Experimental Pancreatitis Model Research Articles

Comparative transcriptomic analysis reveals the molecular changes of acute pancreatitis in experimental models

BACKGROUND Acute pancreatitis (AP) encompasses a spectrum of pancreatic inflammatory conditions, ranging from mild inflammation to severe pancreatic necrosis and multisystem organ failure. Given the challenges associated with obtaining human pancreatic samples, research on AP predominantly relies on animal models. In this study, we aimed to elucidate the fundamental molecular mechanisms underlying AP using various AP models. AIM To investigate the shared molecular changes underlying the development of AP across varying severity levels. METHODS AP was induced in animal models through treatment with caerulein alone or in combination with lipopolysaccharide (LPS). Additionally, using Ptf1α to drive the specific expression of the hM3 promoter in pancreatic acinar cells transgenic C57BL/6J- hM3/Ptf1α(cre) mice were administered Clozapine N-oxide to induce AP. Subsequently, we conducted RNA sequencing of pancreatic tissues and validated the expression of significantly different genes using the Gene Expression Omnibus (GEO) database. RESULTS Caerulein-induced AP showed severe inflammation and edema, which were exacerbated when combined with LPS and accompanied by partial pancreatic tissue necrosis. Compared with the control group, RNA sequencing analysis revealed 880 significantly differentially expressed genes in the caerulein model and 885 in the caerulein combined with the LPS model. Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Set Enrichment Analysis indicated substantial enrichment of the TLR and NOD -like receptor signaling pathway, TLR signaling pathway, and NF-κB signaling pathway, alongside elevated levels of apoptosis-related pathways, such as apoptosis, P53 pathway, and phagosome pathway. The significantly elevated genes in the TLR and NOD -like receptor signaling pathways, as well as in the apoptosis pathway, were validated through quantitative real-time PCR experiments in animal models. Validation from the GEO database revealed that only MYD88 concurred in both mouse pancreatic tissue and human AP peripheral blood, while TLR1 , TLR7 , RIPK3 , and OAS2 genes exhibited marked elevation in human AP. The genes TUBA1A and GADD45A played significant roles in apoptosis within human AP. The transgenic mouse model hM3/Ptf1α(cre) successfully validated significant differential genes in the TLR and NOD -like receptor signaling pathways as well as the apoptosis pathway, indicating that these pathways represent shared pathological processes in AP across different models. CONCLUSION The TLR and NOD receptor signaling pathways play crucial roles in the inflammatory progression of AP, notably the MYD88 gene. Apoptosis holds a central position in the necrotic processes of AP, with TUBA1A and GADD45A genes exhibiting prominence in human AP.

Dynamic Plasma Thiol/Disulfide Balance In Experimental Pancreatitis Model In Rats

Introduction: Dynamic thiol-disulfide balance has critical roles in regulating antioxidant protection and detoxification.Thiol/disulfite levels, which can be easily measured in blood, were investigated in various proliferative and inflammatory diseases. It was observed that the changes in this balance were correlated with oxidant parameters. In previous studies, it has been shown that oxidative stress increases significantly in acute pancreatitis, and it has been shown that these levels are related to the severity of the disease. Owing to acute pancreatitis is a severe inflammatory condition; the thiol/disulfide balance may correlate with the severity of this disease. Our study investigated thiol/disulfide levels in the experimental pancreatitis model. Materials and Methods: In our study, 16 Wistar-Albino male rats were used and divided into two groups as experimental and control groups, each group being eight rats. In the experimental group, we generated acute pancreatitis with Cerulein. Amylase, lipase, and thiol/disulfide balance were determined from inferior vena cava blood sampling with median laparotomy. Results: Amylase and lipase levels were found to be significantly different in the experimental group compared with the control group, but there was no significant difference in the plasma thiol/disulfide balance. Conclusion: The study results showed that the plasma thiol/disulfide level did not reveal the severity of acute pancreatitis. In previous studies, thiol/disulfide levels increased in oxidative stress, but this did not happen in our study. The fact that our study was conducted with a small group may have caused this situation. There is a need for more comprehensive studies to investigate in more detail the plasma thiol/disulfide balance.

