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1 원저 국소대뇌허혈및재관류후생쥐모델에서활성산소제거제 (MnTBAP) 에의한 Endonuclease G 의조기핵내이동방지및뇌경색감소 연세대학교의과대학신경과학교실, 뇌연구소 김현우조경주김현정조양제이병인김경환 Treatment with MnTBAP Protects Against Early Nuclear Translocation of Endonuclease G and Reduces Cerebral Infarction after Focal Cerebral Ischemia/Reperfusion in Mice Hyun-Woo Kim, B.S., Kyoung-Joo Cho, M.S., Hyun-Jeong Kim, M.S., Yang-Je Cho, M.D., Byung-In Lee, M.D., Gyung-Whan Kim, M.D. Department of Neurology, Yonsei University College of Medicine, Brain Research Institute Background: Reactive Oxygen Species (ROS) have been implicated in the pathophysiology of brain injury after ischemia/reperfusion. Recently, it has been reported that endonuclease G (EndoG), a mitochondrial protein, is activated by neuronal excitotoxicity and translocated into nucleus inducing apoptosis. However, it is not elucidated whether ROS are involved in the nuclear translocation of EndoG in focal cerebral ischemia/reperfusion in mice. We investigated whether treatment of manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP) protects against early nuclear translocation of EndoG and reduces cerebral infarction after ischemia/reperfusion in mice. Methods: Adult male mice were subjected to middle cerebral artery occlusion (MCAO) for 60 min, followed by reperfusion. Immunohistochemistry and Western blot analysis for EndoG were performed at various time points after ischemia/reperfusion. Double staining with EndoG and Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) was also performed. MnTBAP was used to determine whether the production of ROS could inhibit translocation of EndoG into the nucleus. Results: Western blot analysis and Immunohistochemistry of EndoG showed that nuclear EndoG was detected as early as 4 hrs after reperfusion, and mitochondrial EndoG was significantly reduced at the same time. Double staining with EndoG and TUNEL showed a spatial relationship between EndoG expression and DNA fragmentation. MnTBAP-treated mice showed that the translocation of EndoG was attenuated in comparison with the vehicletreated mice and decreased infarction volume after ischemia/reperfusion. Conclusions: MnTBAP reduced the generation of ROS, and inhibited the early translocation of EndoG, which was followed by the reduction of infarction volume in the ischemic brain after ischemia/reperfusion. J Korean Neurol Assoc 25(4): , 2007 Key Words: Endonuclease G, DNA fragmentation, Transient focal cerebral ischemia, MnTBAP Received April 9, 2007 Accepted July 6, 2007 *Gyung-Whan Kim, M.D. Department of Neurology College of Medicine Yonsei University, 134 Sinchon-dong, Seodaemun-gu, Seoul, Korea Tel: Fax: gyungkim@yumc.yonsei.ac.kr * 본연구는 2006 년연세대학교의과대학교내연구비 ( ) 와 2006 년정부 ( 교육인적자원부 ) 의재원으로한국학술진흥재단의지원 (KRF E00127) 을받아수행된연구임. 