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1 Journal of Nutrition and Health (J Nutr Health) 2015; 48(5): 451 ~ pissn / eissn Note 화학적저산소증이유도하는뇌신경세포손상에있어서미성숙진귤과피발효추출물의보호효과 고운철 이선령 제주대학교생물학과 Anti-apoptotic effect of fermented Citrus sunki peel extract on chemical hypoxiainduced neuronal injury Ko, Woon Chul Lee, Sun Ryung Department of Biology, Jeju National University, Jeju 63243, Korea ABSTRACT Purpose: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimer's disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride (CoCl 2 )-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. Methods: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2',7'- dichlorofluorescin diacetate (DCF-DA) and 4',6-diamidino-2-phenylindole (DAPI) staining. Results: Exposure to CoCl 2, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the CoCl 2- induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. Conclusion: Based on these data, fermented C. sunki peel extract might have a protective effect against CoCl 2- induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis. KEY WORDS: Citrus sunki, apoptosis, hypoxia, stroke, neuronal cells 서론 의료과학기술의발달로인해노인인구가전체의 20% 이상을차지하는고령화사회로접어들면서퇴행성신경계질환 ( 알츠하이머병, 파킨슨병 ) 이나혈관성뇌질환 ( 뇌졸중 ) 으로고통받는사람이증가하고있다. 현재우리나라에서사망원인이 2위를차지할정도로위험성이높은뇌졸중 (stroke) 은뇌에혈액을공급하는혈관이막히거나터져서생기는뇌손상으로여러합병증의결과물로서도나타날수있다. 1,2 이는뇌혈관의막힘으로오는뇌경색 (ischemic stroke) 과뇌파열에의한뇌출혈 (hemorrhagic stroke) 로구분되며뇌에혈액공급이원활하지않아유발되는저산소환경에의해특이적으로손상받기쉬운해마나대뇌피질의신경세포가사멸하게되고이로인한심각한뇌신경손상은완전한회복을어렵게한다. 3 뇌졸중치료는주로 tissue plasminogen activator (tpa) 를이용하는혈전용해치료법 (thrombolytic therapy) 이효과적인것으로알려져있으나 tpa의사용은 3시간이내에이루어져야되고심각한출혈 (intracerebral hemorrhage) 이나부종 (edema) 같은부작용을야기한다는점에서매우제한적이 Received: August 6, 2015 / Revised: August 24, 2015 / Accepted: September 3, 2015 To whom correspondence should be addressed. tel: , srlee@jejunu.ac.kr 2015 The Korean Nutrition Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 452 / 진귤과피발효추출물의세포사멸보호효과 기때문에 4 해마나대뇌피질을구성하는신경세포의손상을보호하는연구에관심이집중되고있다. 5-7 최근에는여러신경성뇌질환을유발하는모델에서 phytochemical들의효능에대한많은연구가진행되고있으며이들은신경세포보호효능 8 뿐아니라저해된인지와운동기능향상 9 및기억력을회복 10 시키는효능을나타내는것으로보고되어있다. 