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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 44(4), 532~539(15) RANKL 에의해유도되는파골세포분화에대한시금치추출물의영향 김동규 김미혜 강민정 신정혜 ( 재 ) 남해마늘연구소 Effect of Spinach Extract on RANKL-Mediated Osteoclast Differentiation Dong-Gyu Kim, Mi-Hye Kim, Min Jung Kang, and Jung Hye Shin Namhae Garlic Research Institute ABSTRACT Inhibition of osteoclast differentiation is the most important target for prevention of inflammatory bone resorption and bone diseases. Here, we investigated the effect of spinach ethanol extract on osteoclast differentiation in RAW264.7 cells. Spinach was extracted with ethanol at a concentration ranging from to % (, 25, 5, 75, and % ethanol). Inhibitory effects of receptor activator of NF-κB ligan (RANKL)-induced osteoclast differentiation were evaluated using tartrate-resistant acid phosphatase (TRAP) stain assay. The most effective eanol concentration for osteoclast differentiation was %. Spinach extract (% ethanol) suppressed RANKL-induced osteoclast differentiation and TRAP activity. Spinach extract (% ethanol) also suppressed expression of osteoclast differentiationrelated marker genes (NFATc1, c-fos, cathepsin K, and TRAP) and down-regulated RANKL-induced NF-κB and ERK phosphorylation during osteoclast differentiation. Taken together, our results suggest that spinach extract is effective against reducing osteoclast differentiation through the NF-κB-mediated pathway. Key words: spinach, osteoclast, tartrate-resistant acid phosphatase (TRAP), RANKL, c-fos 서 우리몸을지지하는뼈는골격유지기능과칼슘, 인등다양한무기질의저장소역할을담당하는데, 단단한표면적이미지와는달리건강한남녀의경우매년전체골량의 1% 가파괴되고다시만들어진다 (1,2). 뼈는재형성과정 (bone remodeling) 을거쳐건강한뼈가유지되고각종면역세포의발달과성숙에중요한골수구조를형성한다 (3). 이과정에서뼈를파괴하는파골세포 (osteoclast) 와뼈를형성하는조골세포 (osteoblast) 는각각조혈모세포및간엽줄기세포에서발생하여뼈흡착및형성을담당하므로두세포의상호밸런스는건강한골격계형성에필수적이다 (4). 뼈조직에서유일하게뼈의파괴를담당하는파골세포는단핵구 / 대식세포계통의세포이며, 파골세포의분화는골다공증과같은뼈질환에중요하게작용한다 (5). 여러조직에존재하는대식전구세포로부터시험관내에서특정 cytokine인 receptor activator of NF-κB ligand(rankl, RANK ligand) 와 macrophage colony stimulating factor(m-csf) 등을사용하여분화시켜파골세포의생성이가능하다 (6,7). Received 17 December 14; Accepted 4 March 15 Corresponding author: Jung-Hye Shin, Namhae Garlic Research Institute, Gyeongnam , Korea whanbee@hanmail.net, Phone: 론 파골세포는특징적으로 tartrate-resistant acid phosphatase(trap) 와풍부한칼시토닌수용체를가지며실제적으로뼈를흡수할때는산의생성이활발하고 actin ring을형성하며골기질을흡수한다 (8). RANKL은주로뼈모세포에서발현되며, 1α,25-dihydroxyvitamin D3(Vit D3), prostaglandin E2(PGE2), interleukin 1(IL-1), IL-6, TNFα 등에의해유도된다 (9-12). 