Acinar Cell Necrosis Research Articles

TRPM2-mediated extracellular Ca2+ entry promotes acinar cell necrosis in biliary acute pancreatitis.

Acute biliary pancreatitis is a significant clinical challenge as currently no specific pharmaceutical treatment exists. Intracellular Ca2+ overload, increased reactive oxygen species (ROS) production, mitochondrial damage and intra-acinar digestive enzyme activation caused by bile acids are hallmarks of acute biliary pancreatitis. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that has recently emerged as an important contributor to oxidative-stress-induced cellular Ca2+ overload across different diseases. We demonstrated that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar and ductal cells, which can be activated by increased oxidative stress induced by H2 O2 treatment and contributed to bile acid-induced extracellular Ca2+ influx in acinar cells, which promoted acinar cell necrosis in vitro and in vivo. These results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis. Acute biliary pancreatitis poses a significant clinical challenge as currently no specific pharmaceutical treatment exists. Disturbed intracellular Ca2+ signalling caused by bile acids is a hallmark of the disease, which induces increased reactive oxygen species (ROS) production, mitochondrial damage, intra-acinar digestive enzyme activation and cell death. Because of this mechanism of action, prevention of toxic cellular Ca2+ overload is a promising therapeutic target. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that has recently emerged as an important contributor to oxidative-stress-induced cellular Ca2+ overload across different diseases. However, the expression and possible functions of TRPM2 in the exocrine pancreas remain unknown. Here we found that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar and ductal cells, which can be activated by increased oxidative stress induced by H2 O2 treatment. TRPM2 activity was found to contribute to bile acid-induced extracellular Ca2+ influx in acinar cells, but did not have the same effect in ductal cells. The generation of intracellular ROS in response to bile acids was remarkably higher in pancreatic acinar cells compared to isolated ducts, which can explain the difference between acinar and ductal cells. This activity promoted acinar cell necrosis in vitro independently from mitochondrial damage or mitochondrial fragmentation. In addition, bile-acid-induced experimental pancreatitis was less severe in TRPM2 knockout mice, whereas the lack of TRPM2 had no protective effect in cerulein-induced acute pancreatitis. Our results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis.

Functional and histological changes of the pancreas and the liver in the rats after the acute and subacute administration of diazinon

Background/Aim. Organophosphate pesticides (OPs) are used extensively worldwide in agriculture and forestry, and their application represents a major health problem for humans and animals. The aim of this study was to investigate the possibility of the adaptation of an organism to the pro-longed administration of a low dose of diazinon. Methods. The study was conducted on a total of 60 male Wistar rats. The first 30 rats were divided into four equal diazinon groups (n = 6) and the control one (corn oil). Diazinon was orally administered once at doses: 200, 400, 600, 800 mg/kg (one dose ? one group). The concentration of glucose, the activity of ?-amylase and the relative activity of LDH1-LDH5 isoenzymes in the blood were measured 24 hours after the application. The remaining 30 rats were divided into two equal diazinon groups (n = 10) and the control one (corn oil). The first group was treated during 7 days, and the second during 14 days with 55 mg/kg of diazinon (1/10 of previously determined LD50 value). The histopathology of the pancreas and the liver, as well as the relative activities of LDH isoenzymes in the blood, were determined after the completion of both time periods. Results. Single administration of increasing doses of diazinon resulted in a significant increase in the concentrations of glucose, activity of ?-amylase and LDH isoenzymes. Subacute application of a low diazinon dose induced histopathological changes in the pancreas manifested by acinar cell necrosis, and in the liver in the form of portal hepatitis and multifocal necrosis. The cumulative doses resulted in statistically significantly lower activities of LDH isoenzymes compared with the single administration of these doses, indicating a lower degree of the cells damage after the subacute diazinon administration. Conclusion. Subacute administration of a low dose of diazinon leads to a different adaptation degree of organs and organ systems to toxic effects caused by this organophosphate.

Lower Nr5a2 Level Downregulates the β-Catenin and TCF-4 Expression in Caerulein-Induced Pancreatic Inflammation.

