Caerulein Hyperstimulation Research Articles

A novel resveratrol analog upregulates sirtuin 1 and inhibits inflammatory cell infiltration in acute pancreatitis.

Acute pancreatitis (AP), an inflammatory disorder of the pancreas, is a complicated disease without specific drug therapy. (R)-4,6-dimethoxy-3-(4-methoxy phenyl)-2,3-dihydro-1H-indanone [(R)-TML104] is a synthesized analog of the natural product resveratrol sesquiterpenes (±) -isopaucifloral F. This study aimed to investigate the effect and underlying mechanism of (R)-TML104 on AP. The experimental AP model was induced by caerulein hyperstimulation in BALB/c mice. (R)-TML104 markedly attenuated caerulein-induced AP, as evidenced by decreased pancreatic edema, serum amylase levels, serum lipase levels, and pancreatic myeloperoxidase activity. In addition, (R)-TML104 significantly inhibited the expression of pancreatic chemokines C-C motif chemokine ligand 2 and macrophage inflammatory protein-2 and the infiltration of neutrophils and macrophages. Mechanistically, (R)-TML104 activated AMP-activated protein kinase and induced sirtuin 1 (SIRT1) expression. (R)-TML104 treatment markedly induced the SIRT1-signal transducer and activator of transcription 3 (STAT3) interaction and reduced acetylation of STAT3, thus inhibiting the inflammatory response mediated by the interleukin 6-STAT3 pathway. The effect of (R)-TML104 on SIRT1-STAT3 interaction was reversed by treatment with a SIRT1 inhibitor selisistat (EX527). Together, our findings indicate that (R)-TML104 alleviates experimental pancreatitis by reducing the infiltration of inflammatory cells through modulating SIRT1.

Electroacupuncture Ameliorates Acute Pancreatitis: A Role for the Vagus Nerve-Mediated Cholinergic Anti-Inflammatory Pathway.

Organ failure resulting from excessive inflammation is the leading cause of death in the early phase of acute pancreatitis (AP). The autonomic nervous system was reported to be involved in AP, and the vagus nerve could exert anti-inflammatory effects through α7 nicotinic acetylcholine receptor (α7nAChR) signaling. Acupuncture has been widely used in traditional Asian medicine, and recent studies suggested the inflammation modulating effect of electroacupuncture (EA) might be mediated by the autonomic nervous system. In this study, we aimed to investigate the effects of EA in AP animal models. Two independent AP mouse models were used, namely, caerulein hyperstimulation and pancreatic duct ligation. We found that EA at Zusanli acupoint increased vagus nerve activity, suppressed systemic inflammation, and alleviated the histopathological manifestations and leukocyte infiltrations of the pancreas. Induction of AP resulted in a remarkable decrease in the frequency of α7nAchR+ macrophages in the pancreas, while EA counteracted this phenomenon. The anti-inflammatory, pancreatic protective and upregulation of α7nAchR effects of EA were reduced in mice with vagotomy. Moreover, the therapeutic effects of EA were attenuated in mice treated with methyllycaconitine citrate, a selective α7nAChR antagonist. Taken together, EA could modulate inflammation, thereby exerting protective effects in AP. The mechanism may include activating the vagus nerve through the cholinergic anti-inflammatory pathway.

Protective Effects of Necrostatin-1 in Acute Pancreatitis: Partial Involvement of Receptor Interacting Protein Kinase 1.

Acute pancreatitis (AP) is a severe and potentially fatal disease caused predominantly by alcohol excess and gallstones, which lacks a specific therapy. The role of Receptor-Interacting Protein Kinase 1 (RIPK1), a key component of programmed necrosis (Necroptosis), is unclear in AP. We assessed the effects of RIPK1 inhibitor Necrostatin-1 (Nec-1) and RIPK1 modification (RIPK1K45A: kinase dead) in bile acid (TLCS-AP), alcoholic (FAEE-AP) and caerulein hyperstimulation (CER-AP) mouse models. Involvement of collateral Nec-1 target indoleamine 2,3-dioxygenase (IDO) was probed with the inhibitor Epacadostat (EPA). Effects of Nec-1 and RIPK1K45A were also compared on pancreatic acinar cell (PAC) fate in vitro and underlying mechanisms explored. Nec-1 markedly ameliorated histological and biochemical changes in all models. However, these were only partially reduced or unchanged in RIPK1K45A mice. Inhibition of IDO with EPA was protective in TLCS-AP. Both Nec-1 and RIPK1K45A modification inhibited TLCS- and FAEE-induced PAC necrosis in vitro. Nec-1 did not affect TLCS-induced Ca2+ entry in PACs, however, it inhibited an associated ROS elevation. The results demonstrate protective actions of Nec-1 in multiple models. However, RIPK1-dependent necroptosis only partially contributed to beneficial effects, and actions on targets such as IDO are likely to be important.

