This study was to investigate the changes of autophagy in pancreatic tissue cells from hyperlipidemic acute pancreatitis (HLAP) rats and the molecular mechanism of autophagy to induce inflammatory injury in pancreatic tissue cells. Male Sprague Dawley (SD) rats were intraperitoneally injected with caerulein to establish acute pancreatitis (AP) model and then given a high fat diet to further prepare HLAP model. The HLAP rats were treated with autophagy inducer rapamycin or inhibitor 3-methyladenine. Pancreatic acinar (AR42J) cells were treated with caerulein to establish HLAP cell model. The HLAP cell model were treated with rapamycin or transfected with vascular endothelial growth factor (VEGF) siRNA. The inflammatory factors in serum and cell culture supernatant were detected by ELISA method. The histopathological changes of pancreatic tissue were observed by HE staining. The changes of ultrastructure and autophagy in pancreatic tissue were observed by electron microscopy. The expression levels of Beclin-1, microtubule- associated protein light chain 3-II (LC3-II), mammalian target of rapamycin complex 1 (mTORC1), and VEGF were measured by immunohistochemistry and Western blot. The results showed that, compared with control group, the autophagy levels and inflammatory injury of pancreatic tissue cells from HLAP model rats were obviously increased, and these changes were aggravated by rapamycin treatment, but alleviated by 3-methyladenine treatment. In HLAP cell model, rapamycin aggravated the autophagy levels and inflammatory injury, whereas VEGF siRNA transfection increased mTORC1 protein expression, thus alleviating the autophagy and inflammatory injury of HLAP cell model. These results suggest that VEGF-induced autophagy plays a key role in HLAP pancreatic tissue cell injury, and interference with VEGF-mTORC1 pathway can reduce the autophagy levels and alleviate the inflammatory injury. The present study provides a new target for prevention and treatment of HLAP.
Acute Disease ; Animals ; Autophagy ; Ceruletide/adverse effects* ; Male ; Mammals/metabolism* ; Mechanistic Target of Rapamycin Complex 1 ; Microtubule-Associated Proteins/metabolism* ; Pancreatitis ; RNA, Small Interfering/genetics* ; Rats ; Rats, Sprague-Dawley ; Sirolimus/adverse effects* ; Vascular Endothelial Growth Factor A/genetics*

PURPOSE: To determine the potential protective and therapeutic effects and action mechanism of ruscogenin on cerulein-induced acute pancreatitis (AP) model in rats. METHODS: Overall, 32 rats were attenuated to the sham (2-mL/kg/day isotonic solution for 4 weeks), control (20-µg/kg cerulein-induced AP for 12 hours), prophylaxis groups (cerulein-induced AP following 3-mL/kg/day ruscogenin for 4 weeks) and treatment (3-mL/kg/day ruscogenin following cerulein-induced AP for 12 hours). Blood samples were collected for biochemical analysis of nitric oxide synthase 1 (NOS1/neuronal NOS), malondialdehyde (MDA) and intercellular adhesion molecule 1 (ICAM-1). After sacrification, pancreas tissues were collected and prepared for light microscopic (hematoxylin and eosin), immunohistochemical (nuclear factor kappa B) and biochemical analysis (tumor necrosis factor-alpha [TNF-α], interleukin-6 and 1β [IL-6 and IL-1β], CRP, high-sensitivity CRP [hs-CRP] amylase, lipase, and ICAM-1). Ultrastructural analysis was performed by transmission electron microscopy. RESULTS: The protective and therapeutic actions of ruscogenin were accomplished by improvements in histopathology, by decreasing blood cytokine levels of CRP, hs-CRP levels, TNF-α, IL-6, IL-1β, ICAM-1, by reducing the pancreatic enzymes amylase and lipase in blood, and by suppressing the expression of nuclear factor kappa B, ICAM-1, and NOS-1, but not MDA in pancreatic tissues. Ruscogenin also improved cerulein-induced ultrastructural degenerations in endocrine and exocrine cells, especially in treatment group. CONCLUSION: The present findings have demonstrated the beneficial protective and therapeutical effects of ruscogenin, nominating it as a highly promising supplementary agent to be considered in the treatment of AP, and even as a protective agent against the damages induced by disease.
Amylases ; Animals ; Ceruletide ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Lipase ; Malondialdehyde ; Microscopy, Electron, Transmission ; Necrosis ; NF-kappa B ; Nitric Oxide Synthase ; Pancreas ; Pancreatitis ; Rats ; Therapeutic Uses

