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*

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

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

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

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

BACKGROUNDIn patients suffering from acute pancreatitis, the pathogenesis is not completely understood, and several recent studies in vitro suggested that heat shock proteins might play an important role in cell signaling. To investigate the possible role of extracellular heat shock protein 70 (Hsp70) in pancreatitis, toll-like receptor-4 (TLR4)-deficient and wild-type mice were administered with exogenous Hsp70 during the course of cerulein-induced pancreatitis (CIP).

METHODSAcute pancreatitis was induced by 5 intraperitoneal injections of cerulein at hourly intervals, and then treated with recombinant Hsp70 through the caudal vein 4 hours after the start of cerulein injections. Subsequently serum amylase and serum cytokines levels were detected. Histologic alteration of the pancreas was evaluated. Tumor necrosis factor alpha (TNF-alpha) concentrations and myeloperoxidase (MPO) activity in both pancreas and lungs were analyzed. The nuclear factor kappa B (NF-kappaB) activation in pancreatic tissue was measured using a sensitive RelA enzyme-linked immunosorbent assay.

RESULTSTreatment with recombinant Hsp70 to wild-type mice in CIP resulted in significant aggravation of inflammation in pancreas, elevated levels of serum cytokines, up-regulation of pulmonary MPO activity and increase of lung tissues TNF-alpha concentrations. In contrast, treatment with Hsp70 to TLR4-deficient mice had little effect on serum cytokines levels, pancreatic inflammation, pulmonary MPO activity and TNF-alpha concentrations.

CONCLUSIONSThe results suggest that extracellular Hsp70 might induce systemic inflammatory response syndrome (SIRS)-like response in vivo and TLR4 might be involved in the Hsp70-mediated activation of inflammatory reaction in the progression of CIP without infection.

Acute Disease ; Animals ; Ceruletide ; toxicity ; Female ; HSP70 Heat-Shock Proteins ; physiology ; Male ; Mice ; Mice, Inbred C57BL ; Pancreatitis ; etiology ; Systemic Inflammatory Response Syndrome ; etiology ; Toll-Like Receptor 4 ; physiology

BACKGROUND: Many protease inhibitors show a protective effect for acute pancreatitis as seen in animal models. In previous studies, the protease inhibitors were administered before induction of pancreatitis, and there are few published reports examining effects when these agents were administered after induction of pancreatitis. The timing of drug administration may provide an explanation for the ineffectiveness of protease inhibitors for the treatment of patients with acute pancreatitis. Herein, we assessed the protective effect of nafamostat mesilate (NM), a potent protease inhibitor, in a mouse model of cerulean-induced pancreatitis and compared the results of administering the drug before and after the induction of pancreatitis. METHODS: Cerulein, a cholecystokinin analogue, was injected into mice intraperitoneally to induce pancreatitis. The mice received intravenous NM administration before and after the induction of pancreatitis. The serum concentration of amylase and lipase was measured, histological changes were measured, and the tissue expression of myeloperoxidase was measured to assess the degree of inflammation. Expression of p38 MAPK (mitogen-activated protein kinase), phospho-p38 MAPK, and IL-6 (interleukin-6) in tissue was evaluated. RESULTS: Acute pancreatitis was induced successfully by intraperitoneal injection of cerulein. Acute pancreatitis could be prevented when NM was administered before the induction of pancreatits. However, the effect was not guaranteed when given after the induction of pancreatitis. For a group of mice with induced pancreatitis, tissue expression of phospho-p38 MAPK was prominent and there was no marked difference in the expression of IL-6 between groups with or without induced pancreatitits. CONCLUSIONS: Although the efficacy of NM for treatment of acute pancreatitis is doubtful, pretreatment with NM for an expected condition like endoscopic retrograde cholangiopancreatography (ERCP), might be helpful for the prevention of pancreatitis.
Amylases ; Animals ; Ceruletide ; Cholangiopancreatography, Endoscopic Retrograde ; Cholecystokinin ; Humans ; Inflammation ; Injections, Intraperitoneal ; Interleukin-6 ; Lipase ; Mesylates* ; Mice ; Models, Animal ; p38 Mitogen-Activated Protein Kinases ; Pancreatitis* ; Peroxidase ; Protease Inhibitors