서론 뇌졸중은많은나라에서주요사망원인의하나로알려져있으며, 국내에서도전체사망원인 2위, 단일장기질환으로는사망원인 1위로사망률이높은질환이다. 한국사회가점차고령화되면서뇌졸중발생빈도의증가에따른사회경제적인부담이높아지게될것으로예상된바뇌졸중의사망위험을 J Korean Neurol Assoc Volume 25 No. 4,

2 김현우조경주김현정조양제이병인김경환 낮추는인자를찾아내는등치료방법개발이매우절실히요구되고있다. 1 과도하게생성된활성산소종 (reactive oxygen species; ROS) 에의한스트레스는대뇌허혈및재관류후직접적으로세포손상을입히거나간접적신호전달체계를통해세포손상을가져옴으로써, 뇌졸중을포함한여러중추신경계질환에서병인론적으로중요한역할을한다. 2,3 생쥐의허혈및재관류모델실험에서재관류중재공급된산소는다양한효소의산화반응을야기하는생화학적기질을생성하며, 미토콘드리아에서과산화음이온 (superoxide anion) 과과산화수소 (hydrogen peroxide) 와같은활성산소종을발생시킨다고알려졌다. 2,3 이러한활성산소종은세포고사 (apoptosis) 신호전달경로의활성에관여하며대뇌허혈및재관류의중요한병태생리기전으로알려져있다. 2,4-6 대뇌허혈이후에발생하는세포고사에서 caspase 의존성세포고사경로의중요성은잘알려져왔으며, caspase 의존성세포고사유도물질중대표적인것이미토콘드리아내에존재하는 cytochrome c이다. 이로인한 caspase 활성은대뇌허혈및재관류이후에발생하는세포고사에결정적인역할을하는것으로알려져왔다. 7,8 하지만배양된 Rat-1 섬유아세포에 caspase를억제함과동시에 AIF (Apoptosis-Inducing Factor) 를미세주사한후의연구보고에서 caspase 가억제되었음에도불구하고세포고사가일어나는현상으로인해 caspase 비의존성대체경로가존재할것이라는가능성이발표되었으며, 9 본연구팀의이전연구 10,11 에서밝힌바, 생쥐를대상으로한국소대뇌허혈모델에서 AIF 가핵내로이동하여 caspase 비의존성세포고사를일으키며활성산소제거제에의해세포고사가방지되었다. 위와같은연구결과들로뇌경색후발생하는세포고사에 caspase 비의존성세포고사물질이중요한인자로대두되고있다. 또다른 caspase 비의존성세포고사물질로서, 사립체세포고사유도물질인 Endonuclease G (EndoG) 가최근새로이주목받고있는데, 미토콘드리아막사이 (intermembrane) 공간에서핵내로이동한 EndoG 는먼저 50 kbp의대단위 (large-scale) DNA 분절을만든다음, 순차적세포고사과정을통해소단위 (oligonucleosomal) DNA 분절을만든다. 13,16 이러한 EndoG 의작용은 exonuclease 나 DNase I 등의존재에의해촉진되는것으로보고되고있다. 16 이보고는신경세포고사를유도하는데 EndoG 가핵심역할을할것이라는가설을강력하게뒷받침한다. 산화적스트레스로인한척추손상모델에서 SOD1 과발현랫드의 EndoG 의핵내이동과그로인한세포사멸이활성산소종과관련되어있다는보고가있으나, 17 국 소대뇌허혈및재관류유도후활성산소종이과생성된환경에서의 EndoG 와활성산소종제거제와의역할및연관성에대해서는아직확실히규명되어있지않다. 이에본연구에서는국소대뇌허혈및재관류생쥐모델에서활성산소종과 EndoG 의핵내이동과의관계를확인하고자하였다. 이를위해활성산소종제거제인 Manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP) 를사용하여 EndoG 의핵내이동방지여부를확인하고이를통한대뇌허혈부위세포의 DNA 분절현상감소여부와뇌경색병변크기의변화를확인하고자하였다. 대상과방법 1. 일과성대뇌국소허혈모델 본실험에서모든실험동물연구방법은연세의과대학실험동물위원회의지침을준수하였으며, Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) 의승인을받았다. 수컷성체 C57BL/6J 마우스 (3개월령, g) (Daehan Biolink Co., Chunbuk, South Korea) 를나일론봉합사를이용해한시적으로중대뇌동맥을폐색 (middle cerebral artery occlusion, MCAO) 함으로써일과성국소허혈모델을적용하였다 안면마스크를사용하여 2% isoflurane 과함께질소와산소혼합기체 (70%/30%) 로마우스를마취시켰고, 체온은 heating pad 및 lamp 로 37±0.5 를유지하였다. 정중경부를절개하여외경동맥을노출시킨후, 열처리로끝을무디게만든 9.0 mm 길이의 5-0 수술용나일론봉합사 (Ethicon, Edinburg, UK) 를외경동맥을통해내경동맥으로삽입하여중대뇌동맥혈류를차단하였으며, 60분후에나일론을제거하여혈류를회복시켰다. 