Pathomorphological changes in experimental model of chronic alcohol and drug-induced pancreatitis in the rat

Aim of the research: The aim of the study to conduct the histomorphological study of structural and cellular changes in the tissues of the pancreas, adrenal gland, spleen, liver, kidney in experimental alcoholic and drug-induced pancreatitis. Materials and methods. A model of experimental alcoholic and drug-induced pancreatitis was created on white mongrel rats aged 2-3 months, weighing 25-30 g. The animals were divided into 3 groups (control group No. 1, comparison groups No. 2 and No. 3). A control group (n=10) that did not receive any substances. The second comparison group (n=10) was injected daily with 1 ml of 40% ethanol solution for 10 days. The third comparison group (n=10) was administered intragastric tyrosol daily at the rate of 5 mg per 100 g of body weight for 10 days. Withdrawal from the experiment was performed on the 10 th day from the beginning of the experiment. Laboratory animals were slaughtered by decapitation. Pancreas, spleen and liver, kidneys with adrenal glands were taken for pathomorphological examination. Research results and discussion. During histological examination of the pancreas on the 10th day of the experiment, no changes were noted in the control group, in comparison groups No. 2, No. 3, pathomorphological changes were noted, indicating an inflammatory and dystrophic process in the pancreas. During histological examination of tissues, complications in the liver, spleen, kidneys and adrenal glands were also observed on day 10. Conclusions. The above facts indicate that comparison group No. 2 developed chronic experimental alcoholic pancreatitis induced by ethanol solution; comparison group No. 3 induced experimental drug-induced pancreatitis. One of the main ways of endogenous insufficiency is the enteral pathway, the least dependent on pharmacological correction.

Protective Effect of Nigella Sativa on Taurocholate Induced Pancreatitis in Rats

Introduction: Acute necrotizing pancreatitis with a high mortality rate and the search for treatment continues. We investigated the protective effect of Nigella Sativa (NS), with antioxidant and anti-inflammatory effects, in experimental acute necrotizing pancreatitis. Materials and Methods: Thirty six male Wistar albino rats (weights 180-220 g) were randomised into four groups. Group 1 (Control): Rats were given standard mouse chow. No pro-drug or pancreatic intervention was performed. Group 2 (NS): In addition to their standard diet, rats were given 0.1 ml/100gr of NS orally for 3 days prior to the experiment. Group 3: Necrotizing pancreatitis was induced by retrograde administration of 3% Na-Taurocholate through the distal common bile duct of the rats on on experiment day. Group 4 (NS+ANP): Necrotizing pancreatitis was also formed in rats receiving 0.1ml/100 mg of NS for 3 days. Rats were given high-dose anesthesia 8 hours after the onset of pancreatitis. Immunohistochemical (TNF-a, MDA, MPO, Caspase), histological pancreatitis scoring and biochemical (LDH, Lipase, amylase) analyzes were performed from the blood and pancreatic tissue samples obtained. Results: There was no difference in histopathological, immunohistochemical and biochemical values between Group 1 and Group 2 (p>0.05). There were significiant differences between Group 4 and Group 3 in terms of histopathological, immunohistochemical and biochemical parameters (p<0.001). The pancreatitis findings of the Group 4 were found to be significantly milder than Group 3, which did not receive NS. Conclusion: NS pretreatment alleviates NaTaurocholate-induced experimental pancreatitis. NS firstly studied in experimental models of pancreatitis.

Pachymic acid alleviates experimental pancreaticfibrosisthrough repressing NLRP3 inflammasome activation.