Nuclear receptor subfamily 5 group A member 2 (Nr5a2) is widely involved in the physiological and pathological processes of the pancreas. However, the cytological and molecular evidence regarding how Nr5a2 implicated in acute pancreatitis (AP) remains insufficient. Here, we explored this problem by using cellular AP model in both normal and Nr5a2 silenced AR42J pancreatic acinar cells. An in vitro cellular model of AP was established by stimulating AR42J cells with caerulein (CAE) for 24 h. Reduced Nr5a2 expression was observed in the CAE-treated cells. Nr5a2 silencing led to AP-like inflammation, with increased interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α mRNA levels. In the cellular AP model, Nr5a2 silencing further increased IL-1β, IL-6, and TNF-α mRNA levels, as well as amylase activity. In addition, we found that Nr5a2 silencing did not affect IL-10 level under physiological conditions but inhibited the anti-inflammatory response of IL-10 in AP model. Moreover, in CAE-induced pancreatic inflammation, Nr5a2 silencing increased the apoptosis and necrosis of acinar cells and inhibited the proliferation of acinar cells, which has not been shown previously. Further experiments showed, for the first time, that Nr5a2 silencing downregulated the expression of β-catenin and its downstream target gene T-cell factor (TCF)-4 in the cellular AP model but increased the expression of nuclear factor (NF)-κB. In conclusion, in CAE-induced pancreatic inflammation, lower Nr5a2 level leads to downregulation of β-catenin and its downstream target gene TCF-4 and upregulation of NF-κB, which exacerbates the inflammatory response and cell damage and inhibits the proliferation and regeneration of acinar cells.

Docosahexaenoic Acid Inhibits Expression of Fibrotic Mediators in Mice With Chronic Pancreatitis.

BackgroundChronic pancreatitis (CP) is an irreversible progressive disease that destroys exocrine parenchyma, which are replaced by fibrous tissue. As pancreatic fibrosis is a key feature of CP, reducing fibrotic protein content in the pancreas is crucial for preventing CP. Studies suggest that NF-κB facilitates the expression of fibrotic mediators in pancreas and protein kinase C-δ (PKC-δ) regulates NF-κB activation in stimulated pancreatic acinar cells. Docosahexaenoic acid (DHA) is an omega-3 fatty acid having anti-inflammatory and anti-fibrotic effects. It has been shown to inhibit NF-κB activity in cerulein-stimulated pancreatic acinar cells which is a cellular model of CP. In the present study, we investigated if DHA inhibits expression of fibrotic mediators by reducing PKC-δ and NF-κB expression in mouse pancreatic tissues with CP.MethodsFor six weeks, mice were weekly induced for acute pancreatitis to develop CP. Furthermore, acute pancreatitis was induced by hourly intraperitoneal injections of cerulein (50 μg/kg × 7). Mice were administered DHA (10 μM) via drinking water before and after CP induction.ResultsCerulein-induced pancreatic damages like decreased pancreatic weight/total body weight, leukocyte infiltration, necrosis of acinar cells, and vacuolization were found to be inhibited by DHA. Additionally, DHA inhibited cerulein-induced fibrotic mediators like alpha-smooth muscle actin and fibronectin in pancreas. DHA reduced expression of PKC-δ and NF-κB p65 in pancreatic tissues of cerulein-treated mice.ConclusionsDHA may be beneficial in preventing CP by suppressing pancreatic expression of fibrotic mediators.

Chrm3 protects against acinar cell necrosis by stabilizing caspase-8 expression in severe acute pancreatitis mice model.

Acinar cells in acute pancreatitis (AP) die through apoptosis and necrosis, the impacts of which are quite different. Early clinical interference strategies on preventing the progress of AP to severe acute pancreatitis (SAP) are the elimination of inflammation response and inhibition of necrosis. Muscarinic acetylcholine receptor M3 was encoded by Chrm3 gene. It is one of the best-characterized receptors of pancreatic β cells and regulates insulin secretion, but its function in AP remains unclear. In this study, we explored the function of Chrm3 gene in the regulation of cell death in l-arginine-induced SAP animal models. We found that Chrm3 was upregulated in pancreatitis, and we further confirmed the localization of Chrm3 resided in both pancreatic islets and acinar cell membranes. The reduction of Chrm3 decreased the pathological lesion of SAP and reduced amylase activities in serum. Consistently, Chrm3 can suppress acinar cells necrosis markedly, but has no effect on regulating apoptosis after l-arginine treatment. It was shown that Chrm3 attenuated acinar cells necrosis at least in part by stabilizing caspase-8. Thus, this study indicates that Chrm3 is critical participants in SAP, and regulation of Chrm3 expression might be a useful therapeutic strategy for preventing pathologic necrosis.

