Cerulein-stimulated Pancreatic Acinar Cells Research Articles

Suppression of IL-1β expression by the Jak 2 inhibitor AG490 in cerulein-stimulated pancreatic acinar cells

Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the digestive enzyme production and cytoplasmic vacuolization, the death of acinar cells, edema formation, and an infiltration of inflammatory cells into the pancreas. Cytokines are up-regulated in pancreatic acinar cells stimulated with cerulein. In various cells and tissues, Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway mediates inflammatory process. In the present study, we investigated whether the activation of Jak/Stat signaling mediates IL-1β expression in pancreatic acinar AR42J cells stimulated with cerulein in vitro as well as the rats with cerulein pancreatitis in vivo using AG490, the Jak2 inhibitor. Activation of Jak2 and Stat3 were monitored by Western blot analysis for phosphorylated Jak2 and phosphorylated Stat3. mRNA expression and protein level of IL-1β were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). Histological examination of pancreatic tissues were performed and serum IL-1β levels of the rats were determined by ELISA. As a result, cerulein induced the activation of Jak2 and Stat3 as well as IL-1β expression, which was inhibited by the treatment of AG490 in AR42J cells. In cerulein pancreatitis of the rats, edematous and inflammatory changes of the pancreas and increased serum levels of IL-1β were suppressed by AG490 treatment. In conclusion, Jak2/Stat3 pathway may be the underlying mechanism in the pathogenesis of pancreatitis by inducing cytokines such as IL-1β.

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p47 phox and p67 phox to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca 2+ oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca 2+ chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22 phox and p47 phox inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p47 phox and p67 phox to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca 2+-dependent manner.

NADPH oxidase mediates interleukin-6 expression in cerulein-stimulated pancreatic acinar cells

NADPH oxidase produces a large amount of reactive oxygen species (ROS) mainly in phagocytic cells. ROS are involved in NF-κB activation, cytokine expression and thus, pathogenesis of pancreatitis. However, the source of ROS in pancreatic acinar cells has not been clarified. Cerulein rapidly induces acute and edematous form of pancreatitis. We investigated whether pancreatic acinar cells contain NADPH oxidase, and whether NADPH oxidase mediates interleukin-6 (IL-6) in pancreatic acinar AR42J cells stimulated with cerulein. Expression of NADPH oxidase subunits and NADPH oxidase activity were determined in the cells by immunofluorescence staining and lucigenin luminescence, respectively. Oxidant-sensitive nuclear transcription factor NF-κB activation was monitored by electrophoretic mobility shift assay. IL-6 expression was determined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbant assay. NADPH oxidase inhibitor diphenylene iodonium (DPI), antioxidant rebamipide, and antisense oligonucleotides (AS ODNs) for NADPH oxidase subunits p22 phox and p47 phox were used to determine the involvement of NADPH oxidase in NF-κB activation and IL-6 expression in AR42J cells. As a result, pancreatic acinar AR42J cells constitutively express NADPH oxidase subunits p67 phox and p47 phox in the cytosol and Nox1 and p22 phox in the membrane. Cerulein-stimulated NADPH oxidase activity and induced NF-κB activation and IL-6 expression in AR42J cells. Treatment of DPI or rebamipide and transfection of AS ODNs for NADPH oxidase subunits suppressed cerulein-induced NF-κB activation and IL-6 expression compared to S ODNs. In conclusion, NADPH oxidase may mediate the expression of inflammatory cytokines by stimulating NF-κB activation in pancreatic acinar cells during the course of pancreatitis.

Xanthine oxidase-mediated intracellular oxidative stress in response to cerulein in rat pancreatic acinar cells.

Intralobular oxygen radical formation was examined in cerulein-stimulated rat pancreatic acinar cells by digital imaging microscopic fluorography using a hydroperoxide-sensitive fluorescent probe, dichlorofluorescin (DCFH) diacetate. The isolated pancreatic acinar cells loaded with DCFH diacetate were microscopically observed, and the dichlorofluorescein (DCF) fluorescence yielded by DCFH oxidation via hydroperoxides was digitally processed. Within the initial 20 min after the application of cerulein (10 microM), intracellular oxidative stress was observed as indicated by the increase in DCF fluorescence intensity and reached its maximum at 60 min. DCF fluorescence intensity was then gradually decreased until 80 min, followed by a marked increase in propidium iodide (PI) fluorescence, suggesting irreversible cell death. Allopurinol (1 microM), a xanthine oxidase inhibitor, significantly attenuated the early increase of DCF fluorescence intensity as well as the late cell damage. Treatment with hyperbaric oxygen (PO2 300 mm Hg) also significantly attenuated both the increase of DCF fluorescence and the number of PI-positive cells. The results suggest that xanthine oxidase-mediated oxygen radicals may play an important role in cerulein-induced intracellular oxidative stress in pancreatic acinar cells of rats.