pancreatitis through the inhibition of hydrogen sulfide in mice

AimEffective therapy to treat acute pancreatitis (AP) or to prevent its recurrence/complication is still not available. Based on previous results that suggest that: i) hydrogen sulfide (H₂S) levels were significantly increased in pancreatitis and gastritis and ii) Korean red ginseng (KRG) efficiently attenuated Helicobacter pylori-associated gastritis through the suppressive actions of H₂S, we hypothesized that KRG can ameliorate experimental pancreatitis through suppression of H₂S generation.MethodsC57BL/6 mice were pre-administered KRG and then subjected to cerulein injection or pancreatic duct ligation (PDL) to induce pancreatitis. Blood and pancreas tissues were collected and processed to measure serum levels of amylase, lipase and myeloperoxidase and the concentration of H₂S and the levels of various inflammatory cytokine in pancreatic tissues of mice with induced AP.ResultsKRG significantly inhibited NaHS-induced COX-2 and TNF-α mRNA in pancreatic cells, but dl-propargylglycine did not. KRG ameliorated cerulein-induced edematous pancreatitis accompanied with significant inactivation of NF-κB and JNK in pancreatic tissues of C57BL/6 mice (p < 0.001) and also significantly ameliorated PDL-induced necrotizing pancreatitis (p<0.01); in both conditions, the significant suppression of H₂S resulting from KRG pretreatment afforded rescuing outcomes. Along with suppressed levels of H₂S consequent to depressed expressions of CBS and CSE mRNA, KRG administration efficiently decreased the serum level of amylase, lipase, and myeloperoxidase and the expression of inflammatory cytokines in animal models of mild or severe AP.ConclusionsThese results provide evidence for the preventive and therapeutic roles of KRG against AP mediated by H₂S suppression.     

Effect of diabetes mellitus on pancreatic exocrine secretion from isolated perfused pancreas in rats

We studied pancreatic exocrine function in response to cerulein and carbamylcholine in isolated perfused pancreas obtained from control, streptozotocin-induced diabetic, and insulin-treated diabetic rats. The time course of pancreatic juice, protein, amylase, and trypsinogen secretion in response to cerulein or carbamylcholine in diabetic rats was similar to that in control rats. Basal as well as cerulein- or carbamylcholine-stimulated output of amylase from diabetic rat pancreas was significantly reduced, whereas that of trypsinogen was similar to the control. Amylase and trypsinogen outputs in response to 620 pM (1.0 ng/ml) cerulein from insulin-treated diabetic rat pancreas were significantly lower than those from control rat pancreas, although the pancreatic contents of these enzymes were similar to or greater than those in control rats. The dose-response curves of pancreatic juice, protein, amylase, and trypsinogen for cerulein and carbamylcholine were biphasic in both control and diabetic rats. The minimal and the maximal release in response to cerulein occurred with higher concentrations in diabetic rats compared with control rats. In contrast, the maximal responses were obtained with 1 M carbamyl-choline in control rats and with 0.1–1 M carbamylcholine in diabetic rats. The present study demonstrates that the concentration of cerulein required to elicit maximal response was increased, whereas that to carbamylcholine was reduced in diabetic rat pancreas, and that the protein and enzyme outputs in response to cerulein were significantly reduced in insulin-treated diabetic rat pancreas despite restoration of the pancreatic enzyme contents to control levels.     

Heat shock proteins and autophagy in rats with cerulein-induced acute pancreatitis

Background/Aims

Heat shock proteins (HSPs) protect rats from cerulein-induced acute pancreatitis (AP) by preventing the subcellular redistribution of cathepsin B and the activation of trypsinogen. Autophagy plays a critical role in the secretion of digestive enzymes and triggering of cerulein-induced AP via the colocalization of trypsinogen and lysosomes. Therefore, using a rat cerulein-induced AP model, we investigated whether HSPs prevent AP by regulating autophagy.

