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.

Initiation and severity of experimental pancreatitis are modified by phosphate.

Proper mitochondrial function and adequate cellular ATP are necessary for normal pancreatic protein synthesis and sorting, maintenance of intracellular organelles and enzyme secretion. Inorganic phosphate is required for generating ATP and its limited availability may lead to reduced ATP production causing impaired Ca2+ handling, defective autophagy, zymogen activation, and necrosis, which are all features of acute pancreatitis. We hypothesized that reduced dietary phosphate leads to hypophosphatemia and exacerbates pancreatitis severity of multiple causes. We observed that mice fed a low-phosphate diet before the induction of pancreatitis by either repeated caerulein administration or pancreatic duct injection as a model of pressure-induced pancreatitis developed hypophosphatemia and exhibited more severe pancreatitis than normophosphatemic mice. Pancreatitis severity was significantly reduced in mice treated with phosphate. In vitro modeling of secretagogue- and pressure-induced pancreatic injury was evaluated in isolated pancreatic acini using cholecystokinin and the mechanoreceptor Piezo1 agonist, Yoda1, under low and normal phosphate conditions. Isolated pancreatic acini were more sensitive to cholecystokinin- and Yoda1-induced acinar cell damage and mitochondrial dysfunction under low-phosphate conditions and improved following phosphate supplementation. Importantly, even mice on a normal phosphate diet exhibited less severe pancreatitis when treated with supplemental phosphate. Thus, hypophosphatemia sensitizes animals to pancreatitis and phosphate supplementation reduces pancreatitis severity. These appear to be direct effects of phosphate on acinar cells through restoration of mitochondrial function. We propose that phosphate administration may be useful in the treatment of acute pancreatitis.NEW & NOTEWORTHY Impaired ATP synthesis disrupts acinar cell homeostasis and is an early step in pancreatitis. We report that reduced phosphate availability impairs mitochondrial function and worsens pancreatic injury. Phosphate supplementation improves mitochondrial function and protects against experimental pancreatitis, raising the possibility that phosphate supplementation may be useful in treating pancreatitis.

Paeonol protects against acute pancreatitis by inhibiting M1 macrophage polarization via the NLRP3 inflammasomes pathway.

The excessive inflammatory response mediated by macrophage is one of the key factors for the progress of acute pancreatitis (AP). Paeonol (Pae) was demonstrated to exert multiple anti-inflammatory effects. However, the role of Pae on AP is not clear. In the present study, we aimed to investigate the protective effect and mechanism of Pae on AP in vivo and vitro. In the caerulein-induced mild acute pancreatitis (MAP) model, we found that Pae administration reduced serum levels of amylase, lipase, IL-1ß and IL-6 and alleviated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. And Pae decrease the ROS generated, restore mitochondrial membrane potential (ΔΨm), inhibit M1 macrophage polarization and NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) in vitro. In addition, specific NLRP3 inhibitor MCC950 eliminated the protective effect of Pae on AP induced by caerulein in mice. Correspondingly, the inhibitory effect of Pae on ROS generated and M1 polarization was not observed in BMDMs with MCC950 in vitro. Taken together, our datas for the first time confirmed the protective effects of Pae on AP via the NLRP3 inflammasomes Pathway.

MiR-325-3p Alleviates Acute Pancreatitis via Targeting RIPK3.

BACKGROUND AND AIMS: Acute pancreatitis (AP) is an acute inflammatory disease that can lead to death. Mir-325-3p is strongly and abnormally expressed in many diseases, necessitating exploration of its function and mechanism in AP. METHODS: Blood samples from AP patients and mice were analyzed. The expression levels of miR-325-3p in AP patients and mouse were detected. Whether miR-325-3p targets RIPK3 gene was predicted by TargetScan online database and dual luciferase reporter assay. In vitro experiments verified the effect of miR-325-3p overexpression on caerulein-induced MPC83 pancreatic acinar cancer cell line. In vivo experiments verified the effect of overexpression of miR-325-3p on the disease degree of pancreatic tissues in AP mice. RESULTS: Analysis of blood samples from AP patients and experiments in mice demonstrated that expression of miR-325-3p was significantly reduced during the process of AP in humans and mice. Predicted using the TargetScan online database and through dual luciferase reporter assay detection, miR-325-3p directly targets the RIPK3 gene. In vitro experiments revealed that overexpression of miR-325-3p reversed caerulein-induced apoptosis and necroptosis in MPC83 pancreatic acinar cancer cell line. We used Z-VAD-FMK to assess necroptosis and demonstrated that miR-325-3p targets necroptosis to reduce cell damage. In subsequent experiments in mice, we verified that overexpression of miR-325-3p reduces inflammation, edema, hemorrhage, and necrosis in acute pancreatitis. Characteristic western blot, immunohistochemistry, and transmission electron microscopy results revealed that overexpression of miR-325-3p reduces the severity of acute pancreatitis by inhibiting pancreatic necroptosis in AP mice. CONCLUSIONS: The current research results indicate that miR-325-3p directly targets RIPK3 and exerts a protective role in mouse AP. Necroptosis is still the primary mechanism of RIPK3 regulation. MiR-325-3p inhibits acute pancreatitis by targeting RIPK3-dependent necroptosis, which may represent a novel treatment method for acute pancreatitis.

