NADPH oxidase mediates cerulein-induced apoptosis in pancreatic acinar AR42J cells

Abstract

[한글]세포고사는 췌장염에서 산화적 손상과 밀접한 관련이 있다. NADPH oxidase는 췌장 선세포에서 세포고사과정에서 활성산소의 중요한 원천으로서 고려되어지고 있다. 그러나, 췌장 선세포에서의 활성산소의 원천연구는 아직 정확히 알려진 바 없다. Cerulein은 급성, 부종형성을 보이는 췌장염을 빠르게 유발한다.본 연구에서는 췌장 선세포에서 NADPH oxidase가 세포고사를 유도하는지를 보고자 하였다. Cerulein 자극에 의하여 NADPH oxidase의 세포질내 인자인 p47phox와 p67phox 가 세포막으로 이동하여 결합함으로서, NADPH Oxidase가 활성화 되고, 이로 인해 활성산소가 생성되었다. Cerulein은 세포내 칼슘농도를 증가시고, 세포고사성 유전자인 bax, p53, AIF를 발현시키며, DNA 절단화, caspase 3 활성화, 생존 세포수를 감소시킨다. NADPH Oxidase 억제제인 DPI, 칼슘 킬레이터인 BAPTA, NADPH Oxidase의 p47phox와 p22phox 인자의 AS ODN을 세포내 처치하여, cerulein에 의해 생성되는 활성산소, NADPH Oxidase의 활성화, 세포고사성 유전자 발현, DNA 절단화, caspase 3 활성화를 억제하였다. 이러한 결과로 NADPH Oxidase에 의해 생성되는 활성산소가 췌장 선세포에 세포고사를 유도함을 확인하였다.

[영문]Apoptosis linked to oxidative stress has been known to be implicated in pancreatitis. NADPH oxidase has been considered as a major source of reactive oxygen species during apoptosis in pancreatic acinar cells. However, the source of reactive oxygen species in pancreatic acinar cells has not been clarified. Cerulein rapidly induces acute and edematous form of pancreatitis. It was investigated that NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. Treatment of cerulein resulted in the activation of NADPH oxidase, as determined by reactive oxygen species production, translocation of cytosolic subunits p47phox and p67phox to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca++ oscillation, the expression of apoptotic gene bax, p53, AIF and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a NADPH oxidase inhibitor diphenyleneiodonium (DPI), Ca++ chelator BAPTA, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22phox and p47phox inhibited cerulein-induced reactive oxygen species production, translocation of NADPH oxidase cytosolic subunits p47phox and p67phox 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 reactive oxygen species-induced apoptosis in pancreatic acinar cells.