최근관절질환연구에 RANK와 RANKL이파골세포분화에중요한역할을하는것으로알려졌고 RANKL signaling pathway가빠르게밝혀지고있다 (13). RANKL은 TNF family의일원으로다른 adapter molecule을 recruit 하여파골세포분화에서 NF-κB와 MAPKs(mitogen-activated kinase) 가활성화한다 (14). RANK는 c-fos의 induction을통한 AP-1(activating protein-1) 을활성화하는데 NF-κB 와 AP-1은파골세포분화의결정적인조절인자인 NFATc1 (nuclear factor of activated T cell, cytoplasmic 1) 의초기발현을조절한다 (15,16). RANKL에의해활성화된 NFκB는이미존재하고있던 NFATc2와함께 NFATc1 promoter에결합하여 NFATc1의초기발현을주도하며, 이후 NFATc1은다른전사인자와함께 cathepsin K, TRAP, OSCAR(osteoclast-associated receptor), β3-integrin 등의파골세포특이적인유전자의발현을조절한다 (17-). 시금치 (spinach, Spinacia olerecea L.) 는페르시아가원산인명아주과의일년생작물로우리나라에는 15년대에전래되어연중일반가정에서상용하는채소이다 (21). 시금

2 RANKL 에의해유도되는파골세포분화에대한시금치추출물의영향 533 Table 1. Extraction yield of Spinacia olerecea L. from different extraction solvent Extraction solvent ratio (water : ethanol) : 75:25 5:5 25:75 : Yield (%) 치는비타민 A의전구체인카로틴과비타민 C 그리고칼슘, 인, 철분등의무기질을다량함유하고있으며, 클로로필을많이포함하는녹황색엽채류로식이섬유가부드럽고소화가잘되어국민의건강식품으로서꾸준히섭취되고있다 (22). 또한생리학적으로항산화, 항염증등의다양한효과를가지고있다 (23). 최근의보고에따르면건장한 대남성을대상으로한실험에서급격한운동후발생하는산화적스트레스표지인자인 protein carbonyl(pc) 과 uric acid의발현이시금치섭취로감소된다는보고가있었고, 또한고지혈증을유발시킨실험동물에시금치를급이한결과고지방과콜레스테롤에의해유발된산화적스트레스가감소한다는보고도있었다 (24,25). 시금치의생리학적효과에대해서는일정부분연구가이루어져있지만주로부식으로이용되어왔기때문에다양한분야에서의연구는부족한실정이다. 시금치는채소중에서비교적칼슘의함량이높아뼈건강에유용한식품으로인지되고있는데칼슘흡수를방해하는수산이들어있어섭취량의 5% 정도만이체내로흡수되어이용률저하가우려되는반면, 끓는물에데치는작업을통해일정부분의수산을제거할수있기때문에조리된시금치를섭취할경우칼슘의흡수를우려할정도는아니라고보고되어있다 (26). 또한시금치에많이함유된루테인을포함하는 carotenoid 성분들은강력한항산화효과를통해파골세포분화를억제한다는연구결과가있었다 (27,28). 시금치의뼈건강과관련한연구는시금치자체에함유되어있는칼슘이나철분과같은미네랄성분과더불어다른유효성분들도영향을미치므로이와관련한기작을밝히는연구도다양하게추진되어야한다. 본연구에서는이러한연구의일환으로시금치추출물이파골세포의분화억제효능을가진다는것을몇가지실험방법을통해검증하고, 이러한연구결과를토대로시금치가뼈건강에미치는영향을확인하기위한기초자료를얻고자하였다. 재료및방법시료및추출물의제조시금치는경남남해군에서노지재배된것을취하여흐르는물로세척한후자연건조한다음뿌리부분을제거하고동결건조하였다. 동결건조된시금치는분쇄기 (HMF-345S, Hanil, Seoul, Korea) 로분쇄한후 mesh 표준망체로체질하여추출물제조용시료로사용하였다. 시금치동결건조분말에물, 에탄올및이들을비율별로혼합 (75:25, 5:5, 25:75) 한용매를각각 배 (w/v) 씩첨가후상온에서 3분간 2회진탕추출하였다. 