Induction Of Pancreatitis Research Articles

Beneficial effects of aqueous extracts of Mikania glomerata Sprengel and Mikania laevigata Schultz Bip ex Baker on the inflammatory response in rats with acute pancreatitis

ObjectivesAccording to previous studies, aqueous extracts of Mikania glomerata (MG) and Mikania laevigata (ML) possess considerable anti‐inflammatory activity. Altogether, these findings suggest that these species has a potential effect in the treatment of inflammatory conditions, particularly those involving the gastrointestinal tract. In view of this, it is believed that these species may present important effects in the treatment of acute pancreatitis. Since there is no specific pharmacological therapy for acute pancreatitis, the elucidation of this parameter is the main objective of this study, in an attempt to prove the therapeutic efficacy of these plants under this condition.Material and MethodsThe extracts were characterized by Ultra High Efficiency Liquid Chromatography coupled to Mass Spectrometry (UHPLC‐MS). The animals were treated per oral route (p.o.), through the gavage technique, in the final volume of 500 μL, for 7 consecutive days and then submitted to induction of acute pancreatitis (AP). Male Wistar rats were submitted to injection of sodium tauracholate (30 mg/kg) into the common bile duct. After 4 hours the animals were sacrificed, when blood, pancreas and left lung were collected for analysis of pancreatic edema, pancreatic and pulmonary neutrophil infiltrate (MPO), elevation of serum amylase, TNF‐α in serum and interleukins.Results and DiscussionInduction of AP by sodium tauracholate significantly increased all the parameters evaluated when compared with the respective sham group. The pretreatment with MG (400 mg/kg) significantly reduced pancreatic oedema, pancreas and lung MPO activity. The same profile of reduction was observed in the pretreatment with coumarin (75 mg/kg). The pretreatment with ML showed a significant reduction for all doses tested in the pancreatic oedema and lung MPO activity. For pancreas MPO activity, the doses of 200 and 400 mg/kg showed significant reduction of this parameter.ConclusionOur results showed that aqueous extracts of M. glomerata and M. laevigata decreases inflammation acute pancreatitis, making it of interest in future approaches to treat this condition. However, additional studies are being made by our group to establish the effectiveness of this mechanism.Support or Funding InformationCamila S. P. Della Pasqua is a fellowship of National Council for Scientific and Technological Development (CNPQ).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Wilms' tumor suppressor gene regulates pancreas homeostasis and repair.

The Wilms’ tumor suppressor gene (Wt1) encodes a zinc finger transcription factor that plays an essential role in the development of kidneys, gonads, spleen, adrenals and heart. Recent findings suggest that WT1 could also be playing physiological roles in adults. Systemic deletion of WT1 in mice provokes a severe deterioration of the exocrine pancreas, with mesothelial disruption, E-cadherin downregulation, disorganization of acinar architecture and accumulation of ascitic transudate. Despite this extensive damage, pancreatic stellate cells do not become activated and lose their canonical markers. We observed that pharmacological induction of pancreatitis in normal mice provokes de novo expression of WT1 in pancreatic stellate cells, concomitant with their activation. When pancreatitis was induced in mice after WT1 ablation, pancreatic stellate cells expressed WT1 and became activated, leading to a partial rescue of the acinar structure and the quiescent pancreatic stellate cell population after recovery from pancreatitis. We propose that WT1 modulates through the RALDH2/retinoic acid axis the restabilization of a part of the pancreatic stellate cell population and, indirectly, the repair of the pancreatic architecture, since quiescent pancreatic stellate cells are required for pancreas stability and repair. Thus, we suggest that WT1 plays novel and essential roles for the homeostasis of the adult pancreas and, through its upregulation in pancreatic stellate cells after a damage, for pancreatic regeneration. Due to the growing importance of the pancreatic stellate cells in physiological and pathophysiological conditions, these novel roles can be of translational relevance.