Pachymic acid (PA), a natural triterpenoid, possesses the capacity to repress inflammatory and profibrotic responses. However, the role of PA in pancreatic fibrosis remains unclear. Here the effect of PA on anti-fibrogenic response was investigated using in vivo and in vitro pancreatitis models. We demonstrated that PA treatment repressed TGF-β-induced pancreatic stellate cells (PSCs) activation in vitro, as evidenced by decreased expression of Collagen I, α-smooth muscle actin, and fibronectin. PA decreased Cerulein-induced acinar injury and pancreatic fibrosis in an experimental pancreatitis model. Mechanistically, PA repressed Cerulein or (TGF-β)-induced activation of nuclear factor (NF)-κB signaling and thus decreased NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome activation in PSCs. Pharmacological inhibition of NLRP3 repressed TGF-β-induced activation of PSCs. More important, NLRP3 activator partially attenuated the effect of PA on inhibiting PSCs activation. Collectively, these data demonstrate that PA represses PSCs activation and pancreatic fibrosis through repressing NF-κB/NLRP3 signaling.

Differential expressions of miR-223, miR-424, miR-145, miR-200c, miR-139 in experimental rat chronic pancreatitis model and their relationship between oxidative stress, endoplasmic reticulum stress, and apoptosis.

Objective(s):This study aimed to research the roles of miR-139, miR-221, miR-200c, miR-145, miR-223, miR-424, and miR-377 in endoplasmic reticulum stress (ERS), oxidative stress (OS), fibrosis, and apoptosis processes in chronic pancreatitis (CP) rat model. Materials and Methods:Fourteen rats were randomized into 2 groups (Group 1, sham group (n=7) and Group 2, CP group (n=7)). TGF-beta and malondialdehyde concentrations were measured in rat blood samples. qRT-PCR was used to investigate the expression levels of 7 miRNAs in the pancreas tissues. The correlations of mRNA undergoing significant changes with inflammation (TNF-α, IL-6), ERS (Ire1-α, Perk), apoptosis (Caspase 3, Bcl-2), OS (Cat, Gpx1), and fibrosis (α-Sma) were investigated.Results:The biochemical results and histopathological scores in Group 1 were statistically significantly high compared with Group 2 (P<0.5). Expression levels of seven miRNAs (miR-200c, miR-145, miR-223, miR-424) were significantly higher, while miR-139 was significantly lower in CP. In our study, we found that miR-200c, miR-145, and miR-139 may contribute to CP progression and cellular processes based on the correlation between ERS, OS, apoptosis, and inflammation with miRNA expression levels. Conclusion:miR-200c, miR-145, miR-139, miR-223, and miR-424 play roles in the CP model. They may be used as candidate biomarkers for the CP process.

Effect of luteolin on the gene level expression of apoptosis-associated speck-like protein containing a caspase recruitment domain of NLRP3 inflammasome and NF-κB in rats subjected to experimental pancreatitis- influence of HSP70.

Nod-like receptor pyrin domain containing 3 (NLRP3) is one of the well characterized inflammasome that controls the maturation of pro-inflammatory cytokines and thereby the inflammation in pancreas which could be a promising target for anti-inflammatory drugs. The present study is aimed to explore whether luteolin can target the NLRP3 inflammasome and modulate its activity through the signaling protein, HSP70 in the ethanol-cerulein model of experimental pancreatitis. Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats received normal diet. Groups 3 and 4 rats received isocalorically adjusted diet containing ethanol for 5weeks and cerulein (20μg/kg body weight i.p.,thrice weekly for the last 3weeks of the experimental period). Additionally, group 2 and 4 rats received 2mg/kg body weight of luteolin orally from third week. Luteolin co-administration decreased the serum levels of HSP70, oxidative stress markers, myeloperoxidase, GSH/GSSG and GST with concomitant downregulation in the mRNA expression of HSP70, caspase-1, ASC-NLRP3 and NF-κB. Spearman's rank correlation test showed that serum HSP70 has positive correlation with the expression of ASC-NLRP3, caspase-1, NF-κB and 4-hydroxynonenal and negative correlation with GSH:GSSG ratio. The modulating effect of luteolin on the expression of HSP70, NF-κB and thereby on ASC-NLRP3 complex may be claimed for its pancreato-protective activity.

