Cerulein-induced Pancreatitis Research Articles

Activin A signaling stimulates neutrophil activation and macrophage migration in pancreatitis

Acute Pancreatitis (AP) is associated with high mortality and current treatment options are limited to supportive care. We found that blockade of activin A (activin) in mice improves outcomes in two murine models of AP. To test the hypothesis that activin is produced early in response to pancreatitis and is maintained throughout disease progression to stimulate immune cells, we first performed digital spatial profiling (DSP) of human chronic pancreatitis (CP) patient tissue. Then, transwell migration assays using RAW264.7 mouse macrophages and qPCR analysis of “neutrophil-like” HL-60 cells were used for functional correlation. Immunofluorescence and western blots on cerulein-induced pancreatitis samples from pancreatic acinar cell-specific Kras knock-in (Ptf1aCreER™; LSL-KrasG12D) and functional WT Ptf1aCreER™ mouse lines mimicking AP and CP to allow for in vivo confirmation. Our data suggest activin promotes neutrophil and macrophage activation both in situ and in vitro, while pancreatic activin production is increased as early as 1 h in response to pancreatitis and is maintained throughout CP in vivo. Taken together, activin is produced early in response to pancreatitis and is maintained throughout disease progression to promote neutrophil and macrophage activation.

The UBE2F-CRL5ASB11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells

UBE2F, a neddylation E2, neddylates CUL5 to activate cullin-RING ligase-5, upon coupling with neddylation E3 RBX2/SAG. Whether and how UBE2F controls pancreatic tumorigenesis is previously unknown. Here, we showed that UBE2F is essential for the growth of human pancreatic cancer cells with KRAS mutation. In the mouse KrasG12D pancreatic ductal adenocarcinoma (PDAC) model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signals via blocking ubiquitylation of Diras2, a substrate of CRL5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of human pancreatic cancer cells harboring mutant KRAS, and Diras2 deletion largely rescues the phenotypes induced by Ube2f deletion. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in the mouse KrasG12D PDAC model, respectively. The UBE2F-CRL5ASB11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients.

Abstract 4391: The UBE2F-CRL5Asb11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells

Abstract UBE2F is a neddylation E2 that couples with RBX2/SAG E3 to promote CUL-5 neddylation and Cullin-RING ligase-5 (CRL5) activation. Whether and how UBE2F regulates pancreatic tumorigenesis is previously unknown. Here we showed that UBE2F is growth essential for pancreatic cancer cells with KRAS mutant in culture. In KrasG12D mouse PDAC model, Ube2f deletion suppresses cerulein-induced pancreatitis, and progression of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Mechanistically, Ube2f deletion inactivates the Mapk-c-Myc signal pathways via blocking ubiquitylation and degradation of Diras2, which is a new substrate of Cul5Asb11 E3 ligase. Biologically, DIRAS2 suppresses growth and survival of pancreatic cancer cells with KRAS mutant, and Diras2 deletion largely rescued the phenotypes induced by Ube2f deletion, indicating its causal role. Collectively, Ube2f or Diras2 plays a tumor-promoting or tumor-suppressive role in KrasG12D PDAC model, respectively. The Ube2f-CRL5Asb11 axis could serve as a valid target for pancreatic cancer, whereas the levels of UBE2F or DIRAS2 may serve as prognostic biomarkers for PDAC patients. Citation Format: Yi Sun, Yu Chang, Qian Che, Hua Li, Jie Xu, Mingjia Tan, Xiufang Xiong. The UBE2F-CRL5Asb11-DIRAS2 axis is an oncogene and tumor suppressor cascade in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4391.