BACKGROUND: Chronic pancreatitis (CP) is an irreversible progressive disease that destroys exocrine parenchyma, which are replaced by fibrous tissue. As pancreatic fibrosis is a key feature of CP, reducing fibrotic protein content in the pancreas is crucial for preventing CP. Studies suggest that NF-κB facilitates the expression of fibrotic mediators in pancreas and protein kinase C-δ (PKC-δ) regulates NF-κB activation in stimulated pancreatic acinar cells. Docosahexaenoic acid (DHA) is an omega-3 fatty acid having anti-inflammatory and anti-fibrotic effects. It has been shown to inhibit NF-κB activity in cerulein-stimulated pancreatic acinar cells which is a cellular model of CP. In the present study, we investigated if DHA inhibits expression of fibrotic mediators by reducing PKC-δ and NF-κB expression in mouse pancreatic tissues with CP.METHODS: For six weeks, mice were weekly induced for acute pancreatitis to develop CP. Furthermore, acute pancreatitis was induced by hourly intraperitoneal injections of cerulein (50 μg/kg × 7). Mice were administered DHA (10 μM) via drinking water before and after CP induction.RESULTS: Cerulein-induced pancreatic damages like decreased pancreatic weight/total body weight, leukocyte infiltration, necrosis of acinar cells, and vacuolization were found to be inhibited by DHA. Additionally, DHA inhibited cerulein-induced fibrotic mediators like alpha-smooth muscle actin and fibronectin in pancreas. DHA reduced expression of PKC-δ and NF-κB p65 in pancreatic tissues of cerulein-treated mice.CONCLUSIONS: DHA may be beneficial in preventing CP by suppressing pancreatic expression of fibrotic mediators.
Acinar Cells ; Actins ; Animals ; Body Weight ; Ceruletide ; Drinking Water ; Fibronectins ; Fibrosis ; Injections, Intraperitoneal ; Leukocytes ; Mice ; Necrosis ; Pancreas ; Pancreatitis ; Pancreatitis, Chronic ; Protein Kinases

Cerulein-induced pancreatitis is similar to human edematous pancreatitis, characterized by the dysregulation of digestive enzyme production, edema formation, and an infiltration of inflammatory cells into the pancreas. We previously showed that the Janus kinase 2 (JAK2)/STAT3 pathway mediates inflammatory signaling in cerulein-stimulated pancreatic acinar cells. PPAR-γ has been implicated in the regulation of inflammatory responses in several cells. In the present study, we investigated the role of PPAR-γ in cerulein-induced activation of JAK2/STAT3 in pancreatic acinar cells. Treatment with cerulein induced the activation of JAK2/STAT3 and PPAR-γ expression in AR42J cells. Cerulein-induced PPAR-γ expression was inhibited by AG490, a JAK2/STAT3 inhibitor, in AR42J cells. An immunoprecipitation analysis showed that PPAR-γ binds to STAT3 in cerulein-stimulated AR42J cells. Down-regulation of PPAR-γ by siRNA increased STAT3 phosphorylation in AR42J cells stimulated with cerulein. These results show that PPAR-γ inactivates STAT3 by directly interacting with STAT3 in cerulein-stimulated pancreatic acinar cells. Overexpression of PPAR-γ may be beneficial for preventing pancreatitis by suppressing the activation of STAT3 in pancreatic acinar cells.
Acinar Cells* ; Ceruletide ; Down-Regulation ; Edema ; Humans ; Immunoprecipitation ; Janus Kinase 2 ; Pancreas ; Pancreatitis ; Peroxisomes* ; Phosphorylation ; RNA, Small Interfering