이때중대뇌동맥의혈류폐색여부는 Laser Doppler Flowmeter (Transonic Systems Inc., Ithaca NY 14850, USA) 를사용하였다. 2. EndoG 검출을위한 Western blot analysis 조직은 50 mm HEPES (ph 7.4), 150 mm NaCl, 3 mm dithiolthreitol (DTT), 2 mm sodium orthovanadate, 1 mm EGTA, 1 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100 및각각 5 mg/ml 의 leupeptin, pepstatin A와 aprotinin (Sigma, St Louis, MO, USA) 이첨가된차가운균질화완충액에서균질화시켰다. 미토콘드리아 / 세포질층을 4 에서 10분동안 750 g로원심분리한후그침전물을다시 536 대한신경과학회지제 25 권제 4 호, 2007

3 국소대뇌허혈및재관류후생쥐모델에서활성산소제거제 (MnTBAP) 에의한 Endonuclease G 의조기핵내이동방지및뇌경색감소 4 에서 15분동안 1,025 g로원심분리하였다. 핵이포함된침전물을얼음에서 15분동안둔다음, 완충액으로재부유시켜 4 에서 20분동안 16,000 g로원심분리하여핵분획으로사용하였다. 미토콘드리아침전물을얻기위해상층액을 4 에서 15분간 10,000 g 로원심분리한후, 미토콘드리아침전물을 3% Ficoll 용액 (120 mm mannitol, 30 mm sucrose, 25 M EDTA) 에녹이고, 6% Ficoll 용액 (240 mm mannitol, 60 mm sucrose, 50 M EDTA) 으로농도구배후, 다시상층액을채취하여 4 에서 25 분동안 16,000 g로원심분리하여미토콘드리아분획으로사용하였다. 21,22 EndoG 다클론성항체 (1:1000; Oncogene Science, Cambridge, MA, USA) 를가한후, 4 에서하룻밤동안반응시켰다. 이후 0.1% Tween 20이포함된 TBS 로막을 15분간다섯차례세척하고, 이차항체인 horse radish peroxidase 가부착된 anti-rabbit IgG (1:5000; Roche Diagnostics, Indianapolis, IN, USA) 를 37 에서 60분간반응시킨후, chemiluminescence detection system 인 ECL plus kit (Amersham International, Buckinghamshire, England) 로발색시켰다. 3. 면역조직화학염색을통한 EndoG 발현확인 EndoG 분석을위한면역조직화학염색은 avidin-biotin 방법을채택하였다. 23 Urethane 으로마취시킨생쥐를 10 U/ml 헤파린이첨가된 0.9% 생리식염수와 3.7% 포름알데히드로관류시켜, 조직을준비하였다. 뇌를적출하여 3.7% 포름알데히드로 16시간동안후고정을시키고, 사용할때까지 30% sucrose 에저장하였다. 뇌조직을드라이아이스에서얼린후 cryostat section 을이용해, 20 μm 두께로관상 (coronal) 절편을만들었다. 절편을실온에서 1시간동안 20% 의 rabbit serum 으로 blocking 시킨다음, 일차항체로 rabbit anti-endog 다클론성항체 (1:50; Oncogene Science) 를반응시켰다. Vectastatin Elite ABC Kit (Vector laboratories, Burlingame, CA, USA) 를사용하여면역반응을시행하였고 diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) 으로발색시켰으며, methyl green 으로핵대조염색을실시하였다. EndoG 양성세포와 methyl green 양성세포를고배율 (400x) 에서계수한다음, EndoG 양성세포의비로표시하였다 MnTBAP 처리생성된과산화물제거를위해 50 μg/μl 의 MnTBAP 를 MCAO 30 분전에뇌실내로주입하였다 (2 μl, mediolateral=1.0 mm; anteroposterior=0.2 mm; dorsoventral=3.1 mm). 실험군은 MnTBAP 를주입하고대조군은 vehicle( 증류수 ) 을주입하였다. 5. in situ 과산화음이온라디칼검출허혈후발생하는과산화음이온을확인하기위해 oxidized hydroethidine (HEt) 검출법을사용하였다. 24 HEt (Molecular Probes) 를 dimethylsulfoxide (DMSO) 에 100 mg/ml 로용해시킨후 PBS 완충액으로 1:100 비율로희석시켰다. C57BL/6J 마우스를희생시키기 1시간전에 200 μl HEt 을정맥내에주입하고심장을통해 10 U/mL 헤파린이첨가된생리식염수와 3.7% 포름알데히드로관류한후뇌를적출하였다. 적출된뇌를 3.7% formaldehyde 로 16시간동안 4 에서고정시킨다음 vibratome 을사용하여 50 μm의두께로절편을만들었다. 조직절편이준비된즉시뇌절편을유리판위에올리고현미경으로관찰하였다. 