Systemic Bile Acids Affect the Severity of Acute Pancreatitis in Mice Depending on Their Hydrophobicity and the Disease Pathogenesis.

Acute pancreatitis (AP) is a major, globally increasing gastrointestinal disease and a biliary origin is the most common cause. However, the effects of bile acids (BAs), given systemically, on the pancreas and on disease severity remains elusive. In this study, we have investigated the roles of different circulating BAs in animal models for AP to elucidate their impact on disease severity and the underlying pathomechanisms. BAs were incubated on isolated acini and AP was induced through repetitive injections of caerulein or L-arginine; pancreatic duct ligation (PDL); or combined biliopancreatic duct ligation (BPDL). Disease severity was assessed using biochemical and histological parameters. Serum cholecystokinin (CCK) concentrations were determined via enzyme immunoassay. The binding of the CCK1 receptor was measured using fluorescence-labeled CCK. In isolated acini, hydrophobic BAs mitigated the damaging effects of CCK. The same BAs further enhanced pancreatitis in L-arginine- and PDL-based pancreatitis, whereas they ameliorated pancreatic damage in the caerulein and BPDL models. Mechanistically, the binding affinity of the CCK1 receptor was significantly reduced by hydrophobic BAs. The hydrophobicity of BAs and the involvement of CCK seem to be relevant in the course of AP. Systemic BAs may affect the severity of AP by interfering with the CCK1 receptor.

Triptolide Suppresses NF-κB-Mediated Inflammatory Responses and Activates Expression of Nrf2-Mediated Antioxidant Genes to Alleviate Caerulein-Induced Acute Pancreatitis.

Triptolide (TP), the main active ingredient of Tripterygium wilfordii Hook.f., displays potent anti-inflammatory, antioxidant, and antiproliferative activities. In the present study, the effect of TP on acute pancreatitis and the underlying mechanisms of the disease were investigated using a caerulein-induced animal model of acute pancreatitis (AP) and an in vitro cell model. In vivo, pretreatment with TP notably ameliorated pancreatic damage, shown as the improvement in serum amylase and lipase levels and pancreatic morphology. Meanwhile, TP modulated the infiltration of neutrophils and macrophages (Ly6G staining and CD68 staining) and decreased the levels of proinflammatory factors (TNF-α and IL-6) through inhibiting the transactivation of nuclear factor-κB (NF-κB) in caerulein-treated mice. Furthermore, TP reverted changes in oxidative stress markers, including pancreatic glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), in acute pancreatitis mice. Additionally, TP pretreatment inhibited intracellular reactive oxygen species (ROS) levels via upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes expression (HO-1, SOD1, GPx1 and NQO1) in vitro. Taken together, our data suggest that TP exert protection against pancreatic inflammation and tissue damage by inhibiting NF-κB transactivation, modulating immune cell responses and activating the Nrf2-mediated antioxidative system, thereby alleviating acute pancreatitis.

Poly(ADP-Ribose) Polymerase 1 Promotes Inflammation and Fibrosis in a Mouse Model of Chronic Pancreatitis.

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFß, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.

Dopamine D2 receptor activator quinpirole protects against trypsinogen activation during acute pancreatitis via upregulating HSP70.

Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA) receptor 2 (DRD2) activation controls inflammation during AP via PP2A-dependent NF-κB activation. In this study, we sought to examine whether DRD2 signaling mediates trypsinogen activation and the underlying mechanisms. Pancreatic acinar cells were stimulated with cholecystokinin-8 in vitro. AP was induced by intraperitoneal injections of caerulein and LPS or l-arginine. Pancreatitis severity was assessed biochemically and histologically. We found that activation of DRD2 by quinpirole, a potent DRD2 agonist, resulted in the reduction of trypsinogen activation and the upregulation of HSP70 in vitro and in vivo. Mechanistically, we found that quinpirole induced dephosphorylation of heat shock factor 1 (HSF1), a master transcription factor of HSP70, leading to increased nuclear translocation of HSF1 in a PP2A-dependent pathway. Furthermore, DRD2 activation restored lysosomal pH and, therefore, maintained lysosomal cathepsin B activity in a HSP70-dependent manner. VER155008, a potent HSP70 antagonist, abolished the protective effects observed with DRD2 activation in vitro and in two experimental models of AP. Our data showed that besides controlling NF-κB activation, DRD2 activation prevented trypsinogen activation during acute pancreatitis via PP2A-dependent upregulation of HSP70 and further support that DRD2 agonist could be a promising therapeutic strategy for treating AP.NEW & NOTEWORTHY The current study demonstrated that activation of DRD2 by quinpirole protects against trypsinogen activation in the in vitro and in vivo setting of acute pancreatitis by upregulating HSP70 and restoring lysosomal degradation via a PP2A-dependent manner, therefore leading to reduced pancreatic injury. These findings provide a new mechanistic insight on the protective effect of DRD2 activation in acute pancreatitis.

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice expressing oncogenic Kras.

Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.

Multimodal Transgastric Local Pancreatic Hypothermia Reduces Severity of Acute Pancreatitis in Rats and Increases Survival.

BACKGROUND & AIMS: Acute pancreatitis (AP) of different etiologies is associated with the activation of different signaling pathways in pancreatic cells, posing challenges to the development of targeted therapies. We investigated whether local pancreatic hypothermia, without systemic hypothermia, could lessen the severity of AP induced by different methods in rats. METHODS: A urethane balloon with 2 polyurethane tubes was placed inside the stomach of rats. AP was induced in Wistar rats by the administration of cerulein or glyceryl tri-linoleate (GTL). Then, cold water was infused into the balloon to cool the pancreas. Pancreatic temperatures were selected based on those found to decrease acinar cell injury. An un-perfused balloon was used as a control. Pancreatic and rectal temperatures were monitored, and an infrared lamp or heating pad was used to avoid generalized hypothermia. We collected blood, pancreas, kidney, and lung tissues and analyzed them by histology, immunofluorescence, immunoblot, cytokine and chemokine magnetic bead, and DNA damage assays. The effect of hypothermia on signaling pathways initiated by cerulein and GTL was studied in acinar cells. RESULTS: Rats with pancreatic cooling developed less severe GTL-induced AP compared with rats that received the control balloon. In acinar cells, cooling decreased the lipolysis induced by GTL, increased the micellar form of its fatty acid, lowered the increase in cytosolic calcium, prevented the loss of mitochondrial membrane potential (by 70%-80%), and resulted in a 40%-50% decrease in the uptake of a fatty acid tracer. In rats with AP, cooling decreased pancreatic necrosis by 48%, decreased serum levels of cytokines and markers of cell damage, and decreased markers of lung and renal damage. Pancreatic cooling increased the proportions of rats surviving 6 hours after induction of AP (to 90%, from <10% of rats that received the control balloon). In rats with cerulein-induced AP, pancreatic cooling decreased pancreatic markers of apoptosis and inflammation. CONCLUSIONS: In rats with AP, transgastric local pancreatic hypothermia decreases pancreatic necrosis, apoptosis, inflammation, and markers of pancreatitis severity and increases survival.

Measuring digestive protease activation in the mouse pancreas.

Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.

Endocrine and exocrine pancreas pathologies crosstalk: Insulin regulates the unfolded protein response in pancreatic exocrine acinar cells.

