영구적 국소 대뇌 허혈 생쥐 모델에서 활성 산소종에 의한 세포고사 유도인자의 핵내 이동과 세포고사

Abstract

Background: Recently, the mitochondrial proapoptotic protein, apoptosis-inducing factor (AIF), and its nuclear translocation have been reported in caspase-independent neuronal apoptosis. However, it is not elucidated whether oxidative signaling is involved in the nuclear translocation of AIF and subsequent caspase-independent apoptotic cell death. We investigated whether oxidative signaling induces nuclear translocation of AIF and subsequent caspase- independent apoptosis-associated DNA fragmentation after permanent focal cerebral ischemia (FCI). Methods: Adult male ICR mice were subjected to permanent FCI by intraluminal suture blockade of middle cerebral artery. Immunohistochemistry and Western blot analysis were performed. Large-scale DNA fragmentation was evaluated by pulse field gel electrophoresis and apoptotic cell death was quantified. Manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP), which mimics mitochondrial superoxide dismutase, was used to determinewhether the production of reactive oxygen species is required for the induction in AIF translocation. Results: Western blot analysis showed that the nuclear translocation of AIF occurred as early as 2 hours after permanent FCI. Immunostaining for AIF also confirmed early nuclear translocation of AIF after permanent FCI. Large-scale DNA fragmentation was detected 8 hours after permanent FCI. MnTBAP-treatment attenuated AIF translocation and blocked large-scale DNA fragmentation. Caspase-3 activity was similarly inhibited between the pan-caspase inhibitor- and MnTBAP-treated mice, but the amount of apoptosis-associated DNA fragmentation in the MnTBAP-treated mice was less than in the pan-caspase inhibitor-treated mice (P<0.001). In addition, infarction volume was also decreased by MnTBAP. Conclusions: These results suggest that reactive oxygen species induce the caspase-independent nuclear translocation of AIF and subsequent apoptosis-associated DNA fragmentation after permanent FCI.