Inhibition of nicotinamide phosphoribosyltransferase protects against acute pancreatitis via modulating macrophage polarization and its related metabolites

Background & objectivesAcute pancreatitis is a common inflammatory disorder of the exocrine pancreas with no specific therapy. Intracellular nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) salvage pathway, is involved in many inflammatory disorders. In this study, we investigated the role of NAMPT in experimental acute pancreatitis. MethodsAcute pancreatitis was induced in mice using three disparate models: (1) caerulein hyperstimulation, (2) ethanol plus palmitoleic acid, and (3) retrograde biliopancreatic ductal infusion of sodium taurocholate. The NAMPT inhibitor FK866 and NAMPT downstream product nicotinamide mononucleotide (NMN) was administered. Serum and pancreas were collected and analyzed biochemically and histologically. Bone marrow derived macrophages were isolated, cultured with cytokines or pancreatic acini, then analyzed by quantitative PCR and non-targeted metabolomics. ResultsThe levels of pancreatic NAMPT and NAD were down-regulated upon acute pancreatitis. NAMPT inhibitor FK866 suppressed M1 macrophage polarization while NMN boosted it. In co-culture of macrophages with acinar cells, inhibition of NAMPT prevented M1-like macrophage differentiation induced by injured pancreatic acini. The injured pancreatic acinar milieu induced a unique metabolic signature linked to macrophage polarization, and inhibition of NAMPT reversed these metabolites changes. Furthermore, NMN supplementation aggravated caerulein hyperstimulation pancreatitis and alcoholic pancreatitis, and inhibition of NAMPT protected against caerulein hyperstimulation, alcoholic and biliary acute pancreatitis and reducing pancreatic macrophage infiltration in vivo. ConclusionsNAMPT inhibition protects against acute pancreatitis via preventing M1 macrophage polarization and restoring the metabolites related to macrophage polarization and that NAMPT could be a promising therapeutic target for acute pancreatitis.

Butyrate ameliorates caerulein-induced acute pancreatitis and associated intestinal injury by tissue-specific mechanisms.

Acute pancreatitis (AP) is a common acute abdominal condition, frequently associated with intestinal barrier dysfunction, which aggravates AP retroactively. Butyrate exhibits anti-inflammatory effects in a variety of inflammatory diseases. However, its potential beneficial effect on AP and the underlying mechanisms have not been investigated. Experimental AP was induced by caerulein hyperstimulation in wild-type and GPR109A-/- mice. Sodium butyrate was administered intragastrically for 7 days prior to caerulein hyperstimulation. Anti-inflammatory mechanisms of butyrate were further investigated in peritoneal macrophages. Butyrate prophylaxis attenuated AP as shown by reduced serum amylase and lipase levels, pancreatic oedema, myeloperoxidase activity, and improved pancreatic morphology. Amelioration of pancreatic damage by butyrate was associated with reduced levels of TNF-α, IL-6, and CCL2 and suppressed activation of the NLRP3 inflammasome in both pancreas and colon. Further, butyrate ameliorated pancreatic inflammation by suppressing interactions between histone deacetylase 1 (HDAC1) and AP1 and STAT1 with increased histone acetylation at H3K9, H3K14, H3K18, and H3K27 loci, resulting in suppression of NLRP3 inflammasome activation and modulation of immune cell infiltration in pancreas. Additionally, butyrate mediated STAT1/AP1-NLRP3 inflammasome suppression via HDAC1 inhibition was demonstrated in peritoneal macrophage. In colon, butyrate inhibited NLRP3 inflammasome activation via GPR109A. Accordingly, the modulatory effects of butyrate on AP, AP-associated gut dysfunction, and NLRP3 inflammasome activation were diminished in GPR109A-/- mice. Our study dissected tissue-specific anti-inflammatory mechanisms of butyrate during AP, suggesting that increased colonic levels of butyrate may be a strategy to protect against AP.