NQDI-1 protects against acinar cell necrosis in three experimental mouse models of acute pancreatitis

NQDI-1, an inhibitor of ASK1, has been reported to have protective effects in several experimental human disease models. However, the role of NQDI-1 in acute pancreatitis (AP) has not been reported. In this study, we found that NQDI-1 could attenuate histological damage of pancreatic tissue as well as the levels of serum amylase and lipase in a mouse model of AP induced by caerulein. Moreover, the production of reactive oxygen species (ROS) and the expression of necrosis-related proteins (RIP3 and p-MLKL) were also reduced after NQDI-1 administration. Correspondingly, we elucidated the effect of NQDI-1 in vitro and found that NQDI-1 protected against pancreatic acinar cells necrosis via decreasing the ROS production and RIP3 and p-MLKL expression. In addition, we identified the protective effect of NQDI-1 on AP through two other mouse models induced by l-arginine and pancreatic duct ligation. Taken together, these findings showed that NQDI-1 could reduce the acinar cells necrosis and alleviate the severity of AP, which may afford a new therapeutic target on pancreatic necrosis in AP clinically.

MicroRNAs in acute pancreatitis: From pathogenesis to novel diagnosis and therapy.

Acute pancreatitis (AP) is an inflammatory disorder initiated by activation of pancreatic zymogens, leading to pancreatic injury and systemic inflammatory response. MicroRNAs (miRNAs) have emerged as important regulators of gene expression and key players in human physiological and pathological processes. Discoveries over the past decade have confirmed that altered expression of miRNAs is implicated in the pathogenesis of AP. Indeed, a number of miRNAs have been found to be dysregulated in various cell types involved in AP such as acinar cells, macrophages, and lymphocytes. These aberrant miRNAs can regulate acinar cell necrosis and apoptosis, local and systemic inflammatory response, thereby contributing to the initiation and progression of AP. Moreover, patients with AP possess unique miRNA signatures when compared with healthy individuals or those with other diseases. In view of their stability and easy detection, therefore, miRNAs have the potential to act as biomarkers for the diagnosis and assessment of patients with AP. In this review, we provide an overview of the novel cellular and molecular mechanisms underlying the roles of miRNAs during the disease processes of AP, as well as the potential diagnosis and therapeutic biomarkers of miRNAs in patients with AP.

Can we predict recurrence in IAP based on EUS and clinical factors?

Acute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown.To uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function.After the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice.TLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Management of infected pancreatic necrosis: a single center experience

Acute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown.To uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function.After the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice.TLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Inhibition of RIPK1-dependent regulated acinar cell necrosis provides protection against acute pancreatitis via the RIPK1/NF-κB/AQP8 pathway

Currently, preliminary results have confirmed the existence of receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL)-dependent necroptosis of pancreatic acinar cells during early acute pancreatitis (AP), which might be a potential target for the effective regulation of necroinflammatory injury. However, the exact effect of receptor-interacting protein kinase 1 (RIPK1)-dependent regulated acinar cell necrosis on AP is still uncertain. In our study, we first explored the changes in the degree of local and systemic inflammation in AP rats when the activation of acinar cell RIPK1 was inhibited. The RIPK1 inhibitor Nec-1 was used to treat rats, and the levels of related inflammatory markers, necrosis indicators and apoptotic indicators were measured. Changes in pancreatic nuclear factor κB (NF-κB) and aquaporin 8 (AQP8) expression were noted. Next, the expression of AQP8 in AR42J cells was inhibited, and the degree of cell necrosis and inflammatory damage was found to be significantly reduced. Most importantly, we demonstrated that the RIPK1/NF-ĸB/AQP8 axis might be a potential regulatory pathway mediating RIPK1-dependent regulated acinar cell necrosis in early AP. Finally, we used the NF-κB inhibitor PDTC and Nec-1 to treat rats in different groups and measured the degree of pathological pancreatic injury, the activation of RIPK1, and the expression of NF-κB and AQP8. In summary, we hypothesized that there might be a RIPK1/NF-ĸB/AQP8 pathway controlling RIPK1-dependent regulated necrosis of acinar cells in AP, which might be a promising therapeutic target against AP-related injury.