Abstract C089: A novel role of p53 in pancreatic acinar cell identity

Abstract Pancreatitis is an inflammatory disease that affects acinar cells in the exocrine pancreas, and can be triggered by several factors, such as alcohol abuse, smoking and familial genetic alterations. Pancreatic injury associated with pancreatitis is characterized by inflammation, acinar cell death, and acinar-to-ductal metaplasia (ADM), a regenerative process in which metaplastic cells proliferate and re-differentiate into acinar cells. Recurrent pancreatitis can cause persistent ADM, impairing exocrine function and causing precancerous lesions known as pancreatic intraepithelial neoplasia (PanIN) that can initiate pancreatic ductal adenocarcinoma (PDAC), a nearly always fatal disease. Importantly, the lack of treatment options for pancreatitis patients reinforces the need for strategies that could resolve pancreatic damage and stop disease progression. We have recently shown that pancreatic cells harboring a hyperactive variant of p53 display increased resistance to developing acute pancreatitis induced by cerulein (a secretagogue which mimics cholecystokinin). Here, we show that p53 activation using the drug Nutlin-3a can repair the damage caused by cerulein-induced pancreatitis, and reduce the formation of PanIN lesions. This repair is achieved exclusively at low doses of Nutlin-3a, which cause a mild p53 activation, and is entirely dependent on the presence of the p53 gene. We have also identified novel p53 target genes relevant to acinar identity from ChIP-seq and RNA-seq data, and validated their dependence on p53 in mouse embryonic fibroblasts and in pancreatic precancerous lesions. Lastly, we have observed expression of p53 in the developing pancreas at embryonic day 15.5, indicating a potential function of p53 in pancreatic development. Our findings suggest a novel role of p53 in rescuing acinar cells from damage, and that p53 activation may be used as a treatment for pancreatitis and pancreatic cancer prevention. Citation Format: Jennifer J. Twardowski, Stephano S. Mello, Thomas I. Heist. A novel role of p53 in pancreatic acinar cell identity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C089.

Abstract C100: Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis

Abstract Biological stresses that are cell-intrinsic (e.g., oncogene-induced imbalances in protein translation, lipid synthesis, mitochondrial dysfunction) or cell-extrinsic (e.g., nutrient deprivation, hypoxia, drug exposure) disrupt cellular function and viability if not mitigated. The Integrated Stress Response (ISR) consists of upstream regulatory kinases (PERK, GCN2, HRI, PKR) that respond to such stresses in part by activating the ATF4 transcription factor. ATF4 then rewires nutrient uptake, cellular metabolism, mitochondrial function, and protein chaperone networks to restore cellular homeostasis. We have shown that ATF4 is upregulated in mouse and human pancreatic ductal adenocarcinoma (PDAC), even compared to normal acinar cells where ATF4 is expressed at relatively high levels. To investigate the role of ATF4 in PDAC, we generated mice with inducible acinar-specific deletion of Atf4 using Ptf1a-CreERT to delete Atf4 following treatment of adult Atf4-floxed mice with tamoxifen. In contrast to previous studies showing germline deletion of Atf4 in mice caused premature mortality at approximately 3 weeks of age due to pancreatic insufficiency, inducible deletion of Atf4 in adult acinar cells does not cause pancreatic deficiency or animal demise. Rather, adult Ptf1a-CreERT;Atf4fl/fl (CA) mice are viable although more sensitive to cerulein-induced pancreatitis. Specifically, compared to control (WT) mice, CA mice exhibit more extensive acinar-ductal metaplasia (ADM), including more extensive expression of cytokeratin-19 (CK19), increased cell death and take longer to recover normal pancreatic architecture following in vivo cerulein challenge. Using ex vivo acinar explant assays derived from WT and CA mice, we further show that induction of ADM in WT acinar cells with TGF-a is associated with down-regulation of Atf4 and Atf4 target genes in WT acinar explants while CA explants undergo accelerated ADM. Furthermore, expression of exogenous Atf4 in acinar cells inhibited ADM. These in vivo and ex vivo findings are consistent with down-regulation of Atf4 being required for ADM. Given the tumor-promoting role of ADM in the early stages of PDAC, we next crossed the CA mice to LSL-KRasG12D mice to generate LSL-KRasG12D;Ptf1a-CreERT;Atf4fl/fl (KCA) mice to examine how loss of Atf4 affected pancreatic intraepithelial neoplasia (PanIN) and PDAC formation. Surprisingly, given the critical need to down-regulate Atf4 in ADM, we show for the first time that genetic deletion of Atf4 in pancreatic acinar cells blocks both PanIN and PDAC formation in KCA mice compared to age-matched KC mice. Rather, KCA mice developed a fatty pancreas devoid of tumor with very few CK19-positive cells forming. These results suggest that Atf4 is critical for PanIN formation and prevents PDAC. This also suggests that Atf4 plays a different role in PanIN formation than in ADM where its down-regulation was critical and indicates a dynamic role for Atf4 depending on oncogenic versus proteotoxic stress, as will be discussed. Citation Format: Leah M. Ziolkowski, Kay F. Macleod. Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C100.