Inhibition of discoidin domain receptors by imatinib prevented pancreatic fibrosis demonstrated in experimental chronic pancreatitis model

Discoidin domain receptors (DDR1 and DDR2) are the collagen receptors of the family tyrosine kinases, which play significant role in the diseases like inflammation, fibrosis and cancer. Chronic pancreatitis (CP) is a fibro-inflammatory disease in which recurrent pancreatic inflammation leads to pancreatic fibrosis. In the present study, we have investigated the role of DDR1 and DDR2 in CP. The induced expression of DDR1 and DDR2 was observed in primary pancreatic stellate cells (PSCs) and cerulein-induced CP. Subsequently, the protective effects of DDR1/DDR2 inhibitor, imatinib (IMT) were investigated. Pharmacological intervention with IMT effectively downregulated DDR1 and DDR2 expression. Further, IMT treatment reduced pancreatic injury, inflammation, extracellular matrix deposition and PSCs activation along with inhibition of TGF-β1/Smad signaling pathway. Taken together, these results suggest that inhibition of DDR1 and DDR2 controls pancreatic inflammation and fibrosis, which could represent an attractive and promising therapeutic strategy for the treatment of CP.

Experimental pancreatitis is characterized by rapid T cell activation, Th2 differentiation that parallels disease severity, and improvement after CD4+ T cell depletion

BackgroundAcute pancreatitis is a gastrointestinal disorder of high incidence resulting in life threatening complications in up to 20% of patients. Its severe form is characterized by an extensive and systemic immune response. We investigated the role of the adaptive immune response in two experimental models of pancreatitis. MethodsIn C57BI/6-mice mild pancreatitis was induced by 8-hourly injections of caerulein and severe pancreatitis by additional, partial pancreatic duct ligation. T-cell-activation was determined by flow-cytometry of CD25/CD69, T-cell-differentiation by nuclear staining of the transcription-factors Tbet, Gata3 and Foxp3. In vivo CD4+ T-cells were depleted using anti-CD4 antibody. Disease severity was determined by histology, serum amylase and lipase activities, lung MPO and serum cytokine levels (IL-6, TNFα, IL-10). ResultsIn both models T-cells were activated. Th1-differentiation (Tbet) was absent during pancreatitis but we detected a pronounced Th2/Treg (Gata3/Foxp3) response which paralleled disease severity in both models. The complete depletion of CD4+ T-cells via anti-CD4 antibody, surprisingly, reduced disease severity significantly, as well as granulocyte infiltration and pro- and anti-inflammatory cytokine levels. Co-incubation of acini and T-cells did not lead to T-cell-activation by acinar cells but to acinar damage by T-cells. During pancreatitis no significant T-cell-infiltration into the pancreas was observed. ConclusionT cells orchestrate the early local as well as the systemic immune responses in pancreatitis and are directly involved in organ damage. The Th2 response appears to increase disease severity, rather than conferring an immunological protection.

Test of antimicrobial activity of morpholine 2-(5-(3-fluorophenyl) -4-amino-1,2,4-triazol-3-ilthio) acetate (BKP-115) by experimental model of pancreatitis in rats