Combinatory antibiotic treatment protects against experimental acute pancreatitis by suppressing gut bacterial translocation to pancreas and inhibiting NLRP3 inflammasome pathway.

Gut bacterial translocation following impaired gut barrier is a critical determinant of initiating and aggravating acute pancreatitis (AP). Antibiotic combination (ABX; vancomycin, neomycin and polymyxin b) is capable of reducing gut bacteria, but its efficacy in AP prevention and the underlying mechanism have not been investigated yet. AP was induced in BALB/c mice by caerulein (CAE) hyperstimulation. We found that ABX supplementation attenuated the severity of AP as evidenced by reduced pancreatic oedema and myeloperoxidase activity. The protective effect was also confirmed by improved histological morphology of the pancreas and decreased pro-inflammatory markers (IL-1β, TNF-α, MCP-1) in pancreas. ABX administration inhibits the activation of colonic TLR4/NLRP3 inflammasome pathway. Subsequently, down-regulated NLRP3 resulted in decreased colonic pro-inflammation (IL-1β, IL-6, MCP-1) and enhanced gut physical barrier as evidenced by up-regulation of tight junction proteins including occludin, claudin-1 and ZO-1, as well as improved histological morphology of the colon. Together, combinatory ABX therapy inhibited the translocation of gut bacteria to pancreas and its amplification effects on pancreatic inflammation by inhibiting the pancreatic NLRP3 pathway, and inhibiting intestinal-pancreatic inflammatory responses. The current study provides the basis for potential clinical application of ABX in AP.

MiRNA-155 Regulates the Th17/Treg Ratio by Targeting SOCS1 in Severe Acute Pancreatitis.

Acute pancreatitis (AP) is a serious condition associated with intestinal barrier disruption or inflammation of the pancreatic tissue. Specific microRNAs are involved in the pathogenesis of AP, during which IL-17-producing CD4+ T helper (Th17) cells accumulate in the pancreas. In this study, significantly increased levels of miR-155 were detected in clinical samples from patients with AP, and overexpression of miR-155 correlated with severe AP (SAP). To identify the effect of miR-155 on T cell differentiation, we isolated CD4+ T lymphocytes and in vitro experiments showed that inhibition of miR-155 significantly reversed the stress-induced increase in the Th17/Treg ratio. The results also showed that miR-155 increased the Th17-mediated inflammatory response by targeting SOCS1. The interaction between miR-155 and the 3′-UTR of SOCS1 was confirmed by a dual luciferase reporter assay and RT-PCR. Experimental AP of varying severity was induced in BALB/c mice by caerulein hyperstimulation and miR-155 expression was found to increase with disease progression. Inhibition of miR-155 expression significantly improved the pathology of the pancreas. We also observed downregulation of expression of inflammatory factors, IL-17, SOCS1 and phosphorylated STAT1 after miR-155 inhibition. In summary, miR-155 regulates the Th17/Treg ratio by targeting SOCS1, most probably via direct binding to its 3′-UTR region, indicating that this microRNA may be a potential biomarker and/or therapeutic target for AP.

Pancreatic gene expression during recovery after pancreatitis reveals unique transcriptome profiles

It is well known that pancreatic recovery after a single episode of injury such as an isolated bout of pancreatitis occurs rapidly. It is unclear, however, what changes are inflicted in such conditions to the molecular landscape of the pancreas. In the caerulein hyperstimulation model of pancreatitis, the murine pancreas has the ability to recover within one week based on histological appearance. In this study, we sought to characterize by RNA-sequencing (RNA-seq) the transcriptional profile of the recovering pancreas up to two weeks post-injury. We found that one week after injury there were 319 differentially expressed genes (DEGs) compared with baseline and that after two weeks there were 53 DEGs. Forty (12.5%) of the DEGs persisted from week one to week two, and another 13 DEGs newly emerged in the second week. Amongst the top up-regulated DEGs were several trypsinogen genes (trypsinogen 4, 5, 12, 15, and 16). To our knowledge, this is the first characterization of the transcriptome during pancreatic recovery by deep sequencing, and it reveals on a molecular basis that there is an ongoing recovery of the pancreas even after apparent histological resolution. The findings also raise the possibility of an emerging novel transcriptome upon pancreatic recovery.