SHCBP1 Overexpression Aggravates Pancreatitis by Triggering the Loss of Primary Cilia.

Primary cilia are microtubule-based organelles that mediate various biological processes. Pancreatic cells are typically ciliated; however, the role of primary cilia in acute pancreatitis (AP) is largely unknown. Here, we report that the loss of primary cilia, mediated by SHCBP1 (SHC1 binding protein), exerted a provocative effect on AP. Primary cilia are extensively lost in inflamed pancreatic cells in vitro and in mouse tissues with AP in vivo. Abrogation of primary cilia aggravated lipopolysaccharide (LPS)-induced inflammation in pancreatic cells. Mechanistically, AP induced the overexpression of SHCBP1 mitotic factor, which is localized to the base of primary cilia. SHCBP1 deficiency relieved LPS- and cerulein-induced pancreatitis by preventing the loss of primary cilia in vitro and in vivo. Collectively, we reveal that inflammation-induced loss of primary cilia aggravates AP. Furthermore, abrogating SHCBP1 to prevent primary cilia loss is an efficient strategy to combat AP.

Pancreatic Ubap2 deletion regulates glucose tolerance, inflammation, and protection from cerulein-induced pancreatitis

Ubiquitin-binding associated protein 2 (UBAP2) is reported to promote macropinocytosis and pancreatic adenocarcinoma (PDAC) growth, however, its role in normal pancreatic function remains unknown. We addressed this knowledge gap by generating UBAP2 knockout (U2KO) mice under a pancreas-specific Cre recombinase (Pdx1-Cre). Pancreatic architecture remained intact in U2KO animals, but they demonstrated slight glucose intolerance compared to controls. Upon cerulein challenge to induce pancreatitis, U2KO animals had reduced levels of several pancreatitis-relevant cytokines, amylase and lipase in the serum, reduced tissue damage, and lessened neutrophil infiltration into the pancreatic tissue. Mechanistically, cerulein-challenged U2KO animals revealed reduced NF-κB activation compared to controls. In vitro promoter binding studies confirmed the reduction of NF-κB binding to its target molecules supporting UBAP2 as a new regulator of inflammation in pancreatitis and may be exploited as a therapeutic target in future to inhibit pancreatitis.

The role of iron and ferroptosis in the pathogenesis of acute pancreatitis

ABSTRACT Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Iron is an essential element for life and is involved in many metabolic processes. Ferroptosis is a type of regulated cell death that is triggered by iron and oxidative stress. A well-established mouse AP model was adopted to study the role of iron and ferroptosis in the pathogenesis of pancreatitis. Mice were injected with cerulein to induce AP, and pancreatic tissue samples were analyzed to determine the pathology, cell death, iron deposition, expression of iron transporters, and lipid peroxidation. The role of iron was studied by giving mice extra iron or iron chelator. In vitro studies with acinar cells with ferroptosis activator and inhibitor were also performed to assess the inflammatory response. Iron was found accumulated in the pancreatic tissue of mice who suffered cerulein-induced pancreatitis. Cell death and lipid peroxidation increased in these tissues and could be further modulated by iron dextran or iron chelator. Mice given Hemin through gavage had reduced levels of GSH in pancreatic tissue and increased inflammatory response. Studies with acinar cells showed increased levels of lipid peroxidation and ferroptosis-specific mitochondrial damage when treated with ferroptosis inducer and inflammatory cytokines.