Methods

Twelve hours after fed standard laboratory chow and water, the experimental groups (cerulein, water-immersion [WI]-cerulein and heat-shock [HS]-cerulein) and the control groups (control, WI, and HS) received one intraperitoneal injection of cerulein (50 µg/kg) or saline, respectively. All of the rats were sacrificed at 6 hours after injection. The severity of the AP was assessed based on the serum amylase level and the histological and electron microscopy findings. Western blotting was also performed for HSP60/70 and LC3B-II.

Results

WI and HS induced HSP60 and HSP70, respectively. The induced HSP60/70 effectively prevented the development of cerulein-induced AP. Autophagy developed in the rats with cerulein-induced AP and was documented by the expression of LC3-II and electron microscopy findings. The WI-stressed rats and HS-treated rats did not develop cerulein-induced autophagy.

Conclusions

HSPs exert protective effects against cerulein-induced AP in rats by inhibiting autophagy.     

Phosphorylation of the MNK1 substrate eIF4E is not required for response to acute pancreatitis

BackgroundThe MNK1 protein kinase is directly activated by the MAPK pathway and is specifically expressed in pancreatic acinar cells. Both the MNK1 kinase and the MAPK pathway are required for response to pancreatitis, suggesting that their pharmacological targeting would be of therapeutic interest. Because the mRNA cap-binding protein and translation initiation factor eIF4E is the major known MNK1 substrate, one could anticipate that the protective function of MNK1 in pancreatitis is mediated by eIF4E phosphorylation.MethodsAcute pancreatitis was induced by the intraperitoneal administration of cerulein in wild-type mice and in transgenic mice carrying two non-phosphorylatable Eif4e alleles. The expression and phosphorylation of proteins of the MNK1-eIF4E pathway was visualized by western-blotting. The severity of pancreatitis was monitored by the measure of serum amylase levels and by histopathology and immunohistochemistry using apoptosis and immune infiltrate markers.ResultsDespite a strong induction in MNK1 kinase activity in both wild-type and transgenic mice, precluding eIF4E phosphorylation has no impact on the severity of acute pancreatitis. Serum amylase is equally induced in both mouse genotypes and neither acinar cell apoptosis nor immune infiltrate is exacerbated.ConclusioneIF4E phosphorylation is not required for response to pancreatitis indicating that the acinar-cell-specific MNK1 kinase acts in acute pancreatitis via another substrate.     

Acute pancreatitis-induced islet dysfunction in ferrets

Background/Objectives: The pathogenesis of hyperglycemia during acute pancreatitis (AP) remains unknown due to inaccessibility of human tissues and lack of animal models. We aimed to develop an animal model to study the mechanisms of hyperglycemia and impaired glucose tolerance in AP.MethodsWe injected ferrets with intraperitoneal cerulein (50 μg/kg, 9 hourly injections) or saline. Blood samples were collected for glucose (0, 4, 8, 12, 24h); TNF-α, IL-6 (6h); amylase, lipase, insulin, glucagon, pancreatic polypeptide (PP), glucagon-like peptide-1 (GLP-1), and gastric inhibitory polypeptide (GIP) (24h). Animals underwent oral glucose tolerance test (OGTT), mixed meal tolerance test (MMTT) at 24h or 3 months, followed by harvesting pancreas for histopathology and immunostaining.ResultsCerulein-injected ferrets exhibited mild pancreatic edema, neutrophil infiltration, and elevations in serum amylase, lipase, TNF-α, IL-6, consistent with AP. Plasma glucose was significantly higher in ferrets with AP at all time points. Plasma glucagon, GLP-1 and PP were significantly higher in cerulein-injected animals, while plasma insulin was significantly lower compared to controls. OGTT and MMTT showed abnormal glycemic responses with higher area under the curve. The hypoglycemic response to insulin injection was completely lost, suggestive of insulin resistance. OGTT showed low plasma insulin; MMTT confirmed low insulin and GIP; abnormal OGTT and MMTT responses returned to normal 3 months after cerulein injection.ConclusionsAcute cerulein injection causes mild acute pancreatitis in ferrets and hyperglycemia related to transient islet cell dysfunction and insulin resistance. The ferret cerulein model may contribute to the understanding of hyperglycemia in acute pancreatitis.     