Systemic histone release disrupts plasmalemma and contributes to necrosis in acute pancreatitis

BackgroundClinical and experimental acute pancreatitis feature histone release within the pancreas from innate immune cells and acinar cell necrosis. In this study, we aimed to detail the source of circulating histones and assess their role in the pathogenesis of acute pancreatitis. MethodsCirculating nucleosomes were measured in patient plasma, taken within 24 and 48 h of onset of acute pancreatitis and correlated with clinical outcomes. Using caerulein hyperstimulation, circulating histones were measured in portal, systemic venous and systemic arterial circulation in mice, and the effects of systemic administration of histones in this model were assessed. The sites of actions of circulating histones were assessed by administration of FITC-labelled histones. The effects of histones on isolated pancreatic acinar cells were further assessed by measuring acinar cell death and calcium permeability in vitro. ResultsCell-free histones were confirmed to be abundant in human acute pancreatitis and found to derive from pancreatitis-associated liver injury in a rodent model of the disease. Fluorescein isothianate-labelled histones administered systemically targeted the pancreas and exacerbated injury in experimental acute pancreatitis. Histones induce charge- and concentration-dependent plasmalemma leakage and necrosis in isolated pancreatic acinar cells, independent of extracellular calcium. ConclusionWe conclude that histones released systemically in acute pancreatitis concentrate within the inflamed pancreas and exacerbate injury. Circulating histones may provide meaningful biomarkers and targets for therapy in clinical acute pancreatitis.

Aspartae a Novel Treatment for Acute Pancreatitis

Introduction: There is growing evidence that metabolic intermediates and products can modulate inflammatory responses. Acute pancreatitis (AP) affects more than 200,000 people each year in the United States. A hallmark of AP is the frequent rapid progression into a severe inflammatory response which is associated with pancreatic necrosis and multi-organ failure. The ability of non-infectious insults to initiate severe inflammation is a poorly understood feature of AP, and clinical management is currently limited to supportive care Aims: To Test if aspartate has a role in the treatment of acute pancreatitis in wild type mice via modulation of TLR4 pathway and NLRP3 Inflammasome which are the key players of sterile inflammation. Methods: C56BL/6N young adult male mice were administered caerulein sulfate at 100 pg per g body weight in sterile normal saline by intraperitoneal injection for six hourly injections in an injection volume of 10 uL per g body weight. LPS 10mg/kg injection was given with the first dose of cererulein. Concurrent with the first and third dose of caerulein, mice were give intraperitoneal injections of sterile saline, 2.5 mM HEPES, pH 7.40 or 320 mM aspartate, 2.5mM HEPES, pH 7.40 at 30uL per g body weight. Mice were sacrificed one hour after the last injection of caerulein with pancreas and serum then harvested for analysis. Markers of inflammation were quantified by using qPCR, ELISA. Pancreatic tissue was fixed in formalin, paraffin embedded, sectioned, and stained with hemotoxylin and eosin. Necrosis and edema was scored as 0-3 for none, 1-25%, 25-50%, or >50% Results: Aspartate supplementation suppresses inflammation in the LPS caerulein hyperstimulation model of acute pancreatitis. Aspartate supplementation at 9.6 uM per gram body weight by intraperitoneal injection with the first and third dose of six hourly intraperitoneal injections of caerulein sulfate at 100 pg per gram body weight significantly decreased serum amylase and pancreatic pro IL-1beta, NLrp3 and pro caspase 1 induction. On hemotoxylin and eosin stain there is decrease of inflammation, necrosis and edema. Conclusion: Aspartate suppresses TLR4 and NLRP3 mediated pro-inflammatory signaling and acute pancreatitis. Aspartic acid has potential as a therapeutic agent in the treatment of acute pancreatitis.