예로부터한방이나민간요법의약재로사용하고있는진피는제주재래감귤종의하나인진귤 (Citrus sunki Hort. ex Tanaka) 의과피이다. 진귤을포함한감귤은 citrus 속의성분및활성분석을통해과육보다는과피의중요성이강조되었고수확시기 11,12 와발효유무 13 에따라생리활성성분의함유량과효능에있어서차이를보이고있다. 미성숙진귤과피는항염, 11 항비만및지방간개선작용 14,15 에효과적인것으로알려져있으나신경계뇌혈관질환과의연관성에대한연구는미비한실정이다. 따라서본연구는진귤과피가뇌경색과같은혈관성뇌질환에미치는효능을알아보고자수행되었으며뇌경색과가장유사한환경을이루는화학적저산소환경의세포모델을이용하여뇌신경세포손상에따른미성숙진귤과피발효추출물의보호효과를확인하였다. 연구방법 시료본실험에사용된미성숙진귤과피발효추출물은 Cui 등 15 의논문에제시된바와같이추출되었다. 간단히기술하면, 미성숙진귤과피를건조하여분쇄한분말을 80% 에탄올로실온에서추출한후회전농축기로에탄올을제거하고남은수층시료를동결건조하였다. 5% 설탕이첨가된동량의물을첨가하여멸균한후 5% Lactobacillus plantarum ATCC 8014와 5% Saccharomyces cerevisiae IFO 0203으로접종하여 38 o C에서 7일간혐기배양하였고급속냉동후동결건조하여사용하였다. 세포배양 Human neuroblastoma SH-SY5Y 세포는한국세포주은행에서분양받아사용하였다. 세포는 10% fetal bovine serum (FBS), 100 unit/ml penicillin/streptomycin, 20 mm HEPES가포함된 Dulbecco's modified eagle's medium (DMEM, Gibco, Carlsbad, CA, USA) 배지를사용하여 37 o C, 5% CO 2 조건하에서배양하였다. 실험을위해세포는 48시간배양하고 FBS가포함되지않은배지로교환하여 20시간안정화시킨후 CoCl 2 또는추출물을처 리하였다. 세포생존율측정 24 well plate ( cell/ml) 에분주된세포에 CoCl 2 또는추출물을처리하여 24시간배양한후세포를모두수거하여 0.4% trypan blue와섞어 hemocytometer를이용하여살아있는세포와죽은세포를계수하였다. 생존율은아래의식을이용하여산출하였다. 세포생존율 (%) = 살아있는세포수 /( 살아있는세포수 + 죽은세포수 ) 100 세포내 ROS 측정 96 well plate에 CoCl 2 또는추출물이농도별로처리된세포 ( cell/ml) 를준비하여 10 µg/ml의 2',7'-dichlorofluorescin diacetate (DCF-DA) 를 1시간동안처리한후 fluorescence spectrometer (excitation : 485 nm, emission : 520 nm) 에서생성되는형광으로세포내 reactive oxygen species (ROS) 를측정하였다. 핵의형태학적변화관찰 CoCl 2 또는농도별추출물이 24시간처리된세포 ( cell/ml) 에아세톤과메탄올을 1:1로혼합한용액으로 5분동안처리하여고정시킨후 Phosphate buffered saline (PBS) 로수세하였다. DNA에특이적으로결합하는형광색소인 4',6-diamidino-2-phenylindole (DAPI, Sigma, USA) 용액 (3 µg/ml) 을가하여상온에서 30분간반응한후형광현미경 (IX-71, Olympus, Japan) 하에서관찰한후 apoptotic body를가지는세포를계수화하였다. 세포질과미토콘드리아의분획세포질과미토콘드리아분획의단백질을분리하기위해서 mitochondial isolation kit (Pierce, Rockford, IL, USA) 를이용하였다. CoCl 2 또는추출물이 24시간처리된세포를 protease inhibitor가포함된 Sol A (cytoplasmic isolation buffer) 로파쇄하여 750 g에서 10분간원심분리하여얻어진상층액을 10,000 g에서 15분간재원심분리한후상층액을세포질분획으로사용하였다. 남겨진침전물에 protease inhibitor가포함된 Sol B (mitochondial isolation buffer) 를첨가하여현탁시킨후미토콘드리아분획단백질을분리하였고 Bradford protein assay를통해정량하였다. Western blot PBS 수세후수확한세포에 protease inhibitor 가포함된

