Yu Ji Hoon, Kim Kyung Hwan, Kim Dong Goo, Kim Hyeyoung
Department of Pharmacology, Brain Korea 21 Project for Medical Science, College of Medicine, Yonsei University, Seoul, Republic of Korea.
Int J Biochem Cell Biol. 2007;39(11):2063-75. doi: 10.1016/j.biocel.2007.05.021. Epub 2007 Jun 7.
NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.
NADPH氧化酶被认为是吞噬细胞和非吞噬细胞中活性氧的主要来源。与氧化应激相关的细胞凋亡与胰腺炎有关。最近，我们证明NADPH氧化酶亚基Nox1、p27phox、p47phox和p67phox在胰腺腺泡细胞中组成性表达，这些亚基可被胆囊收缩素类似物蛙皮素激活。蛙皮素可诱发急性水肿型胰腺炎。我们通过测定活细胞数量、DNA片段化、TUNEL染色、caspase-3活性以及在蛙皮素刺激的胰腺腺泡AR42J细胞中凋亡诱导因子的表达，研究二苯碘鎓对NADPH氧化酶的抑制是否能抑制活性氧的产生和细胞凋亡。通过NADPH氧化酶活性的改变以及胞质亚基p67phox和p47phox向膜的转位来评估二苯碘鎓对NADPH氧化酶的抑制作用。监测细胞内Ca2+水平以研究在蛙皮素刺激的细胞中NADPH氧化酶与Ca2+之间的关系。结果显示，蛙皮素诱导了NADPH的激活，增加了活性氧的产生，并通过凋亡诱导因子的表达、caspase-3激活、TUNEL染色、DNA片段化和细胞活力来确定凋亡指数。用二苯碘鎓处理可抑制蛙皮素诱导的NADPH氧化酶激活、活性氧的产生和细胞凋亡，但不抑制胰腺腺泡细胞内Ca2+水平的升高。这些结果表明，蛙皮素诱导的细胞内Ca2+水平升高可能是NADPH氧化酶激活的上游事件。二苯碘鎓作为一种NADPH氧化酶抑制剂，可抑制凋亡诱导因子的表达和caspase-3激活，从而抑制胰腺腺泡细胞的凋亡。