Abstract A100: ROBO2 is a stroma suppressor gene in the pancreas through regulation of TGF-β

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, being predicted to become the second leading cause of cancer-related death by 2030. Chronic pancreatitis is a risk factor for PDAC and both diseases are characterized by a strong desmoplastic response, comprised of activated myofibroblasts and immune cell infiltrates. Genomic aberrations in the SLIT-ROBO pathway are frequent in PDAC. Nevertheless, their role in the pancreas is unclear. We have used an integrative approach combining the study of murine models and PDAC patients with the objective of unraveling the function of the SLIT-ROBO signaling pathway in pancreatic disease. RNA expression of SLIT-ROBO genes was analyzed in murine normal pancreas, pancreatitis and PDAC. Primary cell cultures and experimental pancreatitis were studied using pancreas-specific Robo2 (Pdx1-Cre;Robo2F/F) and whole-body Slit1 (Slit1-/-) knockout mice. Gene and protein expression were assessed in a cohort of PDAC patients (n=109). In mouse pancreatitis and PDAC, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Pdx1Cre;Robo2F/F pancreatic cell cultures showed increased activation of Robo1-positive myofibroblasts and induction of TGF-β and Wnt pathways. Likewise, induction of pancreatitis in Pdx1Cre;Robo2F/F mice enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. Similar results were obtained using Slit1-/- animals. Moreover, TGF-β inhibition using galunisertib treatment suppressed Robo2-mediated effects in the microenvironment. In patients, ROBO2 expression is overall low in PDAC, while ROBO1 is variably expressed in epithelium and high in the stroma. ROBO1 expression is correlated with markers of activated stroma, Wnt and TGF-β pathways. ROBO2low;ROBO1high subpopulation of patients present the poorest survival rates. In conclusion, Robo2 acts nonautonomously as a stroma suppressor gene by restraining myofibroblast activation and inflammation in the pancreatic microenvironment. ROBO1/2 expression is prognostic in PDAC patients and may guide therapy with TGF-β inhibitors or immunotherapies, currently being tested in clinical trials for advanced pancreatic cancer. Citation Format: Andreia V. Pinho, Mathias Van Bulck, Lorraine Chantrill, Mehreen Arshi, David Herrmann, Claire Vennin, APGI - Australian Pancreatic Cancer Genome Initiative, Anthony Gill, Paul Timpson, Andrew Biankin, Jianmin Wu, Ilse Rooman. ROBO2 is a stroma suppressor gene in the pancreas through regulation of TGF-β [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A100.

Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine.

Background: Neutrophil extracellular traps (NETs) are generated when activated neutrophils, driven by PAD4, release their DNA, histones, HMGB1, and other intracellular granule components. NETs play a role in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction. The autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore, we investigated the impact of CQ mediated NET inhibition in murine models of pancreatitis and human correlative studies.Methods: L-arginine and choline deficient ethionine supplemented (CDE) diet models of acute pancreatitis were studied in wild type and PAD4−/− mice, incapable of forming NETs. Isolated neutrophils were stimulated to induce NET formation and visualized with fluorescence microscopy. CQ treatment (0.5 mg/ml PO) was initiated after induction of pancreatitis. Biomarkers of NET formation, including cell-free DNA, citrullinated histone H3 (CitH3), and MPO-DNA conjugates were measured in murine serum and correlative human patient serum samples.Results: We first confirmed the role of NETs in the pathophysiology of acute pancreatitis by demonstrating that PAD4−/− mice had decreased pancreatitis severity and improved survival compared to wild-type controls. Furthermore, patients with severe acute pancreatitis had elevated levels of cell-free DNA and MPO-DNA conjugates, consistent with NET formation. Neutrophils from mice with pancreatitis were more prone to NET formation and CQ decreased this propensity to form NETs. CQ significantly reduced serum cell-free DNA and citrullinated histone H3 in murine models of pancreatitis, increasing survival in both models.Conclusions: Inhibition of NETs with CQ decreases the severity of acute pancreatitis and improves survival. Translating these findings into clinical trials of acute pancreatitis is warranted.