Identification of novel and rare recurrent genetic variants in early onset chronic pancreatitis by whole exome sequencing

Acute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown.To uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function.After the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice.TLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Mitochondrial Targeting of Antioxidants Alters Pancreatic Acinar Cell Bioenergetics and Determines Cell Fate

Mitochondrial dysfunction is a core feature of acute pancreatitis, a severe disease in which oxidative stress is elevated. Mitochondrial targeting of antioxidants is a potential therapeutic strategy for this and other diseases, although thus far mixed results have been reported. We investigated the effects of mitochondrial targeting with the antioxidant MitoQ on pancreatic acinar cell bioenergetics, adenosine triphosphate (ATP) production and cell fate, in comparison with the non-antioxidant control decyltriphenylphosphonium bromide (DecylTPP) and general antioxidant N-acetylcysteine (NAC). MitoQ (µM range) and NAC (mM range) caused sustained elevations of basal respiration and the inhibition of spare respiratory capacity, which was attributable to an antioxidant action since these effects were minimal with DecylTPP. Although MitoQ but not DecylTPP decreased cellular NADH levels, mitochondrial ATP turnover capacity and cellular ATP concentrations were markedly reduced by both MitoQ and DecylTPP, indicating a non-specific effect of mitochondrial targeting. All three compounds were associated with a compensatory elevation of glycolysis and concentration-dependent increases in acinar cell apoptosis and necrosis. These data suggest that reactive oxygen species (ROS) contribute a significant negative feedback control of basal cellular metabolism. Mitochondrial targeting using positively charged molecules that insert into the inner mitochondrial member appears to be deleterious in pancreatic acinar cells, as does an antioxidant strategy for the treatment of acute pancreatitis.

Protective effects of HTD4010, a Reg3α/PAP-derived peptide, in mouse model of acute pancreatitis via toll-like receptor 4 pathway

Acute pancreatitis (AP) is one of the most common digestive tract diseases, but effective drug therapy is still lack. Regenerating gene protein 3α (Reg3α) administration significantly reduced the severity of AP in mice. HTD4010 is a new 15 amino acid long synthetic peptide and its biological activities are similar to Reg3α. This study aimed to explore whether HTD4010 could protect pancreatic acinar cells against necrosis and decrease the inflammatory response in AP, and thus to explore underlying mechanisms. It was shown that administration of HTD4010 alleviated significantly the severity of biliary AP (BAP), characterized as less degree of pancreatic histological damage and acinar cell injury (both apoptosis and necroptosis), lower levels of serum amylase and pro-inflammatory cytokines. Moreover, HTD4010 down-regulated the expression of toll-like receptor 4 (TLR4) protein, and TLR4 deficiency eliminated the protective effect of HTD4010 on BAP in mice. In conclusion, these results showed that HTD4010 could alleviate the severity of pancreatitis, reduce the acinar cells necrosis and inflammatory response possibly by TLR4 signaling pathway in AP.

C-Abl kinase regulates neutrophil extracellular trap formation, inflammation, and tissue damage in severe acute pancreatitis

Neutrophil extracellular traps (NETs) are involved in acute pancreatitis (AP) but mechanisms controlling NET expulsion in AP are incompletely understood. Herein, we examined the role of c-Abelson (c-Abl) kinase in NET formation and tissue damage in severe AP. AP was induced by taurocholate infusion into pancreatic duct or intraperitoneal administration of l-arginine in mice. Pancreatic, lung, and blood samples were collected and levels of phosphorylated c-Abl kinase, citrullinated histone 3, DNA-histone complexes, myeloperoxidase, amylase, cytokines, and CXC chemokines were quantified. Citrullinated histone 3, reactive oxygen species (ROS), and NET formation were determined in bone marrow neutrophils. Taurocholate challenge increased phosphorylation of c-Abl kinase and levels of citrullinated histone 3 in the pancreas as well as DNA-histone complexes in the plasma. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase but also decreased levels of citrullinated histone 3 in the pancreas and DNA-histone complexes in the plasma of animals with AP. Moreover, GZD824 decreased plasma levels of amylase, IL-6, and MMP-9 as well as edema, acinar cell necrosis, hemorrhage, CXC chemokine formation, and neutrophil infiltration in the inflamed pancreas. A beneficial effect of c-Abl kinase inhibition was confirmed in l-arginine-induced pancreatitis. In vitro, inhibition of c-Abl kinase reduced TNF-α-induced formation of ROS, histone 3 citrullination, and NETs in isolated bone marrow neutrophils. Our findings demonstrate that c-Abl kinase regulates NET formation in the inflamed pancreas. In addition, inhibition of c-Abl kinase reduced pancreatic tissue inflammation, and damage in AP. Thus, targeting c-Abl kinase might be a useful way to protect the pancreas in severe AP.