3 Journal of Nutrition and Health (J Nutr Health) 2015; 48(5): 451 ~ 456 / 453 lysis buffer (20 mm Tris, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100) 를넣어 30분간반응한후 14,000 rpm에서 15분간원심분리하여단백질을추출하였다. 단백질정량을통해얻어진 30 µg의단백질은 10% polyacrylamide gel에서전기영동하여 nitrocellulose membrane에전이시킨후단백질발현분석을위해 antimouse PARP, anti-mouse cytochrome c (Santa Cruz Biotech, Dallas, TA, USA), anti-rabbit cleaved caspase-3, -7, -9 (Cell signaling, Danvers, MA, USA), anti-mouse beta-actin (Sigma, St. Louis, MO, USA) 항체를이용하여반응하였고 enhanced chemiluminescence kit (ECL) 방법으로확인하였다. 통계처리본실험의결과는 3회반복하여평균 ± 표준편차로표시하였다. 통계처리는 student s t-test에의해수행되었으며 p < 0.05인경우유의한것으로판정하였다. 결과및고찰 화학적저산소증에의한신경세포사멸효과저산소증이유도하는신경세포사멸은뇌경색과같은혈관성뇌손상을야기하는데있어서매우중요한요인이다. 본연구에서사용된 CoCl 2 는산화적스트레스에의해저산소환경을일으킴으로써뇌경색과유사한환경을만드는모델로알려져있다. 16 다양한농도로처리된 CoCl 2 가신경세포인 SH-SY5Y 세포사멸에미치는효과는 Fig. 1에제시하였다. 먼저, trypan blue exclusion으로 CoCl 2 에의한세포생존율을확인한결과, 100 µm 농도에서는세포사멸효과가거의나타나지않았고 55 ± 12% 의생존율을보인 300 µm 처리군에서유의적으로세포생존율이감소하기시작하여 500 µm의농도에서는 22 ± 5% 의낮은생존율을나타내었다 (Fig. 1A). 세포의사멸은활성화된 caspase-3와 poly (ADP-ribose) polymerase (PARP) 의분절화가유도되고세포의응축이나핵의분해등구조적인변화단계를거쳐조절된다. 17 CoCl 2 에의한생존율저해가세포사멸조절단백질에미치는영향을확인하기위해 caspase-3 및 PARP 발현양상을분석하였다. 대조군에비해 300 µm과 500 µm의 CoCl 2 처리군에서핵내에존재하는 PARP의분절현상이관찰되었고세포사멸이진행될수록분절화된형태의활성형 caspase-3의발현이증가되는것을확인할수있었다 (Fig. 1B). 이러한결과는 CoCl 2 가유도하는세포사멸은 caspase-3 의존성경로를통해조절되고있음을보여주는 Fig. 1. Induction of caspase-3 mediated cell death. Effects of CoCl 2 on the cell viability by trypan blue exclusion (A) and expression of apoptosis-related proteins by western blot analysis (B). Data are represented as mean ± SD of three independent experiments. * p < 0.05 vs control. C: control 것이다. 진귤과피발효추출물의신경세포사멸보호효과진귤과피는다른식물에서는발견되지않는감귤특유의플라보노이드로인해다양한생리활성을가진다. 진귤과피에는 tangeretin, nobiletin, hesperidin, rutin, sinensetin 순으로많은플라보노이드성분을가지고있으며성숙되는정도에따라함유량이크게변하여성숙과에비하여미숙과에서 hesperidin을제외한모든플라본성분이 3~5배정도높은함유량을가짐으로써미성숙과피가항염활성을가지는것으로보고되었다. 11 감귤과피에서추출한각각의플라보노이드성분의미미한항신경염증의효능은이들이동시에작용하였을경우시너지효과를발휘하여신경염증을저해하는효과를나타내었다. 18 특히, 미성숙진귤과피에서함유량이높은것으로알려진 nobiletin의경우뇌경색동물모델에서뇌손상을억제 8 하고파킨슨및알츠하이머유발모델에서인지력과기억력을향상 9,10 시키는것으로보고되었다. 따라서플라보노이드의보고인진귤과피가뇌손상에관련된질환제어물질로서기능을수행하는지알아보고자미성숙진귤과피발효추출물을이용하여뇌신경보호효과를확인하였다. CoCl 2 가처리된저산소환경세포사멸모델에서진귤과피발효추출물을 30분전처리하여세포생존율을조사한결과, 진귤과피발