BACKGROUND/AIMS: The aim of this study was to establish a pathogenetic mechanism of pancreatitis in celiac disease and IgG4-related disease using gluten-sensitive human leukocyte antigen (HLA)-DQ8 transgenic mice. METHODS: Transgenic mice expressing HLA-DQ8 genes were utilized. Control mice were not sensitized but were fed gliadin-free rice cereal. Experimental groups consisted of gliadin-sensitized and gliadin-challenged mice; nonsensitized mice with cerulein hyperstimulation; and gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation. RESULTS: Gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation showed significant inflammatory cell infiltrates, fibrosis and acinar atrophy compared with the control mice and the other experimental groups. The immunohistochemical analysis showed greater IgG1-positive plasma cells in the inflammatory infiltrates of gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation compared with the control mice and the other experimental groups. Gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation or gliadin-sensitized and gliadin-challenged mice showed IgG1-stained inflammatory cell infiltrates in the extrapancreatic organs, including the bile ducts, salivary glands, kidneys, and lungs. CONCLUSIONS: Gliadin-sensitization and cerulein hyperstimulation of gluten-sensitive HLA-DQ8 transgenic mice resulted in pancreatitis and extrapancreatic inflammation. This animal model suggests that chronic gliadin ingestion in a susceptible individual with the HLA-DQ8 molecule may be associated with pancreatitis and extrapancreatic inflammation.
Animals ; Animals* ; Atrophy ; Autoimmune Diseases ; Bile Ducts ; Celiac Disease ; Ceruletide ; Eating ; Edible Grain ; Fibrosis ; Gliadin* ; Humans ; Inflammation* ; Kidney ; Leukocytes ; Lung ; Mice* ; Mice, Transgenic ; Models, Animal* ; Pancreatitis* ; Plasma Cells ; Salivary Glands

In the pathogenesis of pancreatitis, oxidative stress is involved in the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and cytokine expression. High serum levels of cholecystokinin (CCK) have been reported in patients with acute pancreatitis, and treatment with cerulein, a CCK analogue, induces acute pancreatitis in a rodent model. Recent studies have shown that cerulein-activated nicotinamide adenine dinucleotide phosphate oxidase elicits reactive oxygen species, which trigger the phosphorylation of the JAK1, STAT1, and STAT3 proteins and induce the production of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6, in pancreatic acinar cells. The JAK/STAT pathway also stimulates cell proliferation and malignant transformation and inhibits apoptosis in the pancreas. This review discusses the possible role of the JAK/STAT pathway in the pathogenesis of pancreatitis and pancreatic cancer in response to oxidative stress.
Acinar Cells ; Apoptosis ; Caerulein ; Cell Proliferation ; Cholecystokinin ; Cytokines ; Humans ; Interleukin-6 ; Interleukins ; NADP ; Oxidative Stress ; Oxidoreductases ; Pancreas ; Pancreatic Neoplasms ; Pancreatitis ; Phosphorylation ; Reactive Oxygen Species ; Rodentia ; STAT3 Transcription Factor ; Transducers ; Tumor Necrosis Factor-alpha

BACKGROUND/AIMS: Heat shock proteins (HSPs) protect rats from cerulein-induced acute pancreatitis (AP) by preventing the subcellular redistribution of cathepsin B and the activation of trypsinogen. Autophagy plays a critical role in the secretion of digestive enzymes and triggering of cerulein-induced AP via the colocalization of trypsinogen and lysosomes. Therefore, using a rat cerulein-induced AP model, we investigated whether HSPs prevent AP by regulating autophagy. METHODS: Twelve hours after fed standard laboratory chow and water, the experimental groups (cerulein, water-immersion [WI]-cerulein and heat-shock [HS]-cerulein) and the control groups (control, WI, and HS) received one intraperitoneal injection of cerulein (50 microg/kg) or saline, respectively. All of the rats were sacrificed at 6 hours after injection. The severity of the AP was assessed based on the serum amylase level and the histological and electron microscopy findings. Western blotting was also performed for HSP60/70 and LC3B-II. RESULTS: WI and HS induced HSP60 and HSP70, respectively. The induced HSP60/70 effectively prevented the development of cerulein-induced AP. Autophagy developed in the rats with cerulein-induced AP and was documented by the expression of LC3-II and electron microscopy findings. The WI-stressed rats and HS-treated rats did not develop cerulein-induced autophagy. CONCLUSIONS: HSPs exert protective effects against cerulein-induced AP in rats by inhibiting autophagy.
Amylases ; Animals ; Autophagy ; Blotting, Western ; Caerulein ; Cathepsin B ; Heat-Shock Proteins ; Hot Temperature ; Injections, Intraperitoneal ; Lysosomes ; Microscopy, Electron ; Pancreatitis ; Rats ; Trypsinogen ; Water