핵대조염색은 Hoechst (Molecular Probes) 을사용하였다. Oxidized HEt 의발현강도와패턴분석을위해 computerized digital camera system 형광현미경 (Ex= nm, Em>580 nm; BX51, Olympus, Tokyo, Japan) 을사용하였고이미지분석프로그램으로분석하였다 (MetaMorpho imaging, version 5.0; Molecular devices, Downington, PA, USA). 6. EndoG와 Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) 이중면역형광염색 EndoG 와 DNA 분절현상이동일위치에발현하는지를확인하기위해이중면역형광염색을시행하였다. 상기방법으로 EndoG 면역조직화학검사를시행한후, PBS 로세척하고절편을 50 μl 의 TUNEL fluorescent 반응혼합액 (terminal deoxynucleotidyl transferase and fluorecein-dutp; Roche Diagnostics, Indianapolis, IN, USA) 과 37 의어두운곳에서 60분동안반응시킨후, Vectashield (Vector laboratories) 로봉입하고 LSM510 confocal laser scanning 현미경 (Carl Zeiss) 으로관찰하여분석하였다. 7. 뇌경색크기측정증류수투여군과 MnTBAP 투여군으로나누어비교하였으며, 투여 15분후에 MCAO 를시행하여일과성국소허혈을유도하였다. 국소대뇌허혈후, 24 시간째에마우스를희생시켜뇌를적출 J Korean Neurol Assoc Volume 25 No. 4,

$김현우조경주김현정조양제이병인김경환 Figure 1. Western blot analysis and Immunohistochemistry of EndoG. (A) EndoG is detected in subcellular fractionation by Western blot analysis.$

4 김현우조경주김현정조양제이병인김경환 Figure 1. Western blot analysis and Immunohistochemistry of EndoG. (A) EndoG is detected in subcellular fractionation by Western blot analysis. EndoG is time-dependently translocated from the mitochondrial fraction to the nuclear fraction after ischemia/reperfusion. (B) The quantitative graphs show the optical density values of EndoG in the mitochondrial and nuclear fractions respectively. COX, cytochrome oxidase; M, mitochondrial fraction; N, nuclear fraction. (C) EndoG is also detected by immunohistochemistry in a time-dependent manner. EndoG is started to appear in cytosol region and showed in nucleus region at 24 hrs after ischemia/reperfusion. a-b) EndoG is not detected in control and 1 hr c-e) Nuclear staining of EndoG became prominent at 4, 8, and 24 hrs after ischemia/reperfusion. f) The percentage of EndoG-positive cells is shown in graph. Scale bar = 20 μm *p<0.05, **p<0.01. 하였다. 적출한뇌는 brain matrix를이용하여전두부부터 1 mm 두께로잘라관상절편화하였다. 각각의절편을 2% 2,3,5-triphenyltetrazolium chloride (TTC) 에담가 37 에서 15분간배양하여염색하였으며, 18 평판스캐너를이용하여 1200 dpi로스캔하였다. 8. 통계학적분석 하였고, 두집단간의차이는 test 를통해분석했으며 (Stat- View; SAS Institute Inc, Cary, NC, USA), p<0.05 를유의한것으로판정하였다. 결과 1. 허혈 / 재관류동안중대뇌동맥의혈류량변화 데이터들은 mean±sd 로표현하였다. 여러집단간의통계학적비교는 ANOVA (with Fischer s post-hoc test) 를사용 중대뇌동맥의혈류량은중대뇌동맥폐색 10분전, 폐색 10분후그리고재관류 10분후에수치를측정하였으며, 세군 538 대한신경과학회지제 25 권제 4 호, 2007

국소대뇌허혈및재관류후생쥐모델에서활성산소제거제 (MnTBAP) 에의한 Endonuclease G 의조기핵내이동방지및뇌경색감소 Figure 2. DNA fragmentation after ischemia/reperfusion. Immunofluorescent double-staining was performed for EndoG and TUNEL.