추출액을여과지 (Whatman No 2, Whatman International Ltd., Maidstone, UK) 로여과한여액각각을회전식감압농축기 (N- 111S-W, EYELA, Tokyo, Japan) 로농축하여추출용매를모두제거하였다. 완전건조된추출물은 dimethylsulfoxide(dmso, Sigma-Aldrich Co., St. Louis, MO, USA) 에재용해시켜 -7 C 에보관하면서실험에사용하였다. 각 용매별추출수율은추출전시금치분말의무게대비완전 건고된추출물의무게비로부터산출하여 Table 1 에제시하 였다. 세포배양및세포독성평가 실험에사용된마우스의대식세포주인 RAW264.7 세포 는한국세포주은행 (KCLB, Seoul, Korea) 에서분양받았으 며, 세포배양을위해 1% fetal bovine serum(fbs) 과 1% penicillin-streptomycin 을포함하는 Dulbecco s modified Eagle s media(dmem; Gibco BRL, Gaithersburg, MD, USA) 를사용하여 CO 2 incubator(37 C, 5% CO 2) 에서 배양하였다. 시금치추출물의세포에대한독성측정은 3- (4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide(mtt) 환원방법을이용하여측정하였다. 세포를 96 well-plate 에 well 당 개가되도록분주하고 24 시 간부착시킨후, 완전건조된용매별시금치추출물을 dimethyl sulfoxide(dmso; Sigma-Aldrich Co.) 에일정한 농도로희석하여, 5, μg/ml 가되도록세포에처리한 다음 3 분후 1 μg/ml lipopolysaccharide(lps, Sigma- Aldrich Co.) 를처리하여 24 시간동안배양하였다. 이후시 료를포함하는배지를제거한후 serum-free 배지와 5 mg/ ml MTT 용액을첨가하여 37 C 에서 2 시간더배양하고 DMSO 를분주하여 sonication 하고 1 분간교반하여용출 시킨뒤 57 nm 에서흡광도를측정해세포생존율을구하였 다. 세포생존율은 LPS 처리군에대한백분율로나타내었다. 파골세포분화유도 마우스유래대식세포주 RAW264.7(KCLB) 을파골전구 세포로하여 1% FBS 와 1% penicillin-streptomycin 이첨 가된 DMEM(Gibco BRL) 을배양액으로 5% CO 2 incubator 를이용하여 37 C 에서계대배양하면서실험에사용하였다. RAW264.7 세포를 culture 용 well plate 에적정수량을분 주한후 4~6 시간배양하여세포가 well 에부착되면배지를 제거하고 1% FBS 가첨가된 α-mem 배지에분화인자인 RANKL 5 ng/ml 와남해용매별시금치추출물을, 5, μg/ml 농도가되도록혼합한배양액을분주하여 2 일에 한번씩배지를교환하면서 4 일또는 7 일간배양하였다.

3 534 김동규 김미혜 강민정 신정혜 Reverse transcription-polymerase chain reaction(rt- PCR) RANKL은파골세포분화단계의주요전사인자인 NFATc1 의분화를유도하므로시금치 % 에탄올추출물이 RANKL에유도된 NFATc1과 c-fos에미치는영향을 RT-PCR로관찰하였다. 그리고파골세포가분화하여기능할때발현되는효소인 TRAP와 cathepsin K의발현에미치는영향을 RT-PCR로관찰하였다. 실험을위해서배양된각각의세포에서 ReliaPrep RNA Cell Miniprep System kit (Promega Corp., Madison, WI, USA) 을이용하여 total RNA를분리하였다. 1-Thioglycerol을포함하는 BL buffer를 plate에첨가하여회수한 cell lysate를 mini-column 에옮긴뒤 14, g에서 3초간원심분리하여 column에흡착시킨다음 DNase와 MnCl 2 혼합액을 column에첨가하여 15분간 incubation 함으로써 DNA를제거하였다. 이후 column wash solution과 RNA wash solution으로 column 을세척하고최종적으로 nuclease free water를첨가하여원심분리를실시해 total RNA 샘플을획득하였다. RNA의정량을위해서 QuantiFlour RNA system(promega Corp.) 을이용하였고, 분리된 RNA로부터 1 μg을취해 GoScript reverse transcription system을이용하여 cdna로합성하였다. 합성된 cdna는 Table 2에서나타낸 primer를이용하여 PCR 하였다. 