The purpose of this research was to study the anti-inflammatory properties of morpholine 2-(5-(3-fluorophenyl) -4-amino-1,2,4-triazol-3-ylthio) acetate (BKP-115) in intramuscular and intragastric introduction on the model of experimental pancreatitis in rats. Determination of the anti-inflammatory activity of the substance morpholine 2- (5- (3-fluorophenyl)-4-amino-1,2,4-triazol-3-ylthio) acetate was performed on 68 male rats with a starting weight of 180-200 g, which were kept in standard vivarium conditions. Since the main chain of pathogenesis of AP is the activation and influence of proteolytic enzymes of the pancreas, the toxic-infectious model was chosen – the introduction of trypsin into the tissue of the pancreas. The introduction of trypsin in animals resulted in pancreatitis with endogenous intoxication, inflammation of the tissue of the pancreas and violation of the liver functional state. As a result, one animal died in the positive control group. Prophylactic i/g and i/v injection of the BKP-115 substance contributed to a significant reduction in the content of MAW (molecules of average weight) and CIM (circulating immune complexes) in animals from control and intact groups, indicated a decrease in the level of endogenous intoxication and detoxification properties of tested substance. The highest effectiveness of BKP-115 was registered after intragastric injection. Animals from this group had similar parameters with animals injected by “Contryven” and from intact control. Intramuscular injection of BKP-115 had therapeutic effect on experimental pancreatitis and endogenous intoxication, but it was less effective after intragastric injection and had weaker efficiency than “Contryven”.

Effects of the Poly(ADP-Ribose) Polymerase Inhibitor Olaparib in Cerulein-Induced Pancreatitis

Activation of the constitutive nuclear and mitochondrial enzyme poly (ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of cell dysfunction, inflammation, and organ failure in various forms of critical illness. The objective of our study was to evaluate the efficacy and safety of the clinically approved PARP inhibitor olaparib in an experimental model of pancreatitis in vivo and in a pancreatic cell line subjected to oxidative stress in vitro. The preclinical studies were complemented with analysis of clinical samples to detect PARP activation in pancreatitis. Mice were subjected to cerulein-induced pancreatitis; circulating mediators and circulating organ injury markers; pancreatic myeloperoxidase and malondialdehyde levels were measured and histology of the pancreas was assessed. In human pancreatic duct epithelial cells (HPDE) subjected to oxidative stress, PARP activation was measured by PAR Western blotting and cell viability and DNA integrity were quantified. In clinical samples, PARP activation was assessed by PAR (the enzymatic product of PARP) immunohistochemistry. In male mice subjected to pancreatitis, olaparib (3 mg/kg i.p.) improved pancreatic function: it reduced pancreatic myeloperoxidase and malondialdehyde levels, attenuated the plasma amylase levels, and improved the histological picture of the pancreas. It also attenuated the plasma levels of pro-inflammatory mediators (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12, IP-10, KC) but not MCP-1, RANTES, or the anti-inflammatory cytokine IL-10. Finally, it prevented the slight, but significant increase in plasma blood urea nitrogen level, suggesting improved renal function. The protective effect of olaparib was also confirmed in female mice. In HPDE cells subjected to oxidative stress olaparib (1 μM) inhibited PARP activity, protected against the loss of cell viability, and prevented the loss of cellular NAD levels. Olaparib, at 1μM to 30 μM did not have any adverse effects on DNA integrity. In human pancreatic samples from patients who died of pancreatitis, increased accumulation of PAR was demonstrated. Olaparib improves organ function and tempers the hyperinflammatory response in pancreatitis. It also protects against pancreatic cell injury in vitro without adversely affecting DNA integrity. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of pancreatitis.

Vacuole Membrane Protein 1 Deficiency Promotes the Development of Pancreatitis Through Autophagy Impairment and Endoplasmic Reticulum Stress