The role of PACAP (pituitary adenylate cyclase activating polypeptide), a peptidergic transmitter, in the pathogenesis of acute pancreatitis is not yet clear. This experiment was conducted to examine the action of exogenous PACAP on rat pancreas and on the course of experimental acute pancreatitis. The results showed that 5-30 microg/kg of PACAP slightly raised the serum amylase level, induced pancreatic edema (23.88% +/- 2.532%-25.86% +/- 1.974% of experiment groups versus 29.21% +/- 5.657% of control group), inflammatory cell infiltration, vacuolization of acinar cells, and occasionally fatty and parenchymal necroses. 15-30 microg/kg of PACAP aggravated cerulein-induced acute pancreatitis; the pancreatic edema became more marked (13.45% +/- 2.045%-17.66% +/- 4.652% of expreiment groups versus 21.83% +/- 3.013% of cerulein group, P<0.05), the serum amylase level became higher; and ascites, pancreatic bleeding, fatty and parenchymal necroses, and extensive vacuolization of acinar cells appeared. For sodium taurocholate-induced pancreatitis, 5-10 microg/kg of PACAP mildly attenuated the pancreatic edema, reduced the serum amylase level (1986.91 +/- 710.97-2944.33 +/- 1182.47 IU/L vs 3690.87 +/- 2277.99 IU/L, P<0.05), whereas it caused multifocal hemorrhage and prominent necrosis in pancreas. Except the cerulein-induced pancreatitis groups, other groups were found to have reduced pancreatic functional capillary density (FCD); when pancreatic edema was taken into consideration and calibrated FCD was introduced (FCD weighted against pancreatic wet/dry ratio), all groups revealed increases in pancreatic functional capillaries when compared with normal control. In conclusion, PACAP is proinflammatory in the pathogenesis of acute pancreatitis, PACAP plus cerulein can induce acute hemorrhagic/necrotizing pancreatitis, and the action of PACAP on cerulein-induced panceatitis may differ from that on sodium taurocholate-induced one. In this experiment, pancreatic FCD was underestimated due to pancreatic edema.
Amylases ; blood ; Animals ; Capillaries ; pathology ; Ceruletide ; Disease Models, Animal ; Male ; Pancreas ; blood supply ; Pancreatitis, Acute Necrotizing ; chemically induced ; enzymology ; pathology ; Rats ; Rats, Wistar

OBJECTIVETo explore the changes in hemorheology and expression of neutrophil adhesion molecules CD18 and CD62L in pancreatic microcirculation of Caerulein induced experimental acute pancreatitis (AP).

METHODSThe Wistar rats (n = 21) were randomized into three groups. The model of AP was established by subcutaneous injection of Caerulein. The changes of apparent viscosity of whole blood were measured by Low- shear 30 rheometer. The expression of adhesion molecules on the surface of neutrophil in duced by shear stress was used with stationary control. CD18 expression was increased on neutrophils treated with shear rate, and andanalyzed using flow cytometry.

RESULTSRat treated with Caerulein showed hyperamyleimia (t = 69.029, t = 79.734, P < 0.05). Blood viscosity of two AP groups were significantly elevated (0.512 s(-1): t = 10.725, t = 16.945; 5.96 s(-1): t = 12.781, t = 11.992, P < 0.05). Compared with stationary control, CD18 expression was increased on neutrophil treated with shear rate, and significantly induced with shear rate >/= 94.5 s(-1) (94.5 s(-1): t = 7.403, t = 13.323, t = 16.655; 128.5 s(-1): t = 10.092, t = 28.531, t = 24.563, P < 0.05). The expression of CD62L was less sensitive to low shear rate, and began to be down-regulated significantly when the shear rate >/= 94.5 s(-1) (94.5 s(-1): t = 10.687, t = 19.376, t = 12.848; 128.5 s(-1): t = 26.152, t = 48.402, t = 56.814, P < 0.05).

CONCLUSIONSThe changes of apparent viscosity of whole blood, and the effect of fluid shear stress on the expression of neutrophil adhesion molecules CD18, CD62L may play an important role in the pancreatic microcirculatory failure of acute pancreatitis.

Acute Disease ; Animals ; Ceruletide ; Flow Cytometry ; Hemorheology ; Microcirculation ; Neutrophils ; Pancreatitis ; Rats, Wistar