Amino acid transporters and enzymes involved in glutathione synthesis are altered in pancreatic acinar cells during acute pancreatitis

s / Pancreatology 15 (2015) S1eS141 S4 activation appears very early in subcellular fractions, that are enriched with zymogen granules and dense lysosomal vesicles but lacking in the autophagosomal marker LC3B-II. Recently we found that zymogens are initial activated as early as 30min caerulein hyperstimulation in dense lysosomal vesicles independent of Atg5-related autophagy. Aims: Here we analyzed effects of autophagy modulation in terms of zymogen activation and its subcellular distribution in relation to lysosomal protease cathepsin B in caerulein hyperstimulated murine pancreas. Materials & methods: NMRI mice were pre-treated with autophagy inducers/inhibitors rapamycin, bafilomycin A1, 3-methyladenine (3-MA) or vinblastine by i.p. injection for up to 27 hours. After 300160 min caerulein hyperstimulation pancreata were homogenized and subcellular fractionated in 46 fractions after Percoll gradient centrifugation. Distribution of enzyme activities and marker proteins were determined by colorand fluorometric measurement or by Western blotting. Results: Whether blocking of autophagosome formation by 3-MA nor inhibition of autophagosome-lysosome fusion by Bafilomycin (VATPase-inhibitor) or autophagy induction by rapamycin affected pancreatic chymotrypsinogen activation in caerulein hyperstimulated mice. However inhibition of microtubule formation and associated blocking of autophagosome fusion with lysosomes by vinblastine decreased significantly chymotrypsinogen activation in caerulein hyperstimulated mice. Conclusion: The early zymogen activation occurs in a dense lysosomal compartment in an ATG5-independent process which requires microtubules formation. Our results give evidence that the activation compartment seems to be generated by fusion events of dense granular vesicles with lysosomes or autolysosomes.

Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis.

Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.

A computer-based automated algorithm for assessing acinar cell loss after experimental pancreatitis.

The change in exocrine mass is an important parameter to follow in experimental models of pancreatic injury and regeneration. However, at present, the quantitative assessment of exocrine content by histology is tedious and operator-dependent, requiring manual assessment of acinar area on serial pancreatic sections. In this study, we utilized a novel computer-generated learning algorithm to construct an accurate and rapid method of quantifying acinar content. The algorithm works by learning differences in pixel characteristics from input examples provided by human experts. HE-stained pancreatic sections were obtained in mice recovering from a 2-day, hourly caerulein hyperstimulation model of experimental pancreatitis. For training data, a pathologist carefully outlined discrete regions of acinar and non-acinar tissue in 21 sections at various stages of pancreatic injury and recovery (termed the “ground truth”). After the expert defined the ground truth, the computer was able to develop a prediction rule that was then applied to a unique set of high-resolution images in order to validate the process. For baseline, non-injured pancreatic sections, the software demonstrated close agreement with the ground truth in identifying baseline acinar tissue area with only a difference of 1%±0.05% (p = 0.21). Within regions of injured tissue, the software reported a difference of 2.5%±0.04% in acinar area compared with the pathologist (p = 0.47). Surprisingly, on detailed morphological examination, the discrepancy was primarily because the software outlined acini and excluded inter-acinar and luminal white space with greater precision. The findings suggest that the software will be of great potential benefit to both clinicians and researchers in quantifying pancreatic acinar cell flux in the injured and recovering pancreas.

Endoplasmic Reticulum Stress Is Chronically Activated in Chronic Pancreatitis

The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T(-/-)), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T(-/-) mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.

Comparative effectiveness of immune-cell depletion and a targeted therapy against LTβR-signaling in the treatment of autoimmune pancreatitis

s / Pancreatology 14 (2014) S1eS129 S18 Aims: In this study, we utilized a novel computer-generated learning algorithm to construct an accurate and rapid method of quantifying acinar content. Materials&methods: HE-stained pancreatic sections were obtained in mice recovering from a 2-day, hourly caerulein hyperstimulation model of experimental pancreatitis. For training data, a pathologist carefully outlined discrete regions of acinar and non-acinar tissue in 21 sections at various stages of pancreatic injury and recovery (termed the “ground truth”). After the expert defined the ground truth, the computer was able to develop a prediction rule which was then applied to a unique set of high resolution images in order to validate the process. Results: For baseline, non-injured pancreatic sections, the software demonstrated close agreement with the ground truth in identifying baseline acinar tissue areawith only a difference of 1% ± 0.05% (p1⁄40.21). Within regions of injured tissue, the software reported a difference of 2.5% ± 0.04% in acinar area compared with the pathologist (p1⁄40.47). Surprisingly, on detailed morphological examination, the discrepancy was primarily because the software outlined acini and excluded inter-acinar and luminal white space with greater precision. Conclusion: The findings suggest that the software will be of great potential benefit to both clinicians and researchers in quantifying pancreatic acinar cell flux in the injured and recovering pancreas.