증폭된 cdna는 1% agarose gel에서분리하였고 gene stain detection solution을이용하여이미지장비 (E-Graph, ATTO, Tokyo, Japan) 로관찰하였다. Western blot 분석 RANKL에의한신호전달과정을관찰하기위해 RAW 세포를 6-well plate에 well당 개가되도록분주하고 1% FBS가포함된 DMEM(Gibco BRL) 을배양액으로 6시간배양한다음, 1% FBS를포함하는 α-mem 에, 5, μg/ml 농도로희석한시금치에탄올추출물과 5 ng/ml의 RANKL을 24시간처리하였다. 배양이끝난세포는 protease inhibitor와 phenylmethane-sulfonyl fluoride를첨가한 radioimmunoprecipitation assay 완충액으로용해하고 13, rpm, 4 C에서 분간원심분리하여얻은상층액을단백질시료로삼았다. Lysate 중의단백 질은 BCA protein assay kit(pierce, Rockford, IL, USA) 을이용하여 albumin 검량곡선에대입하여정량하였고 3 μg의시료를 SDS-PAGE로분리하여 PVDF막에옮긴후특정항체를이용하여반응시키고이미지장비 (Ez-Capture Ⅱ, ATTO, Tokyo, Japan) 를통해단백질밴드를관찰하였다. 통계처리각각의실험군은 3개이상수행하였고결과값은 SPSS 12. package(spss Inc., Chicago, IL, USA) 프로그램을사용하여평균값과표준편차를계산하였다. 모든실험은 3 회이상반복하여동일한실험결과를얻은경우실험결과로사용하였다. 결과의통계는 Student s t-test를이용하여분석하였고 P 값이.5 이하인경우통계적으로유의한것으로간주하였다. 결과및고찰시금치추출물의세포독성평가파골세포는뼈조직에서유일하게뼈의파괴를담당하는단핵구대식세포계통의세포로 RANKL과같은특정 cytokine에의해여러조직에존재하는단핵구대식전구세포로부터분화되어생성이가능하다 (5). 본실험에앞서시금치추출물과 RANKL의세포내처리가세포독성을유발하는지확인하기위해 RAW264.7 세포에 5 ng/ml RANKL과 5 μg/ml 및 μg/ml의시금치추출물을처리하여 7일간배양한후 MTT 방법으로세포독성을평가하였다. 실험결과시금치물추출물과 25~% 에탄올추출물을처리한세포모두에서세포독성을유발하지않았다 (Fig. 1). 파골세포분화에미치는시금치추출물의영향시금치추출물이파골세포분화에미치는영향을평가하기위해서 TRAP 염색을실시하였다. TRAP 염색을하게되면 RANKL 처리를하지않은대조군은 RAW264.7 세포가구모양을유지한채증식하며, TRAP 염색에서도음성반응을보여연한갈색이나황토색을띤다. 이에반해 RANKL을처리한세포에서는진한암갈색내지적갈색으로염색된 TRAP(+) 다핵세포가관찰된다 (29). Table 2. The primer information of gene related to osteoclasts differentiation Primer Primer sequence c-fos sense c-fos antisense NFATc1 sense NFATc1 antisense TRAP sense TRAP antisense Cathepsin K sense Cathepsin K antisense HPRT sense HPRT antisense 5'-CTG GTG CAG CCC ACT CTG GTC-3' 5'-CTT TCA GCA GAT TGG CAA TCT C-3' 5'-CAA CGC CCT GAC CAC CGA TAG-3' 5'-GGC TGC CTT CCG TCT CAT AGT-3' 5'-ACT TCC CCA GCC CTT ACT AC-3' 5'-TCA GCA CAT AGC CCA CAC CG-3' 5'-CTG AAG ATG CTT TCC CAT ATG TGG G-3' 5'-GCA GGC GTT GTT CTT ATT CCG AGC-3' 5'-GTA ATG ATC AGT CAA CGG GGA C-3' 5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'