Rectal administration of tacrolimus protects against post-ERCP pancreatitis in mice

ObjectiveThere is an unmet clinical need for effective, targeted interventions to prevent post-ERCP pancreatitis (PEP). We previously demonstrated that the serine-threonine phosphatase, calcineurin (Cn) is a critical mediator of PEP and that the FDA-approved calcineurin inhibitors, tacrolimus (Tac) or cyclosporine A, prevented PEP. Our recent observations in preclinical PEP models demonstrating that Cn deletion in both pancreatic and hematopoietic compartments is required for maximal pancreas protection, highlighted the need to target both systemic and pancreas-specific Cn signaling. We hypothesized that rectal administration of Tac would effectively mitigate PEP by ensuring systemic and pancreatic bioavailability of Tac. We have tested the efficacy of rectal Tac in a preclinical PEP model and in cerulein-induced experimental pancreatitis. MethodsC57BL/6 mice underwent ductal cannulation with saline infusion to simulate pressure-induced PEP or were given seven, hourly, cerulein injections to induce pancreatitis. To test the efficacy of rectal Tac in pancreatitis prevention, a rectal Tac suppository (1 mg/kg) was administered 10 min prior to cannulation or first cerulein injection. Histological and biochemical indicators of pancreatitis were evaluated post-treatment. Pharmacokinetic parameters of Tac in the blood after rectal delivery compared to intravenous and intragastric administration was evaluated. ResultsRectal Tac was effective in reducing pancreatic injury and inflammation in both PEP and cerulein models. Pharmacokinetic studies revealed that the rectal administration of Tac helped achieve optimal blood levels of Tac over an extended time compared to intravenous or intragastric delivery. ConclusionOur results underscore the effectiveness and clinical utility of rectal Tac for PEP prophylaxis.

Repetitive Cerulein-Induced Chronic Pancreatitis in Growing Pigs-A Pilot Study.

Chronic pancreatitis (CP) is an irreversible and progressive inflammatory disease. Knowledge on the development and progression of CP is limited. The goal of the study was to define the serum profile of pro-inflammatory cytokines and the cell antioxidant defense system (superoxidase dismutase-SOD, and reduced glutathione-GSH) over time in a cerulein-induced CP model and explore the impact of these changes on selected cytokines in the intestinal mucosa and pancreatic tissue, as well as on selected serum biochemical parameters. The mRNA expression of CLDN1 and CDH1 genes, and levels of Claudin-1 and E-cadherin, proteins of gut barrier, in the intestinal mucosa were determined via western blot analysis. The study showed moderate pathomorphological changes in the pigs' pancreas 43 days after the last cerulein injection. Blood serum levels of interleukin (IL)-1-beta, IL-6, tumor necrosis factor alpha (TNF-alpha), C-reactive protein (CRP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGTP), SOD and GSH were increased following cerulein injections. IL-1-beta, IL-6, TNF-alpha and GSH were also increased in jejunal mucosa and pancreatic tissue. In duodenum, decreased mRNA expression of CDH1 and level of E-cadherin and increased D-lactate, an indicator of leaky gut, indicating an inflammatory state, were observed. Based on the current results, we can conclude that repetitive cerulein injections in growing pigs not only led to CP over time, but also induced inflammation in the intestine. As a result of the inflammation, the intestinal barrier was impaired.

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

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

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition

Background and aimsThiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear.MethodsTo model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl– and HCO3– secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization.ResultsWe demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3– and Cl– secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR.ConclusionsAZA impaired the ductal HCO3– and Cl– secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

A hydrolysate of poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) suppresses Cav3.2-dependent pain by sequestering exogenous and endogenous sulfide

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.