Protection of acute renal injury in severe experimental acute pancreatitis

Background: Acute renal failure is a serious early complication in patients with acute pancreatitis (AP) significantly increases mortality rates. Thus, it is essential to protect renal function in patients with severe AP. Methods: Acute necrotizing pancreatitis was induced by L-ornithine in 26 Wistar rats. 16 rats served as control. We administered enoxaparin in 10 rats after induction of acute pancreatitis during 5 days. The effect of enoxaparin was evaluated by determining the levels of serum amylase, creatinine, hydrogen sulfide, and fibrinogen, time of recalcification, activity of NO-synthase and myeloperoxidase (MPO) in pancreas. Results: The concentration of creatinine in serum is increase at 86.86%. It was noticed that the level of creatinine significantly directly correlated with the concentration of MPO and activity of iNOS in the pancreas, amylase in serum blood, and reverse with the concentration of H2S in serum. Renal failure in rats was accompanied by a hypercoagulation, determined by probable direct correlation between the levels of creatinine and fibrinogen, inverse relationship between creatinine concentrations and plasma recalcification time. Enoxaparin ameliorates morphology of the pancreas and kidney. The enoxaparin normalized serum creatinine, fibrinogen, amylase, MPO, H2S, decreased activity of iNOS. Conclusion: Enoxaparin in experimental AP reduces morphological signs of damage in the pancreas and kidneys, the intensity nitrosative stress and inflammation, normalizes hemostasis.

Protection of acute renal injury in severe experimental acute pancreatitis

Background: Acute renal failure is a serious early complication in patients with acute pancreatitis (AP) significantly increases mortality rates. Thus, it is essential to protect renal function in patients with severe AP. Methods: Acute necrotizing pancreatitis was induced by L-ornithine in 26 Wistar rats. 16 rats served as control. We administered enoxaparin in 10 rats after induction of acute pancreatitis during 5 days. The effect of enoxaparin was evaluated by determining the levels of serum amylase, creatinine, hydrogen sulfide, and fibrinogen, time of recalcification, activity of NO-synthase and myeloperoxidase (MPO) in pancreas. Results: The concentration of creatinine in serum is increase at 86.86%. It was noticed that the level of creatinine significantly directly correlated with the concentration of MPO and activity of iNOS in the pancreas, amylase in serum blood, and reverse with the concentration of H2S in serum. Renal failure in rats was accompanied by a hypercoagulation, determined by probable direct correlation between the levels of creatinine and fibrinogen, inverse relationship between creatinine concentrations and plasma recalcification time. Enoxaparin ameliorates morphology of the pancreas and kidney. The enoxaparin normalized serum creatinine, fibrinogen, amylase, MPO, H2S, decreased activity of iNOS. Conclusion: Enoxaparin in experimental AP reduces morphological signs of damage in the pancreas and kidneys, the intensity nitrosative stress and inflammation, normalizes hemostasis.

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.

Dynamic changes of proteasome and protective effect of bortezomib, a proteasome inhibitor, in mice with acute pancreatitis

The proteasome is involved in the activation of NF-κB and can regulate the progression of inflammatory diseases. However, the role of proteasome in acute pancreatitis (AP) has not been demonstrated. In this study, we first observed that the protein level and activity of proteasome 20S were increased significantly in pancreatic injury tissues after caerulein-induced mild acute pancreatitis (MAP) induction, which was in consistent with the expression of the NF-κB nucleoprotein and positively correlated with the severity of AP. Then, bortezomib, a classical proteasome inhibitor, was used to intervene the progression of MAP in mice. The results showed that bortezomib administration reduced the serum amylase and lipase levels and mitigated histopathological manifestation of pancreatic injury in mice. Meanwhile, bortezomib decreased the expression of NF-κB p65 nucleoprotein as well as total proteasome 20S protein, and inhibited the activity of 20S in pancreatic tissues. In addition, we found that bortezomib could protect pancreatic acinar cell against necrosis and mitigate the severity of AP in a severe acute pancreatitis model induced by sodium taurocholate hydrate. Taken together, our study for the first time confirmed that the proteasome participated in the pathogenesis of AP and its inhibitor bortezomib could protect against AP in mice.