Loss of TLR3 and its downstream signaling accelerates acinar cell damage in the acute phase of pancreatitis

BackgroundAcute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown. MethodsTo uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function. ResultsAfter the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice. ConclusionsTLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis.

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl-amidine, a pan-PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and DNA-histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl-amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl-amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl-amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL-6, and MMP-9 in mice with severe AP. This study shows that Cl-amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.

Ximenia americana heteropolysaccharides ameliorate inflammation and visceral hypernociception in murine caerulein-induced acute pancreatitis: Involvement of CB2 receptors

BackgroundThis study aimed to investigate and characterize the anti-inflammatory and anti-hypernociceptive effects of the total polysaccharides of X. americana (TPL-Xa) bark in a mouse model of acute pancreatitis-induced by caerulein and the potential involvement of cannabinoid receptors. MethodsTPL-Xa was characterized by1H and 13C NMR spectroscopy. Animals received TPL-Xa (10 mg/kg, i.v.) 30 min before and after caerulein (50 μg/kg, 10×, i.p.) administration. To evaluate the involvement of cannabinoid receptors, AM281 (3 mg/kg, s.c.) and AM630 (1 mg/kg, s.c.) were administered 30 min before TPL-Xa. Plasma levels of amylase and lipase, pancreatic myeloperoxidase (MPO), histology, visceral hypernociception and motor coordination were evaluated 11 and 24 h after acute pancreatitis (AP) induction. ResultsTPL-Xa, containing a heteropolysaccharide composed of glucose, galactose, arabinose, rhamnose, fucose and galacturonic acid, reduced amylase and lipase levels, MPO activity, acinar cell necrosis, edema and neutrophil infiltration. TPL-Xa increased the threshold of visceral hypernociception, an effect reversed by AM630, an antagonist of cannabinoid receptor type 2 (CB2). In addition, TPL-Xa did not alter the animals’ motor coordination. ConclusionsTPL-Xa contains heteropolysaccharides that inhibit inflammation and hypernociception in the experimental model of caerulein-induced AP, by a mechanism involving type CB2 receptors.