Preclinical testing of dabigatran in trypsin-dependent pancreatitis.

Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis, with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations showed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is probably related to the more severe pathology and insufficient drug concentrations in the pancreas.

Downregulation of miR-146a-5p Promotes Acute Pancreatitis through Activating the TLR9/NLRP3 Signaling Pathway by Targeting TRAF6 In Vitro Rat Model.

Acute pancreatitis (AP) is mainly caused by acinar cells releasing various inflammatory factors, causing inflammatory storms and leading to severe pancreatitis. Detection methods and treatment targets for pancreatitis are lacking, raising the urgency of identifying diagnostic markers and therapeutic targets for AP. MicroRNAs (miRNAs) have recently been identified as molecular markers for various biological processes such as tumors, immunity, and metabolism, and the involvement of miRNAs in inflammatory responses has been increasingly studied. To explore the role of miRNAs in AP is the primary objective of this study. By using qPCR on our cerulein-induced pancreatitis cell model, it is worth noting that the change of miR-146a-5p expression in inflammation-related miRNAs in AP was predominant. Next, ELISA, CCK8, and flow cytometry were used to inspect the impact of miR-146a-5p on pancreatitis. BiBiServ bioinformatics anticipated binding ability of miR-146a-5p and 3′-untranslated region (3′UTR) of TNF receptor-associated factor 6 (TRAF6), and the dual-luciferase assay verified the combination of the two. TRAF6 knockdown verified the effect of TRAF6 on the progression of pancreatitis. Finally, rescue experiments verified the capability of miR-146a-5p and TRAF6 interaction on the Toll-like receptor 9 (TLR9)/NOD-like receptor protein 3 (NLRP3) signaling pathway and cell function. The expression of miR-146a-5p decreased in cerulein-induced AR42J pancreatic acinar cells. Functional experiments verified that miR-146a-5p facilitated the proliferation of AR42J pancreatic acinar cells and inhibited their apoptosis. Bioinformatic predictions and dual-luciferase experiments verified the actual binding efficiency between miR-146a-5p and 3′UTR of TRAF6. Our study confirmed that knockdown of TRAF6 restrained the progression of pancreatitis, and knockdown of TRAF6 rescued pancreatitis caused by miR-146a-5p downregulation by the TLR9/NLRP3 signaling pathway. Therefore, downregulation of miR-146a-5p in the induced pancreatitis cell model promotes the progression of pancreatitis via the TLR9/TRAF6/NLRP3 signaling pathway. There is potential for miR-146a-5p to serve as a diagnostic marker and therapeutic nucleic acid drug for AP.

A Mouse Model of Acute and Chronic Pancreatitis.

Pancreatitis occurs in two forms defined by its chronicity. Acute pancreatitis (AP) occurs suddenly and only lasts for several days. Consequently, most patients with AP recover without permanent damage to the pancreas, and about 20% of patients with AP have severe disease. In contrast, chronic pancreatitis (CP) is a long-lasting inflammation that causes permanent damage to pancreatic tissue; consequently, this form is marked by the emergence of persistent endocrine and exocrine pancreatic insufficiency. Despite these differences, AP and CP share central mechanisms of disease: in both forms, inflammation is initiated and/or sustained by the intrapancreatic activation of pancreatic digestive enzymes followed by the autodigestion of pancreatic tissues. In addition, in both forms enzymatic damage is accompanied by changes in intestinal permeability and entry of commensal organisms into the pancreas where they elicit innate immune responses that ultimately dominate and define pancreatic inflammation. In the murine models of AP and CP described here, both of these elements of pancreatitis pathogenesis are taken into account. Thus, in one approach mice are administered high doses of cerulein, a cholecystokinin analog with the ability at this dose to induce excessive activation of the cholecystokinin receptor expressed in pancreatic acinar cells and the release of active trypsin that causes both direct and indirect acinar damages due to entry of commensal organisms and stimulation of innate immune responses. In a second approach mice are administered low doses of cerulein, which causes little or no damage to the pancreas unless given along with nucleotide-binding oligomerization domain 1 (NOD1) ligand, which in the presence of low-dose cerulein administration induces a pathologic innate immune response mediated by NOD1. These approaches are adopted to produce AP when cerulein or cerulein plus NOD1 ligand is applied only once or to produce CP when a similar regimen is applied multiple times. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cerulein-induced acute pancreatitis Alternate Protocol 1: Acute pancreatitis induced by cerulein and NOD1 ligand Basic Protocol 2: Cerulein-induced chronic pancreatitis Alternate Protocol 2: Chronic pancreatitis induced by cerulein and NOD1 ligand Support Protocol: Isolation of pancreatic mononuclear cells.