4 454 / 진귤과피발효추출물의세포사멸보호효과 Fig. 2. Protective Effect of fermented C. sunki peel extract on the neurotoxicity (A), activities of caspases and PARP (B), formation of apoptotic body (C) in CoCl 2 -treated SH-SY5Y cells. Data are presented as mean ± SD of three independent experiments. * p < 0.05 vs control. # p < 0.05 vs CoCl 2 -treated cells. FCS: Fermented C. sunki peel extract 효추출물이 100~200 µg/ml 농도로처리된세포의생존율은약 69 ± 11% 로나타났으며이는약 46 ± 8% 의생존율을보인 300 µm CoCl 2 단독처리군과비교하였을경우유의성있는결과를보여주었다 (Fig. 2A). 이러한신경세포사멸보호효과는세포사멸관련단백질들의발현양상을확인한결과에서도동일하게나타났다. 생존율이저해된 CoCl 2 단독처리군에비해진귤과피발효추출물처리군에서 PARP의분절화와활성화된 cleaved casase-3, -7, -9 의발현이현저히감소하는양상을보였다 (Fig. 2B). 진귤과피발효추출물의신경세포사멸보호효과를검증하기위해 DAPI staining을이용해세포사멸진행에따른염색체의응축이나 apoptotic body를관찰하였다. Fig. 2C에제시한바와같이 apoptotic body를가지는세포의비율이 23 ± 1.2% 인 CoCl 2 단독처리군에비해진귤과피발효추출물처리군에서는 7 ± 2.0% 로현저히감소하였다. 이러한결과는진귤과피발효추출물이 CoCl 2 가유도하는 caspase- 3 의존성신경세포사멸에대해보호효과가있음을보여주는것으로 epigallocatechin gallate (EGCG), 19 에스트라디올, 20 허브추출물 21 에의해나타나는다양한신경세포손상보호효과들과비교해볼때일정수준이상의보호 효능을발휘하는것으로생각된다. 세포의사멸기전은미토콘드리아매개성경로와사멸수용체매개성경로에의해조절될수있다. 미토콘드리아에의해중재되는세포사멸은 mitochondrial transmembrane potential (MMP, ΔΨm) 를변화시키거나미토콘드리아의시토크롬 c가세포질로의전이에영향을미치는일련의미토콘드리아의 dysfuction을일으킴으로써 caspase-9/-3의활성화와같은 caspase cascade를통해조절된다. 17 CoCl 2 에의해유도되는저산소환경은 HIF-1α 유도에의해 ROS 형성을야기하기도하고 22 활성화된 p38 MAPK signaling 를통한 ROS의생성억제는저산소환경에서의염증을보호하기도하며 16 anti-apoptotic protein인 Bcl-2 family와 proapoptotic protein인 Bax/Bid의상호조절을통해신경세포의사멸을조절한다. 19 따라서진귤과피발효추출물의세포사멸보호효과가산화적스트레스에의한 ROS 생성기전및미토콘드리아매개성경로에의해조절되는지확인하기위해세포내생성되는 ROS의양과 Cytochrome c 의전이양상을확인하였다. 먼저, DCF-DA를이용해생성되는 ROS의양을측정한결과, 대조군에비해 CoCl 2 에의한저산소환경은세포내생성되는 ROS의양을현저히증

5 Journal of Nutrition and Health (J Nutr Health) 2015; 48(5): 451 ~ 456 / 455 가시켰고이는 CoCl 2 처리가산화적스트레스로작용하여 ROS 증가에의해세포사멸에이르게된다는기존의연구와일치하였다. 19,20 반면, 100 µg/ml의진귤과피발효추출물처리에의한 ROS 생성량은저산소환경에의한생성량에비해약 51.8% 로감소되어유의성있는효과를보였으며이는 ROS scavenger인 N-acetyl-cystein (NAC) 를처리하였을경우나타나는효과와유사하였다 (Fig. 3A). Caspase cascade를통해세포사멸을억제하는진귤과피발효추출물이미토콘드리아의존성경로를통해조절되는지검증하기위해미토콘드리아및세포질로분획을나누어 cytochrome c가전이되는양상을비교분석하였다. 