Pancreatitis is a potentially fatal inflammatory disease with significant morbidity and mortality but the exact molecular mechanisms remain largely obscure. We investigated the role of vacuole membrane protein 1 (VMP1), an endoplasmic reticulum (ER) resident protein, in the pathogenesis of pancreatitis. We examined VMP1 expression and ER stress in human pancreatitis samples. We also determined VMP1 level and ER stress in experimental pancreatitis induced by cerulein as well as in pancreatic acinar cell‐specific VMP1 knockout (VMP1Δacinar) mice. We found that VMP1 expression was significantly lower in human pancreatitis samples associated with extensive ER stress. In a mouse experimental pancreatitis model, treatment with ceruleint markedly decreased VMP1 expression but increased levels of several ER stress markers such as p‐elf2α, XBP1s and CHOP. Western blot and immune histochemical analysis of human and experimental mouse pancreatitis samples showed impaired autophagy with accumulation of p62, an autophagy substrate protein that generally is degraded by autophagy. Intriguingly, VMP1Δacinar mice developed spontaneous pancreatitis with pathological features of chronic pancreatitis. In addition, VMP1Δacinar caused accumulation of p62 as well as selective ER‐phagy receptor FAM134B. Moreover, VMP1Δacinar mice had increased ER tress accompanied with massive cell death, suggesting VMP1 may serve as a key mediator of pancreatic ER homeostasis. Collectively, these data indicate a critical role of VMP1 in maintaining ER homeostasis in acinar cells and decreased VMP1 expression may contribute to the development of pancreatitis.Support or Funding InformationR01 AA020518, R01 DK102142, U01 AA024733 and P30GM118247 (W.X.D).

Pentoxifylline has favorable preventive effects on experimental chronic pancreatitis model

Background: To investigate the protective efficacy of pentoxifylline through biochemical parameters and histopathological scores in a caerulein- and alcohol-induced experimental model of chronic pancreatitis in rats.Methods: A model of chronic pancreatitis with caerulein and alcohol was created in female rats of the genus Sprague Dawley. Pentoxifylline was administered in doses of 25 mg/kg (low dose) and 50 mg/kg (high dose) as a protective agent. Each group contained 8 animals. The groups were: group 1 (control group); caerulein + alcohol, group 2 (low-dose pentoxifylline group); caerulein + alcohol + pentoxifylline 25 mg/kg, group 3 (high-dose pentoxifylline group); caerulein + alcohol + pentoxifylline 50 mg/kg, group 4 (placebo); caerulein + alcohol + saline, group 5 (sham group); only saline injection.Rats were sacrificed 12 h after the last injection, and TNF-α, TGF-β, MDA, and GPx concentrations were measured in blood samples. The histopathologic examination was conducted by a pathologist who was unaware of the groups.Results: The biochemical results of the treatment groups (group 2 and group 3) were statistically significantly lower compared with the control group (group 1) (p < .05). The difference between the low-dose treatment group (group 2) and high-dose treatment group (group 3) was significant in terms of biochemical parameters (p < .05). The difference between group 2 and the control group was not significant in terms of histopathologic scores (p > .05), whereas the difference between the group 3 and the control group was statistically significant (p < .05).Conclusions: As a result, pentoxifylline, which has anti-inflammatory and antioxidant properties, was shown to have protective efficacy in an experimentally generated model of chronic pancreatitis.

Novel mitochondrial transition pore inhibitor N-methyl-4-isoleucine cyclosporin is a new therapeutic option in acute pancreatitis.