4 RANKL 에의해유도되는파골세포분화에대한시금치추출물의영향 535 Cell viability (%). 1 Con RANKL DW 25% EtOH 5% EtOH 75% EtOH % EtOH RANKL+spinach extracts (μg/ml) 25%, 5% 및 75% 에탄올추출물처리군에서 TRAP(+) 다핵세포숫자는각각 RANKL 단독처리군대비 31%, 42% 및 % 로유의성있게억제되었으며, % 에탄올추출물에서대조군에비해파골세포분화가 9% 이상억제되었다. 반면시금치물추출물은 RANKL 처리군과유사한수준의파골세포생성을보여분화억제활성을나타내지않았다 (Fig. 2B). 이러한결과를통해시금치에탄올추출물이파골세포의분화를억제하는역할을수행함을확인하였으며, % 에탄올추출물이가장높은파골세포의분화저해효과가있는것을확인하였다. Fig. 1. The effect of spinach extracts on cell viability of RAW cells treated with RANKL (7 days). Each bar represents the mean±sd of three repeated experiments. A RANKL 과함께시금치추출물들을처리한후 2 일에한 번씩배지를교환하며 7 일째에이미지데이터를얻어파골 세포의분화를확인하였다 (Fig. 2A). Positive control 로사 용된 RANKL 처리세포군에서는 3 개이상의핵을가지는 TRAP(+) 다핵세포로분화가유도되었고, RANKL 과시금 치에탄올추출물을처리한세포군에서는추출용매의에탄 올비율이증가할수록파골세포로의분화가억제되었다. B TRAP (+) cells ( 3 nuclear). 1 ** RANKL DW 25% EtOH 5% EtOH 75% EtOH % EtOH RANKL+spinach extracts (μg/ml) Fig. 2. Inhibition of RANKL-induced osteoclastogenesis by spinach extract. (A) The effect of μg/ml spinach extracts on TRAP staining of RAW264.7 cells treated with RANKL (7 days). (B) Each bar represents the mean±sd of three repeated experiments. ** P<.1, P<.1 compared to the RANKL treatment. 시금치에탄올추출물의파골세포분화저해활성검증 % 에탄올추출물이파골세포분화저해활성이있음을확인하였기에이후실험은시금치 % 에탄올추출물을농도별로처리하여 TRAP(+) 다핵세포형성및효소활성을측정하였다. RANKL과함께시금치 % 에탄올추출물을 25, 5, 75 그리고 μg/ml로각각처리하여 7일간배양한결과는 Fig. 3A와같다. 무처리세포군에비해 RANKL 처리군에서는파골세포분화지표인 TRAP(+) 반응의파골세포들이짙은색으로염색되었다. 광학현미경을통해융합되어다핵을지닌파골세포의형성을확인한결과 25~ μg/ml 농도의시금치 % 에탄올추출물처리시분화를억제하여 TRAP(+) 반응특유의짙은색을띠는세포의수가현저히감소되었다. 또한 TRAP(+) 다핵세포수를계수한결과 RANKL 처리에의한 TRAP(+) 다핵세포숫자대비 25~ μg/ml의농도별시금치 % 에탄올추출물은파골세포형성을 % 이상유의하게감소시켰다 (Fig. 3B). 파골세포는특징적으로주석산에내성을가진 TRAP 효소와풍부한칼시토닌수용체를가지며뼈의흡수작용에관여할때는산의생성이활발하게일어나뼈에흡착하여골기질을흡수하며, 이러한 TRAP 효소는파골세포의세포화학적표지효소 (marker enzyme) 로파골세포가골흡수작용을할때분비가증가한다고알려져있다 (8). 이를이용하여파골분화정도를측정한결과 5, 1, 25 μg/ml 농도의시금치 % 에탄올추출물처리시에는효소활성의저해가나타나지않은반면, 5과 μg/ml 농도에서는각각 38% 와 44% 의유의한 TRAP 효소억제활성을나타내었다 (Fig. 3C). 이러한실험결과를통해시금치에탄올추출물이농도의존적으로 TRAP(+) 다핵세포의수뿐만아니라 TRAP 효소활성을감소시켜파골세포의분화를저해함을검증하였다. 시금치에탄올추출물의파골세포분화억제기작 RANKL은 RANK와의결합을통해파골세포의분화에필수적인유전자발현을유도한다 (18). 파골세포의운명에주요인자로알려진 NFATc1, c-fos 그리고 cathepsin K의발현을확인하기위해 RT-PCR을수행하였고, 각각의발현

536 김동규 김미혜 강민정 신정혜 A B C TRAP (+) cells ( 3 nuclear). RANKL 25 5 75 TRAP activity (%). RANKL 5 1 25 5 RANKL+spinach % EtOH extracts (μg/ml) RANKL+spinach % EtOH extracts (μg/ml) Fig. 3. Spinach extract inhibited RANKL-induced osteoclast differentiation of RAW264.