Exocrine pancreas insufficiency is common in diabetic mellitus (DM) patients. Cellular stress is a prerequisite in the development of pancreatic pathologies such as acute pancreatitis (AP). The molecular mechanisms underlying exocrine pancreatic ER-stress in DM are largely unknown. We studied the effects of insulin and glucose (related to DM) alone and in combination with cerulein (CER)-induced stress (mimicking AP) on ER-stress unfolded protein response (UPR) in pancreatic acinar cells. Exocrine pancreas cells (AR42J) were exposed to high glucose (Glu, 25 mM) and insulin (Ins, 100 nM) levels with or without CER (10 nM). ER-stress UPR activation was analyzed at the transcript, protein, immunocytochemistry, western blotting, quantitative RT-PCR and XBP1 splicing, including; XBP1, sXBP1, ATF6, cleaved ATF6, IRE1-p, CHOP, Caspase-12 and Bax. Exocrine acinar cells exposed to high Ins or Ins+Glu concentrations (but not Glu alone) exhibited ER-stress UPR, demonstrated by significant increase of transcript and protein levels of downstream markers in the ATF6 and IRE1 transduction arms, including: sXBP1, cleaved ATF6, XBP1, CHOP, IRE1-p and caspase-12. UPR activation resulted in IRE1-p aggregation and nuclear trans-localization of cleaved activated ATF6 and sXBP1. Ins further aggravated UPR when cells were co-challenged with CER-induced stress, exacerbating the effects of CER alone. High Ins levels, typical to type-2-DM, activate the ER-stress UPR in pancreatic acinar cells, through the ATF6 and IRE1 pathways. This effect of Ins in naïve acinar cells further augments CER-induced UPR. Our data highlight molecular pathways through which DM enhances exocrine pancreas pathologies.

SPOP inhibits mice pancreatic stellate cell activation by promoting FADD degradation in cerulein-induced chronic pancreatitis.

Pancreatic stellate cells (PSCs) have been recognized as key mediators of pancreatic fibrosis, a characteristic feature of chronic pancreatitis (CP). As a cullin-based E3 ubiquitin ligase, speckle-type POZ protein (SPOP) has been identified to participate in tumorigenesis and organ development. However, its biological role in CP remains unknown. Therefore, this study sought to investigate the changed expression of SPOP in CP and to examine the effect on mice PSCs activation of SPOP. We found that SPOP was downregulated in the pancreatic tissues of cerulein-induced CP mice. siRNA-mediated knockdown of SPOP led to significant promotion in primary PSCs activity by activating the nuclear factor-kappaB (NF-κB)/interleukin-6 (IL-6) signaling pathway. In addition, we examined the effects of Fas-associated death domain (FADD), a proven SPOP substrate that activates NF-κB, on the regulation of PSCs activation. We found that FADD was downregulated by SPOP via interaction-mediated degradation, and was upregulated during PSCs activation. The promotion of PSCs activation in knocking down SPOP with siSPOP-1 were counteracted by knocking down FADD. The results suggest that the SPOP-induced inhibition of PSCs activation partially depended on FADD. These results highlight the importance of SPOP in CP and provide a potential target for therapeutic intervention.

Mesna Alleviates Cerulein-Induced Acute Pancreatitis by Inhibiting the Inflammatory Response and Oxidative Stress in Experimental Rats.

BACKGROUND: Acute pancreatitis (AP) is a sudden inflammation of the pancreas that may be life-threatening disease with high mortality rates, particularly in the presence of systemic inflammatory response and multiple organ failure. Oxidative stress has been shown to be involved in the pathophysiology of acute pancreatitis. AIM: This study is designed to investigate the possible effect of mesna on an experimental model of cerulein-induced acute pancreatitis. METHODS: Animals were divided into five groups: Group 1 served as a control group given the saline; group II (mesna group) received mesna at a dose of (100 mg/kg per dose, i.p.) four times; group III (acute pancreatitis group) received cerulein at a dose of (20 µg/kg/dose, s.c.) four times with 1-h intervals; group VI, cerulein + mesna, was treated with mesna at a dose of (100 mg/kg, i.p.) 15 min before each cerulein injection. RESULTS: Animals with acute pancreatitis showed elevated serum amylase and lipase levels. Biochemical parameters showed increased pancreatic tumor necrosis factors-α (TNF-α) and interleukin-1ß (IL-1ß) levels. A disturbance in oxidative stress markers was evident by elevated pancreatic lipid peroxides (TBARS) and decline in pancreatic antioxidants' concentrations including reduced glutathione (GSH); superoxide dismutase (SOD); and glutathione peroxidase (GSH-Px). Histological examination confirmed pancreatic injury. Pre-treatment with mesna was able to abolish the changes in pancreatic enzymes, oxidative stress markers (TBARS, SOD, GSH and GSH-Px), pancreatic inflammatory markers (TNF-α, IL-1ß) as well as histological changes. CONCLUSIONS: Mesna mitigates AP by alleviating pancreatic oxidative stress damage and inhibiting inflammation.

miR-135a deficiency inhibits the AR42J cells damage in cerulein-induced acute pancreatitis through targeting FAM129A.