Transgenic expression of pancreatic secretory trypsin inhibitor-I ameliorates secretagogue-induced pancreatitis in mice

BACKGROUND & AIMS: Endogenous trypsin inhibitors are believed to inhibit protease activity if trypsin becomes inadvertently activated within the acinar cell. However, this action remains unproven, and the role of endogenous pancreatic trypsin inhibitors in acute pancreatitis is unknown. In this study, we tested whether increased levels of pancreatic secretory trypsin inhibitor-I (PSTI-I) in mice could prevent secretagogue-induced pancreatitis. METHODS: Rat PSTI-I expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the rat elastase I enhancer/promoter. Secretagogue-induced pancreatitis was achieved by 12 hourly intraperitoneal injections of caerulein. The severity of pancreatitis was assessed by measurements of serum amylase, histologic grading, and pancreas wet weight-to-body weight ratio. Trypsinogen activation and trypsin activity were measured in pancreatic extracts. RESULTS: Targeted expression of PSTI-I to the pancreas increased endogenous trypsin inhibitor capacity by 190% (P <.01) in transgenic vs. nontransgenic mice. Caerulein administration to nontransgenic mice produced histologic evidence of acute pancreatitis, and significantly elevated serum amylase and pancreas weight ratio. In caerulein-treated transgenic mice, the histologic severity of pancreatitis was significantly reduced. There was no difference in trypsinogen activation peptide levels between caerulein-treated transgenic and nontransgenic mice. However, trypsin activity was significantly lower in transgenic mice receiving caerulein compared with nontransgenic mice. CONCLUSIONS: These data demonstrate that the severity of secretagogue-induced pancreatitis is significantly ameliorated in mice with higher pancreatic levels of trypsin inhibitor. We propose that PSTI-I prevents pancreatitis by inhibiting the activity of trypsin, rather than by reducing trypsinogen activation.     

磷脂酰肌醇3-激酶γ对急性胰腺炎小鼠胰腺腺泡细胞自噬作用的影响

目的 探讨磷脂酰肌醇3-激酶γ(PI3Kγ)对小鼠实验性急性胰腺炎胰腺腺泡细胞自噬作用的影响,并探讨其意义.方法 野生型C57 BL/6小鼠和PI3Kγ基因敲除小鼠各18只,按数字表法随机分为对照组(6只)和急性胰腺炎(AP)组(12只),采用蛙皮素50μg/kg体重腹腔内注射7次、每次间隔1h的方法制备AP模型.首次注射后7h处死小鼠,光镜下观察胰腺病理学变化,免疫荧光检测自噬泡主要组成蛋白LC3颗粒,荧光分光光度计测定胰蛋白酶活性,蛋白质印迹法检测自噬相关蛋白beclin1、LC3-Ⅱ和p62的表达.结果 AP组野生型小鼠和PI3Kγ基因敲除小鼠的胰腺自噬空泡数量分别为(5.14±0.85)、(2.25±0.54)个/每高倍视野(HPF),LC3荧光免疫颗粒数量分别为(78.6±9.38)、(26.4±4.21)个/HPF,胰蛋白酶活性分别为(0.827±0.126)、(0.358±0.098) pmol/mg蛋白,各组间差异均具有统计学意义(P值均＜0.05).野生型小鼠的p62蛋白表达较PI3Kγ基因敲除小鼠显著减弱(0.11比0.92,P＜0.05),面LC3 -Ⅱ、beclin1蛋白表达较PI3Kγ基因敲除小鼠显著增强(1.82比0.93,1.43比1.05,P值均＜0.05).结论 AP时PI3Kγ可能通过增强小鼠胰腺腺泡细胞的自噬作用,促进胰蛋白酶原的活化及诱导腺泡细胞坏死.     