5 536 김동규 김미혜 강민정 신정혜 A B C TRAP (+) cells ( 3 nuclear). RANKL TRAP activity (%). RANKL RANKL+spinach % EtOH extracts (μg/ml) RANKL+spinach % EtOH extracts (μg/ml) Fig. 3. Spinach extract inhibited RANKL-induced osteoclast differentiation of RAW264.7 cells. (A) TRAP staining of RANKL with spinach % EtOH extract treated cells. (B) Inhibition of RANKL-induced TRAP positive cells by spinach % EtOH extract. (C) Inhibition of RANKL-induced TRAP activity by spinach % EtOH extract. Each bar represents the mean±sd of three repeated experiments. P<.1 compared to the RANKL treatment. 량을정량하기위하여 housekeeping gene으로 hypoxanthine phosphoribosyltransferase(hprt) 의발현을확인하였다. HPRT gene은다른여타 housekeeping gene에비해경제적이고정확하다고알려져있다 (3). RAW264.7 세포에 RANKL을처리했을때 c-fos, NFATc1, TRAP mrna 발현이증가되었지만시금치 % 에탄올추출물을처리한세포군에서는 RANKL에의해증가된 NFATc1과 c-fos 유전자발현의증가가감소되었다 (Fig. 4). 또한시금치 % 에탄올추출물은 RANKL에의해발현이증가된 cathepsin K와 TRAP 유전자의발현도유의적으로감소시켰다. RANKL은파골세포분화단계의전사인자인 NFATc1의발현을유도한다 (18). 이 NFATc1은다른파골세포분화에중요한단백질의발현을조절하는데이때 MAPK 중 ERK의활성이파골세포분화에기여한다고보고된바있고, 더욱이 ERK는분화뿐만아니라파골세포의생존에도관여함이알려져있다 (31-33). RANKL에의해유도되는 NFATc1과 c-fos 단백질의유도형및 c-fos의활성형에대한시금치 % 에탄올추출물의영향을 western blot법으로확인하였다. 그결과유전자수준에서의분석결과와일치하는경향으로 RANKL을처리하지않은무처리대조군에서도 endogenous한 NFATc1 단백질의발현이확인되었다. 더욱이 RANKL 처리에의해 NFATc1의양이일부증가되는양상을나타내었으나 μg/ml 농도의시금치 % 에탄올추출물에의해 NFATc1의발현감소가유도되었다 (Fig. 5A). 또다른분화에중요한전사인자인 c-fos의발현역시 RANKL에의해현저하게증가하였고, 시금치 % 에탄올추출물은 RANKL에의한 c-fos의발현증가를무처리대조군의 c-fos 유도형과활성형수준으로현저하게감소시켰다 (Fig. 5B, D). 분화신호를전달하는시그널단백질중하나인 ERK의불활성형에대해서는영향을미치지않았으나활성형의발현은감소시키는것을확인하였다 (Fig. 5C, D). 시금치 % 에탄올추출물은 ERK의발현에대한저해효과가낮은수준이었으나, 파골세포분화에필수적인전사인자 c-fos의유도형과활성형의발현을저해함으로써파골세포분화의신호전달과정을억제하는것으로추정된다. 요약파골세포의분화에대한시금치추출물의영향을확인하고자 RANKL을처리한 RAW264.7 세포에서세포독성, TRAP

RANKL 에의해유도되는파골세포분화에대한시금치추출물의영향 537 A B NFATc1 expression (%). c-fos expression (%). Control RANKL Spinach Control RANKL Spinach RANKL - + + RANKL - + + % EtOH % EtOH Ext. (μg/ml) - - Ext.