Acute pancreatitis (AP) is a common clinical critical disease with high mortality and the exact pathogenesis is not fully elucidated. The present study aimed to uncover the function of miR-135a in the proliferation, apoptosis, and inflammatory characteristics of diseased pancreatic cells and the potential molecular mechanisms. The expression patterns of miR-135a and family with sequence similarity 129 member A (FAM129A) in patients with AP were analyzed on the basis of the GEO database. The transfection efficiency and expression level of miR-135a in AR42J cells were determined by qRT-PCR. The biological characteristics of AR42J cells treated with cerulein were detected by cell counting kit-8 (CCK-8), flow cytometry, and western blot assays. The potential interaction between miR-135a and FAM129A was confirmed by bioinformatics prediction softwares and luciferase reporter assay. MiR-135a inhibitor and pcDNA3.1-FAM129A were co-transfected to determine the regulation of miR-135a/FAM129A on inflammatory AR42J cell injury. We observed that miR-135a was highly expressed in AP samples. Depletion of miR-135a could alleviate the condition so that the AR42J cells proliferation increased, apoptosis decreased, and the expression of inflammatory cytokines enhanced. In addition, mRNA and protein expression of FAM129A were negatively regulated by miR-135a, and over-expression of FAM129A could strengthen the relief effect of miR-135a inhibitor in AP induced by cerulein. In summary, our data demonstrates that silencing miR-135a reduces AR42J cells injury and inflammatory response in AP induced by cerulein through targeting FAM129A.

Inhibiting role of rosiglitazone in the regulation of inflammatory response and protective effects for severe acute pancreatitis in mice.

OBJECTIVES: Our study aimed to probe the effects of rosiglitazone treatment on a severe acute pancreatitis (SAP) model induced by caerulein and investigate the underlying mechanism. METHODS: Differentially expressed messenger RNAs (mRNAs) in the mice of a SAP group were screened out by microarray analysis. The inflammatory response pathway was obtained from the online website DAVID Bioinformatics Resources 6.8. The interactions of caerulein and its target proteins were shown by search tool for interactions of chemicals (STITCH). Functional interactions of the genes associated with pancreatitis and the target proteins of caerulein were obtained with search tool for interactions of chemicals (STRING). SAP mice were established by hourly intraperitoneal injection of caerulein. Rosiglitazone was used as treatment drug, and pancreatic inflammation was assessed. The expression of Socs3 was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of interleukin (IL)-6, IL-1b, and Egr1 were studied by RT-PCR and Western blot analysis. RESULTS: The GSE77983 data were analyzed, and the results showed that Socs3 was overexpressed in SAP tissues. The inflammation response pathway in pancreas was selected by DAVID, STITCH, and STRING. After injection of rosiglitazone in mice, the serum levels of amylase and lipase were decreased. Furthermore, the mRNA and protein levels of Socs3 and inflammatory cytokines in pancreatic tissues were downregulated. CONCLUSIONS: Rosiglitazone could protect mice with SAP from injury by downregulating Socs3 and inhibiting the inflammatory response pathway.

Spautin-A41 Attenuates Cerulein-Induced Acute Pancreatitis through Inhibition of Dysregulated Autophagy.

Autophagy plays key roles in the development of acute pancreatitis (AP) and the regulation of impaired autophagy has therapeutic potential. The objective of the present study was to investigate whether pharmacological inhibition of autophagy could ameliorate AP in mice and examine the underlying mechanisms. In current study, by imaging-based high-throughput screening, a novel spautin-1 derivative spautin-A41 was identified as a potent autophagy inhibitor. Mice treated with spautin-A41 were resistant to the cerulein-induced elevation of serum pancreatic enzyme activities and pancreatic apoptosis. Mechanistically, spautin-A41 effectively reduced the expression levels of Class III phosphatidylinositol 3 (PI3) kinase complexes and subsequently ameliorated pancreatitis by inhibiting the formation of autophagosome. Therefore, pharmacological inhibition of autophagy by spautin-A41 may serve as new target for treating or lessening the severity of AP.

The Effects of Lycium Barbarum Polysaccharide (LBP) in a Mouse Model of Cerulein-Induced Acute Pancreatitis.