The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for >30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler ɑ-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS’s ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic KRAS.MethodsMice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion.ResultsMice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients.ConclusionsOur results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS’s ability to promote pancreatic intraepithelial lesion formation.

Fetal microchimerism in mouse caerulein-induced pancreatitis model.

Objective(s):Fetal microchimerism is the persistence of allogeneic cell population that transfer from the fetus to the mother. The aim of this study was to evaluate the presence of fetal microchimerism in the pancreas of the mouse with acute pancreatitis (AP).Materials and Methods:In this experimental study, female wild-type mice were mated with male EGFP+. AP model was obtained by injection of caerulein two days after delivery. Sixty mice were divided into 3 groups: the virgin pancreatitis-induced animals, pregnant pancreatitis-induced animals mated with transgenic EGFP mice, and pregnant sham animals. To prove pancreatitis induction, the blood amylase and lipase were assessed; and pancreas was removed from a subpopulation of each group for histopathological examinations after 6 hr. The remaining mice were kept for 3 weeks and histopathological exanimation, immunohistochemistry, and PCR were performed.Results:EGFP+ cells were found in acini and around the blood vessels in the pancreas of pregnant pancreatitis-induced animals. They differentiated to acinar, adipocyte-like, and mesenchymal-like cells. PCR showed that 20% of the pregnant pancreatitis-induced animals were EGFP+. The histopathological study showed improvement in pancreatitis scores in the mice with history of pregnancy. Conclusion:It seems that pregnancy has a beneficial impact on caerulein-induced pancreatitis and improves the pancreatitis score in mouse.

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.

The protective effect of nano-curcumin in experimental model of acute pancreatitis: The involvement of TLR4/NF-kB pathway

Objective(s): The objective of the present study is to explore whether Nanocurcumin improves pancreatic inflammation through the inhibition of the TLR4/NFkB signaling pathway in cerulein-induced acute pancreatitis. Methods: Acute pancreatitis was induced by five intraperitoneal (i.p.) injection of cerulein (50 μg/kg) with 1h intervals. Vehicle and nanocurcumin (100mg/kg/day) were given to the animals by oral gavage six days before the induction of pancreatitis. The last dose was administered 1 hour before pancreatitis induction. The serum level of amylase and lipase and the tissue level of MPO enzymes were assessed by biochemical analysis. Microscopic lesions were examined. In addition, the expression level of TLR4, NF-kB p65 and TNF-α proteins were measured by western blotting analysis. Results: Nanocurcumin reduced the microscopic lesions. In addition, the drug decreased the level of amylase, lipase and MPO enzymes. Furthermore, nanocurcumin inhibited the cerulein-induced expression of TLR4, NF-kB p65 and TNF-α proteins. Conclusion: It is suggested that the anti-inflammatory effect of nanocurcumin on cerulein-induced acute pancreatitis may involve the inhibition of the TLR4/NFkB signaling pathway.

