일과성 국소 대뇌 허혈 생쥐 모델에서 Apoptosis Signal-Regulating Kinase 1의 증가 및 Ask1 표적 siRNA 처치에 의한 대뇌 허혈 감소
김현우, 조경주·김현정·조양제·이병인·김경환
연세대학교 의과대학 신경과학교실, 뇌졸중 및 신경-혈관집중치료실
Increase of Apoptosis Signal-Regulating Kinase 1 and Inhibitory Effect of Ask1 Targeted Small Interfering RNA on Infarction after Ischemia/Reperfusion in Mice
Hyun-Woo Kim, BS Kyoung-Joo Cho, MS, Hyun-Jeong Kim, MS, Yang-Je Cho, MD, Byung-In Lee, MD and Gyung Whan Kim, MD, PhD
Stroke and Neurocritical Care Section, Department of Neurology, Yonsei University College of Medicine, Seoul, Korea
ABSTRACT
Background: Oxidative signal transduction has been crucial for the ischemic neuronal cell death. Apoptosis signal-regulating kinase 1 (Ask1), mitogen-activated protein MAP kinase kinase kinase 5, activated by oxidative stress which induces apoptotic signal pathway. We investigated the expression of Ask1 after ischemia/reperfusion. And siRNA targeting Ask1 could efficiently attenuate the cerebral infarction volume. Methods: Adult male ICR mice were subjected to middle cerebral artery occlusion (MCAO) for 60 minutes followed by reperfusion. The expression and mRNA of Ask1 were evaluated by Western blot, RT-PCR and immunohistochemistry at various time points after reperfusion. Ask1 and cell death were also investigated by Western blot analysis, immunohistochemistry and TUNEL assay to evaluate the effect of Ask1 on cerebral infarction by treatment with siRNA targeting Ask1. Results: The expression of Ask1 was increased as early as 1 hour after ischemia/reperfusion and continued to 24 hours in ischemic brain compared to nonischemic brain. Double immunofluorescent staining demonstrated that Ask1 expression was colocalized in DNA damaged cells after ischemia/reperfusion. After treatment of Ask1-siRNA, down-regulation was confirmed by that the level of mRNA and protein of Ask1 was decreased and subsequently led to reduce cerebral infarction volume. Conclusions: Our results suggest the increased Ask1 expression induce apoptotic DNA damage after ischemia/reperfusion. And also we demonstrated that Ask1-siRNA attenuates upregulation of Ask1, which was followed by the reduction of infarction volume in ischemic brain after ischemia/reperfusion. Ask1-siRNA could represent a molecular target for prevention of ischemic stroke.
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