BACKGROUND Acute pancreatitis is an inflammatory disease of the pancreas associated with high patient morbidity. Lycium barbarum polysaccharide (LBP), a traditional Chinese medicine with an active component extracted from the goji berry, has previously been reported to have anti-inflammatory effects. This study aimed to investigate the effects of LBP in a mouse model of cerulein-induced acute pancreatitis. MATERIAL AND METHODS Acute pancreatitis was induced by intraperitoneal injection of cerulein in C57BL/6 wild-type mice or nuclear factor erythroid-2-related factor 2 (NRF2) gene knockout mice. LBP or normal saline was administrated by gavage once daily for one week before the induction of acute pancreatitis. At 12 hours after the first intraperitoneal injection of cerulein, the mice were euthanized. Blood and pancreatic tissue were sampled for histology and for the measurement of pro-inflammatory cytokines, serum amylase, and lipase. RESULTS In the untreated mouse model of cerulein-induced acute pancreatitis, amylase and lipase levels were increased, and these levels were reduced by LBP treatment when compared with vehicle treatment. In the untreated mouse model, histology of the pancreas showed edema and inflammation, which were reduced in the LBP-treated mice. In the untreated mouse model, increased levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were found, which were reduced in the LBP-treated mice. NRF2 gene knockout mice with cerulein-induced acute pancreatitis showed reduced anti-inflammatory effects of LBP treatment. LBP increased the expression of NRF2 and heme oxygenase-1 (HO-1). CONCLUSIONS In a mouse model of cerulein-induced acute pancreatitis, LBP reduced inflammation by upregulating NRF2 and HO-1.

Maresin-1 Inhibits Oxidative Stress and Inflammation and Promotes Apoptosis in a Mouse Model of Caerulein-Induced Acute Pancreatitis.

BACKGROUND This study aimed to investigate the effects of maresin-1 (MaR1) in a mouse model of caerulein-induced acute pancreatitis (AP). MATERIAL AND METHODS Fifty C57BL/6 mice with caerulein-induced AP were divided into the untreated control group (N=10), the untreated AP model group (N=10), the MaR1-treated (low-dose, 0.1 µg) AP model group (N=10), the MaR1-treated (middle-dose, 0.5 µg) AP model group (N=10), and the MaR1-treated (high-dose, 1 µg) AP model group (N=10). Enzyme-linked immunoassay (ELISA) measured serum levels of amylase, lipase, tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß (IL-1ß), and IL-6 and mRNA was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Malondialdehyde (MDA), protein carbonyls, superoxide dismutase (SOD), and the ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) were measured. Histology of the pancreas included measurement of acinar cell apoptosis using the terminal-deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay. Western blot measured Toll-like receptor 4 (TLR4), MyD88, and phospho-NF-kappaB p65, and apoptosis-associated proteins Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9. RESULTS Following treatment with MaR1, serum levels of amylase, lipase, TNF-alpha, IL-1ß, and IL-6 decreased, MDA and protein carbonyl levels decreased, SOD and the GSH/GSSG ratio increased in a dose-dependent manner. In the MaR1-treated AP mice, inflammation of the pancreas and the expression of inflammatory cytokines, pancreatic acinar cell apoptosis, Bcl-2 expression, and expression of TLR4, MyD88, and p-NF-kappaB p65 were reduced, but Bax, cleaved caspase-3, and cleaved caspase-9 expression increased. CONCLUSIONS In a mouse model of caerulein-induced AP, treatment with MaR1 reduced oxidative stress and inflammation and reduced apoptosis.

Dihydromyricetin inhibits caerulin-induced TRAF3-p38 signaling activation and acute pancreatitis response.

Acute pancreatitis (AP) is a common inflammatory disease in gastrointestinal tract. Our previous study has shown that caerulin induces TNF receptor-associated factor 3 (TRAF3)-p38 signaling activation and pro-inflammatory response in macrophages, causing damage to co-cultured pancreatic acinar cells. Dihydromyricetin (DHM) is a flavonoid extracted from Ampelopsis grossedentata, which has displayed anti-inflammation and anti-oxidant functions. Our results here show that DHM potently inhibited caerulin-induced expression and productions of multiple pro-inflammatory cytokines (IL-1ß, TNF-α and IL-17) in murine bone marrow-derived macrophages (BMDMs). DHM significantly inhibited caerulin-induced TRAF3 protein stabilization, TRAF3-mitogen-activated protein kinase kinase 3 (MKK3) association and following MKK3-p38 activation in BMDMs. Significantly, DHM was ineffective against caerulin in TRAF3-silenced BMDMs. Importantly, DHM supplement attenuated the cytotoxicity of caerulin-activated BMDMs to co-cultured pancreatic acinar cells, resulting in significantly decreased acinar cell death and apoptosis. In vivo, DHM co-administration largely attenuated pancreatic and systemic inflammation in caerulin-injected AP mice. Together, DHM inhibits caerulin-induced TRAF3-p38 signaling activation and AP response. DHM could be further studied as a potential anti-AP agent.