•Bile acids, ethanol and fatty acids affect pancreatic ductal fluid and bicarbonate secretion via mitochondrial damage, ATP depletion and calcium overload. •Pancreatitis-inducing factors open the membrane transition pore (mPTP) channel via cyclophilin D activation in acinar cells, causing calcium overload and cell death; genetic or pharmacological inhibition of mPTP improves the outcome of acute pancreatitis in animal models. •Here we show that genetic and pharmacological inhibition of mPTP protects mitochondrial homeostasis and cell function evoked by pancreatitis-inducing factors in pancreatic ductal cells. •The results also show that the novel cyclosporin A derivative NIM811 protects mitochondrial function in acinar and ductal cells, and it preserves bicarbonate transport mechanisms in pancreatic ductal cells. •We found that NIM811 is highly effective in different experimental pancreatitis models and has no side-effects. NIM811 is a highly suitable compound to be tested in clinical trials. Mitochondrial dysfunction plays a crucial role in the development of acute pancreatitis (AP); however, no compound is currently available with clinically acceptable effectiveness and safety. In this study, we investigated the effects of a novel mitochondrial transition pore inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), in AP. Pancreatic ductal and acinar cells were isolated by enzymatic digestion from Bl/6 mice. In vitro measurements were performed by confocal microscopy and microfluorometry. Preventative effects of pharmacological [cylosporin A (2µm), NIM811 (2µm)] or genetic (Ppif-/- /Cyp D KO) inhibition of the mitochondrial transition pore (mPTP) during the administration of either bile acids (BA) or ethanol+fatty acids (EtOH+FA) were examined. Toxicity of mPTP inhibition was investigated by detecting apoptosis and necrosis. In vivo effects of the most promising compound, NIM811 (5 or 10mgkg-1 per os), were checked in three different AP models induced by either caerulein (10×50µgkg-1 ), EtOH+FA (1.75gkg-1 ethanol and 750mgkg-1 palmitic acid) or 4% taurocholic acid (2mlkg-1 ). Both genetic and pharmacological inhibition of Cyp D significantly prevented the toxic effects of BA and EtOH+FA by restoring mitochondrial membrane potential (Δψ) and preventing the loss of mitochondrial mass. In vivo experiments revealed that per os administration of NIM811 has a protective effect in AP by reducing oedema, necrosis, leukocyte infiltration and serum amylase level in AP models. Administration of NIM811 had no toxic effects. The novel mitochondrial transition pore inhibitor NIM811 thus seems to be an exceptionally good candidate compound for clinical trials in AP.

EIF4A inhibition circumvents uncontrolled DNA replication mediated by 4E-BP1 loss in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) relies on hyperactivated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in pancreatic cancer cells. We identified a functional enrichment for mRNAs encoding DNA replication and repair proteins, including RRM2 and CDC6. Consequently, 4E-BP1 depletion favors DNA repair and renders DNA replication insensitive to mTOR inhibitors, in correlation with a sustained protein expression of CDC6 and RRM2, which is inversely correlated with 4E-BP1 expression in PDAC patient samples. DNA damage and pancreatic lesions induced by an experimental pancreatitis model uncover that 4E-BP1/2-deleted mice display an increased acinar cell proliferation and a better recovery than WT animals. Targeting translation, independently of 4E-BP1 status, using eIF4A RNA helicase inhibitors (silvestrol derivatives) selectively modulates translation and limits CDC6 expression and DNA replication, leading to reduced PDAC tumor growth. In summary, 4E-BP1 expression loss during PDAC development induces selective changes in translation of mRNA encoding DNA replication and repair protein. Importantly, targeting protein synthesis by eIF4A inhibitors circumvents PDAC resistance to mTOR inhibition.

Protective effects of urocortin 2 against caerulein-induced acute pancreatitis.

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

Preclinical Evaluation of 68Ga-DOTA-NT-20.3: A Promising PET Imaging Probe To Discriminate Human Pancreatic Ductal Adenocarcinoma from Pancreatitis.

Neurotensin receptor 1 (NTSR1) is overexpressed in human pancreatic ductal adenocarcinoma (PDAC). Specific noninvasive positron-emission tomography (PET) imaging probes may improve the diagnostic accuracy and the monitoring of therapy for patients with PDAC. Here, we report the use of the 68Ga-labeled neurotensin (NTS) analogue DOTA-NT-20.3 to image human PDAC in animal models and to discriminate tumors from pancreatitis. In addition to the preclinical study, two tissue microarray slides, constructed by small core biopsies (2-5) from standard paraffin-embedded tumor tissues, were used to confirm the high (78%) positivity rate of NTSR1 expression in human PDAC. PET imaging, biodistribution, blocking, and histology studies were performed in subcutaneous AsPC-1 pancreatic tumor-bearing mice. 68Ga-DOTA-NT-20.3 PET images showed rapid tumor uptake and high contrast between the tumor and background with a fast blood clearance and a moderate accumulation in the kidneys. Ex vivo biodistribution showed low uptake in normal pancreas (0.22% IA/g) and in the remaining organs at 1 h postinjection, kidney retention (5.38 ± 0.54% IA/g), and fast clearance from blood and confirmed high uptake in tumors (5.28 ± 0.93% IA/g), leading to a tumor-to-blood ratio value of 6 at 1 h postinjection. The significant decrease of tumor uptake in a blocking study demonstrated the specificity of 68Ga-DOTA-N-T20.3 to target NTSR1 in vivo. PET imaging was also conducted in an orthotopic xenograft model that allows tumors to grow in their native microenvironment and in an experimental pancreatitis model generated by caerulein injections. As opposed to 2-[18F]fluoro-deoxyglucose, 68Ga-DOTA-NT-20.3 distinguishes PDAC from pancreatitis. Thus, 68Ga-DOTA-NT-20.3 is a promising PET imaging probe for imaging PDAC in humans.