Gamma-enolase predicts lung damage in severe acute pancreatitis-induced acute lung injury

Severe acute pancreatitis (SAP) associated acute lung injury (ALI) accounts for about 70% mortality of SAP patients. However, there are no precise biomarkers for the disease currently. Herein, we evaluated the potential of gamma-enolase (ENO2), against its universal isoform alpha-enolase (ENO1), as a marker of SAP-ALI in a rat model. Firstly, 16 male Sprague-Dawley rats were randomly divided into two groups, Sham (n = 8) and SAP-ALI (n = 8), for pancreatitis induction. Ultra-structure examination by electron microscopy and HE staining were used for lung injury assessment. Lung tissue expressions of alpha-enolase and gamma-enolase were evaluated by qRT-PCR and immunohistochemistry. In a prospective validation experiment, 28 rats were used: sham (n = 8), SAP-ALI at 3h (3h, n = 10), and SAP-ALI at 24h (24h, n = 10). Lung tissue damage, tissue expression and circulating alpha-enolase and gamma-enolase levels were evaluated. Elevated serum levels of α-amylase and TNF-α were observed in SAP rats but not in sham-operated rats. Histological examination of pancreatic and lung tissues indicated marked damage in SAP rats. While alpha-enolase was universally expressed, gamma-enolase was expressed only in damaged lung tissues. Gamma-enolase was detected in lung tissues, BALF, and serum as early as 3h post-surgery when physical pathological damage was not apparent. Unlike alpha-enolase, secreted and/or circulating gamma-enolase level progressively increased, especially in serum, as lung damage progressed. Thus, gamma-enolase may signal and correlate lung tissue damage well before obvious physical pathological tissue damage and might be a candidate diagnostic and/or prognostic marker.

Resveratrol Ameliorates the Severity of Fibrogenesis in Mice with Experimental Chronic Pancreatitis.

Resveratrol is generally considered beneficial to health-span and longevity since this dietary stilbenoid has been scrutinized for its activating property on the "rescue gene" sirtuin-1 that promotes cellular survival under stress. In addition to its antiaging property, our previous in vitro studies revealed that resveratrol notably inhibits the production of extracellular matrix (ECM) proteins in pancreatic stellate cells (PSCs), the classic effector cells against pancreatic injury. We aim to extrapolate resveratrol intervention to the management of fibrogenesis in mice with chronic pancreatitis. C57/BL6 mice are given repetitive injections of cerulein (50 μgkg-1 h-1 ) for 6 weeks for the induction of chronic pancreatitis. We demonstrate that the oral administration of resveratrol (20 mgkg-1 d-1 ) effectively attenuated PSC activation, ECM deposition, fibrogenesis, and acinar atrophy in the pancreatitic parenchyma of cerulein-induced mice, as the damage index score was improved by 45.5%. The enhanced cell survival and preserved acinar integrity by resveratrol plausibly involves a perpetuated nuclear accumulation of Mist1 and a negative modulation of the Akt and p38 MAPK pathways. We suggest that resveratrol is potentially a nutraceutical for the mitigations of pancreatic fibrosis in chronic pancreatitis for which no effective therapeutic measure is currently available.

Dexmedetomidine attenuates pancreatic injury and inflammatory response in mice with pancreatitis by possible reduction of NLRP3 activation and up-regulation of NET expression

ObjectivePrevious studies have shown that acute inflammation is associated with increased sympathetic activity, which in turn increases the inflammatory response and leads to organ damage. The present study aimed to investigate whether dexmedetomidine administration during acute pancreatitis (AP) lessens pancreatic pathological and functional injury and the inflammatory response, and to explore the underlying mechanisms. MethodsMild pancreatitis was induced in mice with caerulein, and severe pancreatitis was induced with caerulein plus lipopolysaccharide (LPS). After pancreatitis induction, dexmedetomidine at 10 or 20 μg/kg was injected via the tail vein. Pancreatic pathological and functional injury was assessed by histology and serum levels of amylase and lipase, respectively. The inflammatory response was evaluated by determining serum levels of inflammatory factors. The expression of myeloperoxidase (MPO) was examined by immunohistochemistry. The expression of norepinephrine transporter (NET), NLRP3, pro-IL-1β, and interleukin (IL)-1β in pancreatic tissue was detected by Western blot and real-time PCR. ResultsDexmedetomidine at 20 μg/kg significantly attenuated pancreatic pathological injury, reduced serum levels of amylase, lipase, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, and decreased the expression of MPO in pancreatic tissue in both mouse models of pancreatitis. In addition, dexmedetomidine at 20 μg/kg significantly down-regulated the expression of NLRP3, pro-IL-1β, and IL-1β in pancreatic tissue, but up-regulated the expression of NET in both mouse models. ConclusionDexmedetomidine attenuates pancreatic injury and inflammatory response in mice with pancreatitis possibly by reducing NLRP3 activation and up-regulating NET expression.