Skeletal muscle-specific Sidt2 knockout in mice induced muscular dystrophy-like phenotype

BackgroundSidt2 is an integral lysosomal membrane protein. Previously, we generated a Sidt2 global knockout mouse and found impaired insulin secretion, along with skeletal muscle pathology.MethodsA mouse model with a muscle-specific knockout of the Sidt2 gene (Sidt2^f/fCre) had been generated, to which extensive morphologic study as well as functional study was applied to investigate the direct role of Sidt2 on skeletal muscle tissue in vivo. Secondly, the autophagy-lysosomal pathway was examined by Western blot and immunostaining. Additionally, RNA expression changes in Sidt2^f/fCre mice were analyzed by genechip.ResultsSidt2 deficiency in skeletal muscle results in pathognomonic hallmarks of muscular dystrophy, including muscle mass decrease, muscle weakness, fibrosis, central nucleation, fiber regeneration, mildly elevated serum creatine kinase, and dramatically elevated sarcolipin mRNA. Along with accumulation of autophagolysomes, LC3-II, adaptor protein p62, ubiquitinated aggregates, and Lamp2-positive vacuoles were increased significantly in Sidt2^f/fCre skeletal muscle fibers. However, only lysosomal-related genes were upregulated, while the genes upstream of the autophagy pathway were unchanged. Simultaneously, the proteasome chymotryptic activity and the lysosomal soluble enzyme activity were unimpaired, which largely excluded the possibility of proteasome chymotryptic activity defect and the lysosomal soluble enzyme defect leading to ubiquitinated aggregates accumulation.ConclusionWe concluded that Sidt2 deficiency leads to muscular dystrophy-like phenotype in mice and Sidt2 plays a critical role in the late stage of autophagy.     

Simvastatin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer

Background and aimsPancreatic cancer is among the most dismal of human malignancies. There are no chemopreventive strategies for pancreatic cancer or its precursor lesions, pancreatic intraepithelial neoplasia (PanINs). Recent evidence suggests that statins have potential chemopreventive abilities. In this study, we used a genetically engineered mouse model of pancreatic cancer to evaluate the chemopreventive potential of this drug.MethodsSimvastatin was injected i.p. in LsL-Kras^G12D; Pdx1-Cre or LsL-Kras^G12D;LsL-Trp53^R172H;Pdx1-Cre mice. After five months, animals were sacrificed. The effect of simvastatin was evaluated by histopathological analyses, immunostaining, and real-time PCR.ResultsAfter five months of treatment, simvastatin was able to significantly delay progression of mPanINs in LsL-Kras^G12D; Pdx1-Cre mice. Furthermore, formation of invasive pancreatic cancer in LsL-Kras^G12D; LsL-Trp53^R172H; Pdx1-Cre transgenic mice was partially inhibited by simvastatin. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-Kras^G12D; LsL-Trp53^R172H;Pdx1-Cre untreated control mice. In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p = 0.004) developed murine pancreatic cancer during the study. Using real-time PCR we found a significant up-regulation of Hmgcr as sign of blocking HMG-CoA reductase, a key enzyme in the cholesterol biosynthesis. This shows our ability to achieve effective pharmacologic levels of simvastatin during pancreatic cancer formation in vivo.ConclusionUsing a transgenic mouse model that recapitulates human pancreatic cancer, this study provides first evidence that simvastatin is an effective chemopreventive agent by delaying the progression of PanINs and partially inhibit the formation of murine pancreatic cancer.     

Effects of Mast-Cell Stabilization in Cerulein-Induced Acute Pancreatitis in Rats