5 국소대뇌허혈및재관류후생쥐모델에서활성산소제거제 (MnTBAP) 에의한 Endonuclease G 의조기핵내이동방지및뇌경색감소 Figure 2. DNA fragmentation after ischemia/reperfusion. Immunofluorescent double-staining was performed for EndoG and TUNEL. EndoG is mostly colocalized with TUNEL at 24 hrs after ischemia/reperfusion. EndoG in control group is exclusively detected in the cytosol by confocal microscopy. In contrast, EndoG is colocalized with TUNEL (arrow heads) in the nucleus at 24 hrs after ischemia/reperfusion. Scale bar = 10 μm. 간의유의적차이는없었다 (%: 폐색 10분전, 100±0, 폐색 10분후, 20.6±14.1, 재관류 10분후, 96.6±9.4, n=13, mean ±SD). 2. Western blot 분석에서 EndoG 의핵내발현증가 Western blot 을이용하여, EndoG 의시간에따른발현변화양상을관찰하였는데, 허혈및재관류이후허혈뇌조직에서 ~ 35 kda 분자량의단백이미토콘드리아와핵분획에서뚜렷이관찰되었다 (Fig. 1-A). Figure 1-B 에서보이듯이많은양의 EndoG 가정상뇌조직의미토콘드리아분획에서도관찰되었으며, 허혈및재관류후시간이지남에따라통계적으로유의한감소를보였다 (optical density (O.D.). : 0 hr, 10.0±0.19; 4 hrs, 7.1±0.53; 8 hrs, 5.6±0.38; 24 hrs, 4.4±0.56; n=5, ANOVA, p<0.01). 동일뇌조직의핵분획을조사하였는데, 허혈및재관류후시간이지남에따라미토콘드리아 EndoG 의면역반응이감소하는것과는대조적으로시간에따른핵내 EndoG 의면역반응은정상뇌조직에비해통계학적으로유의한증가를보였다 (OD: 0 hr, 1.5±0.5; 4 hrs, 5.1±1.0; 8 hrs, 6.4±0.9; 24 hrs, 10.7±1.0; n=5, ANOVA, p < 0.01) (Fig. 1-B). 3. 면역조직화학염색에서의 EndoG 의핵내발현증가면역조직화학염색을이용하여, 허혈및재관류이후 EndoG 의발현을조사하였다 (Fig. 1). 일차항체를제외하고처리한조 직에서는면역반응이전혀관찰되지않았고 (data not shown), 정상뇌조직 (Fig. 1-Ca) 과허혈및재관류 1시간후에허혈뇌조직 (Fig. 1-Cb) 에서는 EndoG 면역반응이거의관찰되지않았다. 반면허혈및재관류 4시간째부터허혈뇌조직에서는핵내 EndoG 의면역반응이나타났고 (Fig. 1-Cc: open arrow heads), 세포질에도역시 EndoG 의면역반응이관찰되었다. 그러나이러한면역반응에양성을보인세포들은형태학적으로는아직특이사항이관찰되지않았다 (Fig. 1-Cc). 허혈및재관류후 8시간째부터 EndoG 에대한핵내양성반응이점점증가하였고 (Fig. 1-Ad, black arrow heads), 24시간째에는형태학적으로허혈성변화를보이는거의모든신경세포핵에서 EndoG 에대한양성반응이뚜렷이관찰되었다 (Fig. 1-Ce). Figure 1-Cf 는단위면적당전체세포에대한 EndoG 양성세포의수를백분율로표시한그래프이며, 허혈및재관류이후 24 시간까지핵내 EndoG 의발현양은시간이지남에따라증가함을확인할수있었다 (1 hr, 7.2±0.3; 4 hrs, 26±0.5; 8 hrs, 38±0.8; 24 hrs, 74±0.9, the percentages of nuclear EndoG-positive cells, n=4) (Fig. 1-Cf). 4. EndoG 와 TUNEL 이중면역형광염색 EndoG 와 TUNEL 이중면역형광염색을통해, 허혈및재관류 24시간후조직에서핵내 EndoG 양성세포와 TUNEL 양성세포가일치하여나타나는것을확인할수있었으며 (Fig. 2), 반면대조군인반대쪽정상뇌세포에서는 EndoG 양성세포와 TUNEL 양성세포도관찰되지않았다. J Korean Neurol Assoc Volume 25 No. 4,

6 김현우조경주김현정조양제이병인김경환 Figure 3. The effect of MnTBAP on ROS production and on an apoptotic cell death. (A) The representative photomicrographs show the production of superoxide radicals by the detection of oxidized HEt in both the MnTBAP- and vehicle-treated mouse brain at 4 hrs after ischemia/reperfusion. Oxidized HEt signals express in red spots at perinuclear sites and profound increase of oxidized HEt signals in the cytosol are observed in the ischemic brain of vehicle-treated mice. In MnTBAP-treated mice, oxidized HEt signals are observed slightly increased cytosolic expression in contrast to the vehicle-treated mice. The lowest panel shows quantified values of HEt signals. MnTBAP, manganese tetrakis (4-benzoic acid) porphyrin; HEt, hydroethidine. Scale bar = 10 μm. (B) Inhibition against translocation of EndoG and cell death after MnTBAP treatment. EndoG expression was compared in the MnTBAP- and vehicle-treated mice at 24 hrs after ischemia/reperfusion by Immunohistochemistry and EndoG nuclear translocation was decreased in MnTBAP treated group. Lower panels of vehicle- and MnTBAPtreated group in higher magnification. Scale bar, 50 μm (lower magnification); 10 μm (higher magnification). 