Abstract 1249: Animal model in the prevention of alcoholic pancreatitis

Abstract Background: Alcohol abuse is a major risk factor for pancreatitis. Alcohol addiction-induced molecular pathogenesis of pancreatitis remains obscure, and no current effective treatment exists. Therefore, approaches to investigate pathogenesis, prevention and cellular mechanisms by which alcohol causes pancreatitis are necessary for establishing therapeutics. Our efforts demonstrate that alcohol induces activation of a major intracellular transcriptional factor, cyclic AMP response element-binding protein (CREB). We further investigated the functional role of CREB in alcohol-induced pathogenesis of pancreatitis using cellular and genetic mouse models of pancreas. Materials and Methods: Human tissue microarrays were immunostained to determine the significance of pCREB expression among pancreatic tissues obtained from normal and chronic pancreatitis. Rat acinar cell line AR42J and mouse PSCs (mPSCs) were exposed to alcohol (50 mMol/L). Inducible Ptf1aCreERTM knockin mice and Ptf1aCreERTM;CREBfl/fl (iPC) mice were fed with Lieber Decarli diet alcohol or regular diet for 14 weeks with or without caerulein (50 µg/kg). Mice were then euthanized 24 hours after the last caerulein injection, and pancreas tissues were processed for morphometric analysis (necrosis, vacuolization, hemorrhage, edema and inflammation) and immunohistochemical analysis of amylase, trichrome blue, SMA, collagen 1, fibronectin and pCREB expression. To determine whether alcohol accelerated morbidity in mice, we evaluated pathogenesis of chronic pancreatitis by analyzing acinar atrophy and pancreatic fibrosis. Results: Expression of pCREB was significantly higher (p <0.001) in chronic pancreatitis vs. normal patient tissues, confirming the role of activated CREB in pancreatitis. Activated CREB levels were very high in alcohol-fed Ptf1aCreERTM mice when compared with control diet-fed mice. Pancreatic sections from alcohol-fed mice challenged with caerulein revealed significantly higher score of acinar cell vacuolization and necrosis, inflammatory infiltrate and hemorrhage compared with minimal lesions in control diet-fed animals receiving caerulein. Pancreatic sections from alcohol-fed Ptf1aCreERTM animals showed higher score of histologic injury, extracellular matrix deposition, collagen deposition and increased pancreatic fibrosis when compared with control-fed mice. iPC mice showed decrease in the pathogenesis of chronic pancreatitis when compared to Ptf1aCreERTM mice with alcohol. Conclusion: CREB is overexpressed in pancreatitis and alcohol activates CREB, which then drives pathogenesis of pancreatitis. Severity of pancreatitis in response to alcohol is diminished in the absence of CREB. Therefore, we conclude that targeting CREB represents a promising treatment for alcohol-induced pancreatitis. Citation Format: Supriya Srinivasan, Tulasigeri Totiger, Michael VanSaun, Fanuel Messaggio, Chanjuan Shi, Austin Dosch, Eric Nestler, Nipun Merchant, Nagaraj Nagathihalli. Animal model in the prevention of alcoholic pancreatitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1249.

Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

Severe acute pancreatitis is a lethal inflammatory disease frequently accompanied by pancreatic necrosis. We aimed to identify a key regulator in the development of pancreatic necrosis. A cytokine/chemokine array using sera from patients with acute pancreatitis (AP) revealed that serum CXCL16 levels were elevated according to the severity of pancreatitis. In a mouse model of AP, Cxcl16 expression was induced in pancreatic acini in the late phase with the development of pancreatic necrosis. Cxcl16−/− mice revealed similar sensitivity as wild-type (WT) mice to the onset of pancreatitis, but better resisted development of acinar cell necrosis with attenuated neutrophil infiltration. A cytokine array and immunohistochemistry revealed lower expression of Ccl9, a neutrophil chemoattractant, in the pancreatic acini of Cxcl16−/− mice than WT mice. Ccl9 mRNA expression was induced by stimulation with Cxcl16 protein in pancreatic acinar cells in vitro, suggesting a Cxcl16/Ccl9 cascade. Neutralizing antibody against Cxcl16 ameliorated pancreatic injury in the mouse AP model with decreased Ccl9 expression and less neutrophil accumulation. In conclusion, Cxcl16 expressed in pancreatic acini contributes to the development of acinar cell necrosis through the induction of Ccl9 and subsequent neutrophil infiltration. CXCL16 could be a new therapeutic target in AP.

INT-777, a bile acid receptor agonist, extenuates pancreatic acinar cells necrosis in a mouse model of acute pancreatitis

Bile acids receptor TGR5 and its agonist INT-777, which has been found to be involved in the NLRP3 inflammasome pathway, play an important role in inflammatory diseases. However, the role of INT-777 in acute pancreatitis (AP) has not been reported. In this present study, we found that TGR5 was expressed in pancreatic tissue and increased after AP onset induced by caerulein and further evaluated the impact of INT-777 on the severity of AP. The results showed that INT-777 could reduce the severity of AP in mice, which was manifested as decreased pancreatic tissue damage as well as the decrease of serum enzymes (amylase and lipase), pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and the expression of necrosis related proteins (RIP3 and p-MLKL). Furthermore, we found that INT-777 reduced the reactive oxygen species (ROS) production in pancreatic acinar cells and inhibited the activation of NLRP3 inflammasome pathway. In conclusion, our data showed that INT-777 could protect pancreatic acinar cell against necrosis and reduce the severity of AP, which may be mediated by inhibiting ROS/NLRP3 inflammasome pathway.