IP3 Receptor Type 2 Deficiency Is Associated with a Secretory Defect in the Pancreatic Acinar Cell and an Accumulation of Zymogen Granules

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca2+. These aberrant Ca2+ elevations are triggered by release of Ca2+ from apical Ca2+ pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca2+ release channel (IP3R2−/−). Using live acinar cell Ca2+ imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca2+ signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2−/− and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca2+ signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

Caerulein or taurocholate induced enzymatic and histologic alterations in the isolated perfused rat pancreas

Early events in the pathogenesis of experimental acute pancreatitis are intensively studied using isolated cells or animal models. However, the results and their interpretations are dependent on the complexity of biological structures. Therefore, we proposed that studies on isolated perfused pancreas can give additional information about processes leading to acinar cell injury. This hypothesis was examined adapting the well-established caerulein hyperstimulation model and the taurocholate model of acute pancreatitis to the extracorporeal perfused isolated rat pancreas. The pancreas was removed with the duodenum including the arterial supply. A continuous perfusion of the organ was performed with a modified Krebs-Ringer bicarbonate buffer. Intraarterial caerulein application or an intraductal taurocholate (3.5%) application were used to induce acinar cell injury which was determined as the release of amylase, lipase and lactate dehydrogenase into the portal outflow medium and into the transudation fluid and by examination of histological alterations. Trypsinogen release and activation was followed by analysis of trypsinogen activation peptide (TAP) in the transudation fluid and in pancreatic tissue. Perfusion of isolated rat pancreas with supramaximal concentrations of caerulein or retrograde injection of taurocholate (3.5%) resulted in acinar cell injury indicated by elevated levels of amylase and lipase into the perfusate and into the transudation fluid. TAP levels in the transudation fluid significantly increased after perfusion with caerulein or retrograde injection of taurocholate (3.5%). The histological alterations after taurocholate application include oedema and necrosis and show significant differences to the control perfusion. Extensive pancreatic necroses were not observed after caerulein hyperstimulation. The isolated perfused rat pancreas is a useful model to investigate pathophysiological mechanisms which are relevant for the early phase of acute pancreatitis. The caerulein and the taurocholate models are transferable to the isolated rat pancreas. Studies on isolated perfused rat pancreas enable pathophysiological investigations of the exocrine pancreas without influence of systemic components, but with preserved morphology.

Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via the activation of NF-κB

Acinar cell injury early in acute pancreatitis leads to a local inflammatory reaction and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death. Inflammatory mediators, including chemokines and substance P (SP), are known to play a crucial role in the pathogenesis of acute pancreatitis. It has been shown that pancreatic acinar cells produce the chemokine monocyte chemoattractant protein-1 (MCP-1) in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Similarly, SP levels in the pancreas and pancreatic acinar cell expression of neurokinin-1 receptor, the primary receptor for SP, are both increased during secretagogue-induced experimental pancreatitis. This study aims to examine the functional consequences of exposing mouse pancreatic acinar cells to SP and to determine whether it leads to proinflammatory signaling, such as production of chemokines. Exposure of mouse pancreatic acini to SP significantly increased synthesis of MCP-1, macrophage inflammatory protein-1alpha (MIP-1alpha), as well as MIP-2. Furthermore, SP also increased NF-kappaB activation. The stimulatory effect of SP was specific to chemokine synthesis through the NF-kappaB pathway, since the increase in chemokine production was completely attenuated when pancreatic acini were pretreated with the selective NF-kappaB inhibitor NF-kappaB essential modulator-binding domain peptide. This study shows that SP-induced chemokine synthesis in mouse pancreatic acinar cells is NF-kappaB dependent.

Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis

Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.