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Deficiency of cathepsin C ameliorates severity of acute pancreatitis by reduction of neutrophil elastase activation and cleavage of E-cadherin

Acute pancreatitis is characterized by premature intracellular protease activation and infiltration of inflammatory cells, mainly neutrophil granulocytes and macrophages, into the organ. The lysosomal proteases cathepsin B, D, and L have been identified as regulators of early zymogen activation and thus modulators of the severity of pancreatitis. Cathepsin C (CTSC, syn. dipeptidly-peptidase I) is a widely expressed, exo-cystein-protease involved in the proteolytic processing of various other lysosomal enzymes. We have studied its role in pancreatitis. We used CTSC-deleted mice and their WT littermates in two experimental models of pancreatitis. The mild model involved eight hourly caerulein injections and the severe model partial duct ligation. Isolated pancreatic acini and spleen-derived leukocytes were used for ex vivo experiments. CTSC is expressed in the pancreas and in inflammatory cells. CTSC deletion reduced the severity of pancreatitis (more prominently in the milder model) without directly affecting intra-acinar cell trypsin activation in vitro The absence of CTSC reduced infiltration of neutrophil granulocytes impaired their capacity for cleaving E-cadherin in adherens junctions between acinar cells and reduced the activity of neutrophil serine proteases polymorphonuclear (neutrophil) elastase, cathepsin G, and proteinase 3, but not neutrophil motility. Macrophage invasion was not dependent on the presence of CTSC. CTSC is a regulator and activator of various lysosomal enzymes such as cathepsin B, D, and L. Its loss mitigates the severity of pancreatitis not by reducing intra-acinar cell zymogen activation but by reducing infiltration of neutrophil granulocytes into the pancreas. In this context one of its key roles is that of an activator of neutrophil elastase.

Genistein protects against acute pancreatitis via activation of an apoptotic pathway mediated through endoplasmic reticulum stress in rats

Acute pancreatitis (AP) is a severe and frequently lethal disorder, but the precise mechanisms are not well understood and there is lack of effective drugs. Therefore, our study examined the in vivo intervention effects of genistein and elucidated its mechanism in acute experimental pancreatitis models. We used cerulein or taurocholate to induce acute pancreatitis (AP) in Sprague-Dawley rats with prior genistein treatment. Histological examination of the pancreas was performed and the expression of unfolded protein response (UPR) components and apoptotic mediators like caspase 12 and c-Jun N-terminal protein kinase (JNK) were measured. The amount of apoptosis in pancreatic acinar cells was also determined. Our studies found that the severity of cerulein- or taurocholate-induced AP was rescued by prior genistein treatment. Genistein stimulated the activation of multiple endoplasmic reticulum (ER) stress-related regulators like GRP78, PERK, eIF2α, and upregulated the expression of the apoptotic genes, caspase 12 and CHOP. Moreover, TUNEL assays showed that genistein treatment promoted acinar cell apoptosis. Taken together, we speculated that ER stress-associated apoptotic pathways in AP are induced by genistein, which showed cytoprotective capacity in the exocrine pancreas. These data suggest novel therapeutic strategies that employ genistein in the prevention of AP.