To study the expression of X-linked inhibitor of apoptosis protein (XIAP) and cell apoptosis in vitro model of acute pancreatitis (AP), we carried out experiments to stimulate AR42J cell line with caerulein (10(-8) mol/L) for 12 hours, then collected cells at various time points (0 h, 4 h, 8 h, 12 h, and 24 h, respectively). We then observed the morphologic changes of AR42J cells with the stimulation of caerulein with electronic microscope. The gene expression of XIAP, caspase-3 and caspase-9 was detected using real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), and the protein expression of XIAP was assessed by western blot. The activation of nuclear factor-kappa B (NF-kappaB) was measured by flow cytometry (FCM). With the stimulation of caerulein, the expression of XIAP and the NF-kappaB activation could first decrease and then increase, but the change of caspase-3 and caspase-9 expressions were opposite. XIAP may inhibit the cell apoptosis in rat pancreatic acinus AR42J cell lines at first with the stimulation of caerulein, then NF-kappaB can upgrade the expression of XIAP and increase the cell apoptosis.
Acinar Cells ; cytology ; metabolism ; Animals ; Apoptosis ; physiology ; Cell Line ; Ceruletide ; pharmacology ; NF-kappa B ; metabolism ; Pancreas ; cytology ; metabolism ; Pancreatitis ; metabolism ; Rats ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

BACKGROUND/AIMS: Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the production and secretion of digestive enzymes, edema formation, cytoplasmic vacuolization and the death of acinar cells. We hypothesized that membrane proteins may be altered as the early event during the induction of acute pancreatitis. Present study aims to determine the differentially expressed proteins in the membranes of cerulein-treated pancreatic acinar cells. METHODS: Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 1 hour. Membrane proteins were isolated from the cells and separated by two-dimensional electrophoresis using pH gradients of 5-8. Membrane proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. The differentially expressed proteins, whose expression levels were more or less than three-fold in cerulein-treated cells, were analyzed. RESULTS: Two differentially expressed proteins (mannan-binding lectin-associated serine protease-2, heat shock protein 60) were up-regulated while four proteins (protein disulfide isomerase, gamma-actin, isocitrate dehydrogenase 3, seven in absentia homolog 1A) were down-regulated by cerulein treatment in pancreatic acinar cells. These proteins are related to cell signaling, oxidative stress, and cytoskeleton arrangement. CONCLUSIONS: Oxidative stress may induce cerulein-induced cell injury and disturbances in defense mechanism in pancreatic acinar cells.
Acinar Cells ; Actins ; Caerulein ; Cytoplasm ; Cytoskeleton ; Edema ; Electrophoresis ; Heat-Shock Proteins ; Humans ; Isocitrate Dehydrogenase ; Isocitrates ; Mass Spectrometry ; Membrane Proteins ; Membranes ; Oxidative Stress ; Pancreatitis ; Protein Disulfide-Isomerases ; Proteins ; Proteome ; Proton-Motive Force ; Serine

Acute pancreatitis is a multifactorial disease associated with the premature activation of digestive enzymes. The genes expressed in pancreatic acinar cells determine the severity of the disease. The present study determined the differentially expressed genes in pancreatic acinar cells treated with cerulein as an in vitro model of acute pancreatitis. Pancreatic acinar AR42J cells were stimulated with 10(-8) M cerulein for 4 h, and genes with altered expression were identified using a cDNA microarray for 4,000 rat genes and validated by real-time PCR. These genes showed a 2.5-fold or higher increase with cerulein: lithostatin, guanylate cyclase, myosin light chain kinase 2, cathepsin C, progestin-induced protein, and pancreatic trypsin 2. Stathin 1 and ribosomal protein S13 showed a 2.5-fold or higher decreases in expression. Real-time PCR analysis showed time-dependent alterations of these genes. Using commercially available antibodies specific for guanylate cyclase, myosin light chain kinase 2, and cathepsin C, a time-dependent increase in these proteins were observed by Western blotting. Thus, disturbances in proliferation, differentiation, cytoskeleton arrangement, enzyme activity, and secretion may be underlying mechanisms of acute pancreatitis.
Acinar Cells ; Animals ; Antibodies ; Blotting, Western ; Caerulein ; Cathepsin C ; Cytoskeleton ; Gene Expression ; Guanylate Cyclase ; Lithostathine ; Myosin-Light-Chain Kinase ; Oligonucleotide Array Sequence Analysis ; Pancreatitis ; Proteins ; Rats ; Real-Time Polymerase Chain Reaction ; Ribosomal Proteins ; Trypsin