Cathepsin B-Mediated Activation of Trypsinogen in Endocytosing Macrophages Increases Severity of Pancreatitis in Mice

BACKGROUND & AIMS:Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice.METHODS:Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)- knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens.RESULTS:Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines.CONCLUSIONS:Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.

Class I histone deacetylase inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar‐to‐ductal metaplasia

Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease. We analysed HDAC regulation during cerulein-induced acute, chronic and autoimmune pancreatitis using different transgenic mouse models. The functional relevance of class I HDACs was tested with the selective inhibitor MS-275 in vivo upon pancreatitis induction and in vitro in activated macrophages and primary acinar cell explants. HDAC expression and activity were up-regulated in a time-dependent manner following induction of pancreatitis, with the highest abundance observed for class I HDACs. Class I HDAC inhibition did not prevent the initial acinar cell damage. However, it effectively reduced the infiltration of inflammatory cells, including macrophages and T cells, in both acute and chronic phases of the disease, and directly disrupted macrophage activation. In addition, MS-275 treatment reduced DNA damage in acinar cells and limited acinar de-differentiation into acinar-to-ductal metaplasia in a cell-autonomous manner by impeding the EGF receptor signalling axis. These results demonstrate that class I HDACs are critically involved in the development of acute and chronic forms of pancreatitis and suggest that blockade of class I HDAC isoforms is a promising target to improve the outcome of the disease.

Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.

Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3-/- pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to-duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.

Morphine worsens the severity and prevents pancreatic regeneration in mouse models of acute pancreatitis

BackgroundOpioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways...

Ibuprofen and diclofenac treatments reduce proliferation of pancreatic acinar cells upon inflammatory injury and mitogenic stimulation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are administered to manage the pain typically found in patients suffering from pancreatitis. NSAIDs also display anti-proliferative activity against cancer cells; however, their effects on normal, untransformed cells are poorly understood. Here, we evaluated whether NSAIDs inhibit the proliferation of pancreatic acinar cells during the development of acute pancreatitis. The NSAIDs ibuprofen and diclofenac were administered to C57BL/6 mice after induction of pancreatitis with serial injections of cerulein. In addition, ibuprofen was administered concomitantly with 3,5,3-L-tri-iodothyronine (T3), which induces acinar cell proliferation in the absence of tissue inflammation. The development of pancreatic inflammation, acinar de-differentiation into metaplastic lesions and acinar proliferation were quantified by histochemical, biochemical and RT-PCR approaches. Therapeutic ibuprofen treatment selectively reduced pancreatic infiltration of activated macrophages in vivo, and M1 macrophage polarization and pro-inflammatory cytokine expression both in vivo and in vitro. Reduced macrophage activation was accompanied by reduced acinar de-differentiation into acinar-to-ductal metaplasia. Acinar proliferation was significantly impaired in the presence of ibuprofen and diclofenac, as demonstrated at both the level of proliferation markers and expression of cell cycle regulators. Ibuprofen also reduced acinar cell proliferation induced by mitogenic stimulation with T3, a treatment that does not elicit pancreatic inflammation. Our study provides evidence that the NSAIDs ibuprofen and diclofenac inhibit pancreatic acinar cell division. This suggests that prolonged treatment with these NSAIDs may negatively affect the regeneration of the pancreas and further studies are needed to confirm these findings in a clinical setting. This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.