세포사멸이유도된 CoCl 2 처리군은높은발현량의 cytochrome c가세포질분획에서관찰된반면, 진귤과피발효추출물의처리는미토콘드리아분획에서 cytochrome c의발현양이높게나타나는것으로보아진귤과피발효추출물은미토콘드리아에서세포질로의 cytochrome c의방출을억제하였다 (Fig. 3B). 이상의결과는진귤과피발효추출물이 CoCl 2 에의한 ROS 생성을저해하여 cytochrome c의방출을억제함으로서 caspase-3 불활성화을유도하여세포사멸의보호효과를발휘하고있음을보여주는것으로이전연구에서보고한저산소환경이유도하는신경세포세포모델에서플라보노이드성분에의해조절되는작용기전과유사한결과를보여주었다. 녹차에다량포함되어있는 EGCG 는 MMP 와 cytochrome c 의변화및 Bcl-2/Bax 의발현조절을통해 caspase-3 의존성세포사멸을억제하였고 19 또다른플라보노이드의하나인 scutellarin의경우, ROS 저해및 p38 MAP kinase의인산화억제를통해 caspase-3 활성을조절함으로써세포사멸을조절하였다. 23 본연구결과역시진귤과피발효추출물이 ROS 저해및 cytochrome c의변화에의한 caspase cascade 경로에의해조절되는것으로보아식물이가지는플라보노이드성분의작용기전과마찬가지로미성숙과피에포함된다량의플라보노이드성분이미토콘드리아의존성경로를통해저산소환경에대한세포사멸을억제하는데중요한역할을수행했을것으로추측된다. 본연구에사용된미성숙진귤과피에는플라보노이드중 tangeretin과 nobiletin의성분이높은것으로알려져있고 11 최근보고된뇌경색동물모델에서 nobiletin의뇌신경손상보호효능이뛰어난것으로보아 8 미성숙진귤과피가함유하고있는보다많은플라보노이드성분의시너지효과로인해저산소성세포사멸을보호하는데있어서유의적인결과가도출되었을것으로사료된다. 그러나본연구는미토콘드리아가매개하는사멸기전에관련된일부분만을살펴본것으로 Bcl-2 family와같은또다른신호전달경로와의상호조절및사멸수용체를통한조절기전은추후계속연구되어야할부분이다. 혈액흐름의감소로인해일어나는뇌경색은결국뇌손상으로이어지게되고이러한손상은산화적인스트레스나염증, glutamate nerotoxicity 및부종에의해일어나는복합적인현상이다. 뇌손상의저해는뇌졸중을포함한뇌질환의좋은예후를기대할수있고뇌졸중환자들의삶을회복시킬수있다는점에서매우중요한일이다. 본연구결과에서보여준진귤과피발효추출물의보호효과는뇌졸중유발원인저산소증에의한뇌신경손상을억제할수있는 phytochemical로서향후뇌질환의예방및치료를위해활용이가능한잠재적후보로서의가능성을제시하였다. 그러나보다더정확한효능검증을위해동물모델에서의적용및뇌질환증상을완화시키는약물과의비교분석을통한지속적인연구가진행되어야할것으로판단된다. References Fig. 3. Effect of fermented C. sunki peel extract on the CoCl 2 - induced ROS generation (A) and release of mitochondrial cytochrome c to cytosol (B). Data are represented as mean ± SD of three independent experiments. * p < 0.05 vs control. # p < 0.05 vs CoCl 2 -treated cells. FCS: Fermented C. sunki peel extract, NAC: N-acetyl-cystein, Cyt c: Cytochrome c 1. World Health Organization (CH). The top 10 causes of death [Internet]. Geneva: World Health Organization; 2013 [updated 2014 May; cited 2015 Mar 11]. Available from: int/mediacentre/factsheets/fs310/en/. 2. Statistics Korea Cause of death statistics [Internet]. Daejeon: Statistics Korea; 2014 [cited 2014 Sep 23]. Available from: kostat.go.kr/portal/korea/kor_nw/2/6/2/index.board.

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Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,