5. MnTBAP 처리군에서의과산화음이온생산의억제 mean±sd, n=5, ANOVA, p<0.01). 과산화음이온은세포질에서산화된 hydroethidine 이빨간입자형태로관찰되는데 (Fig. 3), 일시적허혈유도후 4시간상태에서 vehicle 처리군의허혈부위에서는산화된 hydroethidine이정상대조군 (Fig. 3, Normal control) 과비교시현저히증가된반면 (Fig. 3, Vehicle+ I/R 4 hrs), MnTBAP 를처리한군 (Fig. 3, MnTBAP+ I/R 4 hrs) 의병변부위산화 hydroethidine는정상부위에비해별로증가하지않아 MnTBAP 처리군과 vehicle 처리군사이에의미있는차이를보였다 (normal control, 50.82±8.6; vehicle+i/r 4 hrs, ± ; MnTBAP+I/R 4 hrs, ±797.4; 6. MnTBAP 처리군에서의 EndoG 의핵내이동감소와세포고사및뇌경색감소일시적허혈유도 24시간후에, MnTBAP 처리군과 vehicle 처리군 (Fig. 3) 에서면역조직화학염색과 TUNEL 을통해 EndoG 단백질발현과세포고사를비교하였다. MnTBAP 처리군에서는 EndoG 핵내이동이 vehicle 처리군 (Fig. 3, Vehicle+ I/R 24 hrs) 에비해현저히감소하였으며, 세포고사를나타내는 TUNEL 양성세포는발견되지않았다 (Fig. 3, MnTBAP+ I/R 24 hrs). 대조군인정상뇌조직에서의 EndoG 핵내이동은 MnTBAP 처 540 대한신경과학회지제 25 권제 4 호, 2007

7 국소대뇌허혈및재관류후생쥐모델에서활성산소제거제 (MnTBAP) 에의한 Endonuclease G 의조기핵내이동방지및뇌경색감소 Figure 4. The comparison of infarction area between vehicle- or MnTBAP-treated mice. (A) Scanned representative images of an entire TTC were stained in ischemic brain sections. The infarcted areas remain unstained (white). (B) The graph represents quantified infarction area in two groups. 리군과 vehicle 처리군모두에서발견되지않았다 (data not shown). TTC 염색을통해서각군간의병변의크기를비교한결과 MnTBAP 처리군이 vehicle 을처리한군보다병변의크기가작았다 (Fig. 4). 고찰 이번연구를통해국소대뇌허혈및재관류이후이른시기에 EndoG 가미토콘드리아에서핵내로이동하여 DNA 분절을유도하며뇌경색에이르게하는것을밝혔으며, 활성산소제거제인 MnTBAP 에의한활성산소종제거를통하여 EndoG 의핵내이동및뇌경색을방지함을입증하였다. 본연구의결과를요약하면, 첫째, 미토콘드리아분획내 EndoG 의양은국소대뇌허혈및재관류후 4시간이경과된시점에서부터감소하기시작하였으며, 핵분획내 EndoG 의양은미토콘드리아분획에서의감소시점부터유의하게증가하였음을보여주었다 (Fig. 1). 또한 24시간후에 DNA 손상을의미하는 TUNEL 양성세포가핵내 EndoG 양성세포와일치하여나타나는것을보여주었다 (Fig. 2). 둘째, MnTBAP 를처리한군에서국소대뇌허혈및재관류로인한과산화음이온의생산이감소됨을 oxidized hydroethidine 감소를통해확인하였다 (Fig. 3). 셋째, 활성산소제거제인 MnTBAP 를처리한군에서허혈및재관류후 EndoG 의핵내이동은 vehicle 처리군보다유의하게감소하였 으며뇌경색의크기가감소하였음을확인하였다 (Fig. 3, 4). 이는과도하게발생한과산화음이온과같은활성산소종이 EndoG 의핵내이동을유도하며, 최종적으로는활성산소종을제거함으로써뇌경색을감소시킬수있음을시사한다. 이번실험에서 EndoG 가생쥐대뇌의허혈및재관류유도후, 4시간만에미토콘드리아에서감소하고핵내에서증가함을 Western blot 분석과면역조직화학염색 (Fig. 1) 을통해명확하게보여줌으로써국소대뇌허혈및재관류이후 EndoG 의조기핵내이동이일어남을증명하였는데, 이는정상세포고사과정에서 EndoG 의조기핵내이동이일어난다는 in vitro 실험들 13,25,26 과도잘부합하는것이다. 또한본결과는국소대뇌허혈및재관류후 24시간째에 EndoG 가핵내로이동된세포에서 TUNEL 양성세포가나타남을 EndoG 와 TUNEL 의이중면역형광염색을통해확인하였다 (Fig. 2). 이는 EndoG 가핵내이동후세포고사를유도한다는것을보여줄뿐만아니라, 세포고사메커니즘에 EndoG 가관여함을보여주는하나의단서를제공해준다. 최근뇌허혈을일으키는세포흥분독성 (excitotoxicity) 유발제인 kainic acid 처치모델연구에서, 야생형생쥐에비해 EndoG 결핍 (knockout) 생쥐의 EndoG 핵내이동이감소되었음을보였고, 뇌허혈흥분독성에대한방어효과가있다는보고는 27 EndoG 가핵내이동후세포고사를유도한다는본연구의가설을뒷받침해준다. 특히활성산소제거제인 MnTBAP 가허혈및재관류후에 J Korean Neurol Assoc Volume 25 No. 4,