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

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

Analysis of Pain and Analgesia Protocols in Acute Cerulein-Induced Pancreatitis in Male C57BL/6 Mice.

Pancreatitis is known to be painful in humans and companion animals. However, the extent of pain in experimental mouse models of acute pancreatitis is unknown. Consequently, the severity classification of acute pancreatitis in mice is controversially discussed and standardized pain management is missing. In this study, we investigated acute Cerulein-induced pancreatitis with pain-specific and well-being orientated parameters to detect its impact on mice. Male C57BL/6J male mice were injected with Cerulein; animals that received saline injections served as control group. The animals were observed for weight change and water intake. To assess pain, behaviors like stretch-and-press and reduced rearing, the Mouse Grimace Scale, and von Frey hypersensitivity were assessed. Fecal corticosterone metabolites and burrowing behavior were assessed to detect changes in the animal’s well-being. Pancreatitis severity was evaluated with amylase and lipase in the blood and pancreas histology. To investigate whether different analgesics can alleviate signs of pain, and if they influence pancreas inflammation, animals received Buprenorphine, Paracetamol in combination with Tramadol, or Metamizole in the drinking water. The calculated intake of these analgesics via drinking reached values stated to be efficient for pain alleviation. While pancreatitis did not seem to be painful, we detected acute pain from Cerulein injections that could not be alleviated by analgesics. The number of inflammatory cells in the pancreas did not differ with the analgesic administered. In conclusion: (1) Cerulein injections appear to be acutely painful but pain could not be alleviated by the tested analgesics, (2) acute pancreatitis induced by our protocol did not induce obvious signs of pain, (3) analgesic substances had no detectable influence on inflammation. Nevertheless, protocols inducing more severe or even chronic pancreatitis might evoke more pain and analgesic treatment might become imperative. Considering our results, we recommend the use of Buprenorphine via drinking water in these protocols. Further studies to search for efficient analgesics that can alleviate the acute pain induced by Cerulein injections are needed.

Glyoxalase-I Is Upregulated in Acute Cerulein-Induced Pancreatitis: A New Mechanism in Pancreatic Inflammation?

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

SETD4-expressing cells contribute to pancreatic development and response to cerulein induced pancreatitis injury

In the adult pancreas, the presence of progenitor or stem cells and their potential involvement in homeostasis and regeneration remains unclear. Here, we identify that SET domain-containing protein 4 (SETD4), a histone lysine methyltransferase, is expressed in a small cell population in the adult mouse pancreas. Genetic lineage tracing shows that during pancreatic development, descendants of SETD4+ cells make up over 70% of pancreatic cells and then contribute to each pancreatic lineage during pancreatic homeostasis. SETD4+ cells generate newborn acinar cells in response to cerulein-induced pancreatitis in acinar compartments. Ablation of SETD4+ cells compromises regeneration of acinar cells, in contrast to controls. Our findings provide a new cellular narrative for pancreatic development, homeostasis and response to injury via a small SETD4+ cell population. Potential applications may act to preserve pancreatic function in case of pancreatic disease and/or damage.