Spinach extract down-regulated the expression of osteoclast-specific genes. RT-PCR analysis to measure the gene expression of NFATc1 (A), c-fos (B), cathepsin K (C), and TRAP (D).

물과 25, 5, 75 및 % 에탄올시금치추출물의세포독성을측정한결과모든추출물들이 µg/ml 이하의농도에서 RAW264.7 세포에독성을유발하지않았다. TRAP 염색을통해 TRAP(+) 다핵세포의수와효소활성을측정한결과물추출물을제외한모든추출물이대조군에비해분화억제및효소활성저해효과가있었다.

또한단백질수준에서시금치에탄올추출물은 RANKL에의해증가된 NFATc1의발현을현저히감소시키는것으로확인되었고, 또한 c-fos의활성화형태인인산화된 c-fos의발현뿐만아니라인산화되지않은비활성의 c-fos 발현도감소시켰다.

6 RANKL 에의해유도되는파골세포분화에대한시금치추출물의영향 537 A B NFATc1 expression (%). c-fos expression (%). Control RANKL Spinach Control RANKL Spinach RANKL RANKL % EtOH % EtOH Ext. (μg/ml) - - Ext. (μg/ml) - - C D Cathepsin K expression (%). TRAP expression (%). Control RANKL Spinach Control RANKL Spinach RANKL RANKL % EtOH % EtOH Ext. (μg/ml) - - Ext. (μg/ml) - - Fig. 4. Spinach extract down-regulated the expression of osteoclast-specific genes. RT-PCR analysis to measure the gene expression of NFATc1 (A), c-fos (B), cathepsin K (C), and TRAP (D). Each bar represents the mean±sd of three repeated experiments. P<.1 compared to the RANKL treatment. (+) 다핵세포의형성, 파골세포분화관련유전자의발현, 그리고단백질발현을확인하였다. 물과 25, 5, 75 및 % 에탄올시금치추출물의세포독성을측정한결과모든추출물들이 µg/ml 이하의농도에서 RAW264.7 세포에독성을유발하지않았다. TRAP 염색을통해 TRAP(+) 다핵세포의수와효소활성을측정한결과물추출물을제외한모든추출물이대조군에비해분화억제및효소활성저해효과가있었다. 특히 µg/ml 농도의 % 에탄올추출물은 RANKL만처리한대조군과비교해 % 의유의한 TRAP(+) 다핵세포숫자감소와 44% 의 TRAP 효소활성저해율을보였다. 시금치에탄올추출물은 RANKL에의한파골세포분화의지표가되는관련유전자인 NFAT, c-fos, cathepsin K 및 TRAP의발현을억제하였다. 또한단백질수준에서시금치에탄올추출물은 RANKL에의해증가된 NFATc1의발현을현저히감소시키는것으로확인되었고, 또한 c-fos의활성화형태인인산화된 c-fos의발현뿐만아니라인산화되지않은비활성의 c-fos 발현도감소시켰다. 반면파골세포의분화에직간접적인영향을미친다고알려진 MAPK 중 ERK의활성에는거의영향을미치지않는것으로보아시금치에탄올추출물은 c-fos의활성, 비활성형전체를감소시킴으로파골세포분화를감소시키는것으로확인되었다. REFERENCES 1. Titorencu I, Pruna V, Jinga VV, Simionescu M. 14. Osteoblast ontogeny and implications for bone pathology: an overview. Cell Tissue Res 355: Rodan GA, Martin TJ.. Therapeutic approaches to bone diseases. Science 289: Walsh MC, Kim N, Kadono Y, Rho J, Lee SY, Lorenzo J, Choi Y. 6. Osteoimmunology: interplay between the

538 김동규 김미혜 강민정 신정혜 A NFATc1 B p-c-fos β-actin c-fos RANKL - - + + % EtOH Ext. (μg/ml) - - - + C h 24 h RANKL - - + + % EtOH Ext. (μg/ml) - - - + h 24 h p-erk ERK β-actin RANKL - - + + % EtOH Ext.

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