8 김현우조경주김현정조양제이병인김경환 과발현한과산화음이온을제거하여 EndoG 의핵내이동을상당히억제하였으며, 동시에세포고사성 DNA 손상지표인 TUNEL 양성세포가줄어들었음을보여주었는데 (Fig. 3), 이는 human pulmonary artery endothelial cells 배양연구 28 와 3-amino-1,2,4-triazole+mercaptosuccinic acid 에의해유도된쥐간세포사멸연구 29 등과같은 in vitro 실험에서제시한 EndoG 의핵내이동이활성산소종에의해유도된다는사실과부합한다. 또한배양된망막세포와신경세포에항산화제인비타민 E와아이디비논 (Idebenone) 처치후효과를보고한연구와 30 형질전환된쥐에서전사인자인 Nrf2 의항산화적효과에관한보고등 31 에서보여주는것과같이과도한활성산소종에의한손상을치료하기위한연구들이이루어지고있다. 또한활성산소제거효소인 SOD1 (Copper/Zinc Superoxide Dismutase) 이과발현된생쥐및랫드연구에서는허혈및재관류후과도한산화적스트레스에의한대뇌손상후뇌손상보호에효과가있음이보고되었는데, 8,32 이는본연구에서 MnTBAP 처리여부에따른뇌경색크기를비교한결과, MnTBAP 처리군에서뇌경색감소에효과가있으며 (Fig. 4) 활성산소종제거가뇌경색에치료적효과가있음을뒷받침해준다. 결론적으로본연구결과는국소허혈및재관류이후 EndoG 의조기핵내이동이후세포고사가일어났으며, 활성산소제거제인 MnTBAP 를투여하여활성산소종을제거하면, EndoG 의핵내이동이차단되며, 세포고사및뇌경색이현저히감소함을보였다. 다만 EndoG 가세포고사과정에서작용하는정확한기전과 MnTBAP 에의한과산화음이온억제에따른 EndoG 의핵내이동방지기전에대한연구가좀더이루어진다면, 향후뇌경색을비롯한여러뇌신경질환을이해하고치료하는데도움이될것이다. REFERENCES 1. Korea National Statistical Office. Cause of death Statistics Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 2001:21: Chan PH. Oxygen radicals in focal cerebral ischemia. Brain Pathol 1994:4: Kim GW, Sugawara T, Chan PH. Involvement of oxidative stress and caspase-3 in cortical infarction after photothrombotic ischemia in mice. J Cereb Blood Flow Metab 2000:20: Fujimura M, Morita-Fujimura Y, Kawase M, Copin JC, Calagui B, Epstein CJ, et al. Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice. J Neurosci 1999:19: Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, et al. Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci 1998:18: Schulz JB, Weller M, Moskowitz MA. Caspases as treatment targets in stroke and neurodegenerative diseases. Ann Neurol 1999:45: Sugawara T, Noshita N, Lewen A, Gasche Y, Ferrand-Drake M, Fujimura M, et al. Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation. J Neurosci 2002:22: Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, et al. Molecular characterization of mitochondrial apoptosisinducing factor. Nature 1999:397: Cho YJ, Lee YH, Cho KJ, Lee DJ, Kim HW, Kim HJ, et al. Nuclear translocation of apoptosis inducing factor and subsequent DNA fragmentation by reactive oxygen species after permanent focal cerebral ischemia in mice. Korean J Stroke 2005:7: Lee BI, Chan PH, Kim GW. Metalloporphyrin-based superoxide dismutase mimic attenuates the nuclear translocation of apoptosisinducing factor and the subsequent DNA fragmentation after permanent focal cerebral ischemia in mice. Stroke 2005:36: Lee BI, Lee DJ, Cho KJ, Kim GW. Early nuclear translocation of endonuclease G and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Neurosci Lett 2005:386: Li LY, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 2001:412: Saelens X, Festjens N, Vande Walle L, van Gurp M, van Loo G, Vandenabeele P. Toxic proteins released from mitochondria in cell death. Oncogene 2004:23: Schafer P, Scholz SR, Gimadutdinow O, Cymerman IA, Bujnicki JM, Ruiz-Carrillo A, et al. Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis. J Mol Biol 2004:338: Widlak P, Li LY, Wang X, Garrard WT. Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I. J Biol Chem 2001:276: Yu F, Sugawara T, Nishi T, Liu J, Chan PH. Overexpression of SOD1 in transgenic rats attenuates nuclear translocation of endonuclease G and apoptosis after spinal cord injury. J Neurotrauma 2006:23: Kim GW, Kondo T, Noshita N, Chan PH. Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide radicals. Stroke 2002:33: Kim GW, Noshita N, Sugawara T, Chan PH. Early decrease in dna repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Stroke 2001:32: Tsuchiya D, Hong S, Kayama T, Panter SS, Weinstein PR. Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice. Brain Res 2003:970: Zhu C, Qiu L, Wang X, Hallin U, Cande C, Kroemer G, et al. Involvement of apoptosis-inducing factor in neuronal death after hypoxia-ischemia in the neonatal rat brain. J Neurochem 2003:86: 542 대한신경과학회지제 25 권제 4 호, 2007

9 국소대뇌허혈및재관류후생쥐모델에서활성산소제거제 (MnTBAP) 에의한 Endonuclease G 의조기핵내이동방지및뇌경색감소 Cao G, Clark RS, Pei W, Yin W, Zhang F, Sun FY, et al. Translocation of apoptosis-inducing factor in vulnerable neurons after transient cerebral ischemia and in neuronal cultures after oxygenglucose deprivation. J Cereb Blood Flow Metab 2003:23: Kim GW, Gasche Y, Grzeschik S, Copin JC, Maier CM, Chan PH. Neurodegeneration in striatum induced by the mitochondrial toxin 3-nitropropionic acid: role of matrix metalloproteinase-9 in early blood-brain barrier disruption? J Neurosci 2003:23: Arnoult D, Gaume B, Karbowski M, Sharpe JC, Cecconi F, Youle RJ. Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. Embo J 2003:22: Parrish J, Li L, Klotz K, Ledwich D, Wang X, Xue D. Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature 2001:412: Zhang J, Dong M, Li L, Fan Y, Pathre P, Dong J, et al. Endonuclease G is required for early embryogenesis and normal apoptosis in mice. Proc Natl Acad Sci U S A 2003:100: Wu Y, Dong M, Toepfer NJ, Fan Y, Xu M, Zhang J. Role of endonuclease G in neuronal excitotoxicity in mice. Neurosci Lett 2004: 364: Liu PL, Chen YL, Chen YH, Lin SJ, Kou YR. Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG. Am J Physiol Lung Cell Mol Physiol 2005:289:L Ishihara Y, Shimamoto N. Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress. J Biol Chem 2006:281: Rego AC, Santos MS, Oliveira CR. Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress. Free Radic Biol Med 1999: 26: Shih AY, Li P, Murphy TH. A small-molecule-inducible Nrf2- mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo. J Neurosci 2005:25: Fujimura M, Morita-Fujimura Y, Noshita N, Sugawara T, Kawase M, Chan PH. The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice. J Neurosci 2000:20: J Korean Neurol Assoc Volume 25 No. 4,

신하영 이두재 조경주등 Kainate 유발간질모델 - - 크레질바이올렛 (cresyl violet) 염색 Kainate 유발간질모델에서의뇌파검사 대상과방법 산소음이온라디칼 (superoxide anion radical, O.- 2 ) 검출 in situ- - 대한간질학

신하영 이두재 조경주등 Kainate 유발간질모델 - - 크레질바이올렛 (cresyl violet) 염색 Kainate 유발간질모델에서의뇌파검사 대상과방법 산소음이온라디칼 (superoxide anion radical, O.- 2 ) 검출 in situ- - 대한간질학 KISEP Original Article 대한간질학회지 2004;8(2):108 8(2):108-115 115 Kainate 유발간질모델에서 Apurinic/Apyrimidinic Endonuclease 의빠른소실과이에따른세포고사 신하영 이두재 조경주 김미애 이용현 허경 김경환 이병인 Rapid Loss of Apurinic/Apyrimidinic Endonuclease