Summary Aim. In this study we aimed to clarify the role of mast cells in the development and progression of inflammation in cerulein-induced acute pancreatitis (AP) in rats. We have also examined the effects of ketotifen; a mast-cell stabilizing agent in the treatment of acute pancreatitis and its relation with nitric oxide (NO) synthesis. Methods. In the first part of the study we planned to examine the effects mast cell stabilization in acute pancreatitis, while the second part was focused on examining the relation between NO synthesis and the potential effects of ketotifen in AP. Wistar albino rats were randomly divided into 6 groups (n: 10). In the first part of the study, AP was induced by four subcutaneous (sc) injections of 20 μg/kg body weight of cerulein at hourly intervals in Groups A and B while Group C was treated with saline as the control group. Group B was pretreated with ketotifen 1 mg/kg (ip). In the second part, the study design was similar except for the inhibition of nitric oxide synthesis by N-nitro L-arginine methyl ester (L-NAME) 30 mg/kg (ip) in Groups D, E and F. Group D was treated with L-NAME and cerulein and Group E was treated with ketotifen, L-NAME and cerulein. Group F was treated with L-NAME and saline as the control group. Serum amylase activity and pancreatic myeloperoxidase activity (MPO) were measured. Pancreatic histology and mast-cell count in pancreatic tissue were evaluated. Results. Mast cell count was found to be increased in the pancreatic tissue in cerulein-induced AP. (2.93±0.26 vs 1.98±0.26; p<0.001). Ketotifen treatment significantly reduced cerulein induced edema (1.30±0.21 vs 0.70±0.15; p<0.001), neutrophil infiltration (1.50±0.16 vs 0.60±0.16; p<0.001) and attenuated the increase in amylase (4394.0±149.5 U/L vs 3350.5±216.9 U/L; p<0.05) and MPO activity (1.14±0.13 U/gr tissue vs 0.54±0.08 U/gr tissue; p<0.001). Mast-cell count in pancreatic tissue was also decreased significantly with ketotifen pretreatment (2.93±0.26 vs 1.70±0.21; p<0.05). Inhibition of NO synthesis with L-NAME treatment decreased the beneficial effects of ketotifen. Conclusion. It seems likely that mast cell activity may play an important role in the initiation and progression of acute pancreatitis. Ketotifen treatment may reduce the severity of AP in rats. The protective action of ketotifen in cerulein-induced acute pancreatitis is most probably owing to mast cell stabilization and stimulation of NO synthesis.     

Expression of Nitric Oxide Synthase Isoforms and Nitric Oxide Production in Acute Pancreatitis and Associated Lung Injury

Background/Aims: The role of nitric oxide (NO) has been increasingly implicated in the pathophysiology of acute pancreatitis (AP). Studies have shown increased NO production in AP although not all are agreeable on whether NO is beneficial or detrimental in AP. This study aims to profile NO production and NO synthase (NOS) expression in the pancreas and lungs in the progression of AP in mice to gain insights to the role played by different NOS isoforms. Methods: AP was induced in mice by hourly administration of cerulein. NO production was determined by measuring the total nitrite and nitrate (NOx) content while NOS expression was measured by Western blot. Results: Pancreatic NO production increased sharply and was sustained throughout AP. iNOS expression was greatly increased while eNOS was downregulated at the later stages. In the lungs, there was an unexpected early increase in the constitutive NOS expression; however iNOS was also significantly overexpressed at the later time point along with a significant increase in NO. Acinar cells were found to overproduce NO in response to cerulein hyperStimulation with iNOS again being the major contributor. Conclusion: These data show that NO production and NOS expression are differentially regulated temporally and in magnitude in the pancreas and lungs in response to cerulein hyperstimulation which suggests differing roles for each NOS isoform.     

Activation of proteases in cerulein-induced pancreatitis

The activation of zymogen proteases and lysosomal enzyme cathepsin B in the pancreas was investigated in cerulein-induced pancreatitis in rats. Acute pancreatitis was induced by two intraperitoneal injections of 40 micrograms/kg of body weight of cerulein at intervals of 1 h. After the first cerulein injection, the active trypsin and elastase contents in the pancreas tissues significantly increased, and reached the highest level at 3 h after the first injection, followed by peaks at 5 h in the serum amylase and lipase levels and the pancreas wet weight. Cathepsin B contents in pancreas tissues showed a parallel increase with active zymogen enzymes during the first 3 h of pancreatitis. These findings may suggest that the intracellular activation of trypsinogen is an important step in the development of cerulein-induced acute pancreatitis and that cathepsin B plays a role in the activation of trypsinogen in pancreatic acinar cells.