동의생리병리학회지제 23 권 6 호 Korean J. Oriental Physiology & Pathology 23(6):1299 1304, 2009 파골세포의골흡수에미치는녹용의억제효과 김윤경 최윤홍 1 송정훈 2 장성조 3 김현정 4 이창훈 4 안선호 4 이지은 4 김정중 1 최민규 1 * 원광대학교약학대학한약학과, 1 : 의과대학해부학교실, 2 : 성형외과학교실, 3 : 신경외과학교실, 4 : 내과학교실 Inhibitory Effect of Deer Antler on Osteoclastic Bone Resorption Yun Kyung Kim, Yun Hong Choi 1, Jeong Hoon Song 2, Sung Jo Jang 3, Hyun Jung Kim 4, Chang Hoon Lee 4, Ho Seon Ahn 4, Ji Eun Lee 4, Jeong Joong Kim 1, Min Kyu Choi 1 * Department of Oriental Pharmacy, School of Pharmacy, 1:Department of Anatomy, 2:Department of Plastic Surgery, 3:Department of Neurosurgery, 4:Department of Internal Medicine, School of Medicine, Wonkwang University We have previously shown that water extract of deer antler (WEDA) inhibited RANKL-mediated osteoclast differentiation from bone marrow macrophages by suppressing c-fos and NFATc1 expression. Thus, we examined the effect of WEDA in inflammation-induced bone loss in vivo. Here we found that WEDA inhibited osteoblast-supported osteoclast differentiation induced by lipopolysaccharide (LPS). However, WEDA did not suppress the expression of receptor activator of NF-κB ligand (RANKL) in response to LPS in osteoblasts. WEDA also inhibited the bone resorptive activity of mature osteoclasts. To examine the effect of WEDA on bone loss, when LPS injected subcutaneously in mice, bone loss was greatly increased, but WEDA treatment inhibited LPS-mediated bone loss. Taken together, we conclude that WEDA inhibited osteoclast differentiation and bone resorption in vitro and in vivo. Thus WEDA may be useful in the treatment of bone-related disorders. Key words : osteoclast, deer antler, lipopolysaccharide 서론 골항상성 (Bone homeostasis) 은골형성과골흡수사이의균형에의해유지된다 1). 이러한균형은폐경기여성들의에스트로젠결핍과만성염증에의해쉽게파괴되는데골다공증과류마티스관절염과같은골손실은골을형성하는조골세포 (osteoblast) 의골형성억제보다는파골세포 (osteoclast) 의과도한활성에의해유도된다 2,3). 또한골석화증은파골세포의형성및기능의장애로비정상적인뼈가형성되는질환으로이모든질병은파골세포와깊은관련이있다 4). 파골세포는조혈모세포에서유래된세포로골을흡수하는유일한세포이며단핵구 / 대식세포계열의세포가융합되어파골세포로분화된다 5). 단핵구 / 대식세포는파골세포의전구세포로 receptor activator of NF-κB (RANK) 수용체를발현하고조골세포에서발현하는 RANK ligand (RANKL) 와 macrophage-colony * 교신저자 : 최민규, 익산시신용동 344-2, 원광대학교의과대학해부학교실 E-mail : mkchoi@wku.ac.kr, Tel : 063-850-6761 접수 : 2009/11/16 수정 : 2009/11/30 채택 : 2009/12/11 stimulating factor (M-CSF) 의자극에의해서분화된다 1,6). RANKL은 tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), osteoprotegerin (OPG) ligand라고도불리며 TNF 계열의사이토카인으로활성화된 T 세포에서발현되는사이토카인으로처음밝혀졌다 1). RANKL는파골세포의형성에중요한사이토카인으로 RANKL이결핍된생쥐에서파골세포의형성이억제되었고심각한골석화증이유도되었다 7). RANKL은대부분조골세포에서발현되며 interleukin 1 (IL-1), prostaglandin E2 (PGE2) 와 1α, 25-dihydroxyvitamin D3 (VitD3) 등의자극에의해발현이유도된다 8-10). 전구세포에서발현되는 RANK와 RANKL의결합은파골세포의분화에중요한여러신호전달물질을활성화시킨다. 특히 mitogen-activated protein kinase (MAPK) 인 p38과 JNK 그리고전사인자인 NF-κB 등은파골세포분화에필수적인전사인자 c-fos와 nuclear factor of activated T cells (NFAT)c1의발현을촉진한다 11,12). 또한전사인자 Mi transcription factor (MITF) 와 PU.1이파골세포의분화에중요하다고알려져있다. 이전사인자들은파골세포의지표인 tartrate resistant acid phosphatase (TRAP), calcitonin - 1299 -
김윤경 최윤홍 송정훈 장성조 김현정 이창훈 안선호 이지은 김정중 최민규 receptor, osteoclast-associated receptor (OSCAR) 등의발현을유도한다 11,13). 현재사용되고있는골다공증치료제및골질환치료제인 selective estrogen receptor modulator(serm) 와 bisphosphonate 는합성약품으로많은부작용이대두되고있다 14,15). 최근단삼 ( 丹蔘 ) 에서추출한 tanshinone IIA, 노각나무추출물, 녹용의 chloroform 추출물이파골세포의분화및기능에탁월한효과를나타낸다고보고되었다 16-18). 저자는최근녹용, 커큐민, 두충, 토사자등이파골세포의분화및기능을억제한다고보고하였다 19-22). 특히이들은고대부터현대까지널리섭취되고있으며부작용이없어골다공증치료제로이용될수있을것이다. 녹용은면역질환치료제로사용되고관절염치료에좋은효과를나타낸다고보고되었다 23). 따라서본연구에서저자는생체내골질환모델에서녹용의효과를검증하고자하였다. 생체내파골세포는조골세포에서발현하는 RANKL과염증부위의 T 세포에서발현되는 RANKL에의해파골세포형성이유도되어골흡수가증가된다. 저자는강력한염증유도제인 lipopolysaccharide (LPS) 를이용하여조골세포에의존적인파골세포분화에미치는녹용의효과를규명하고자하였으며골질환생쥐모델에서녹용의효과를검증하고자하였다. 재료및방법 1. 시료녹용 ( 옴니허브, 한국 ) 은예전에발표한논문에서와같이추출하여시료를얻었다 19). VitD3, PGE2, LPS, TRAP 용액은 Sigma Aldrich(St. Louis, MO, U.S.A) 사에서구입하였다. Hydroxyapatite-coated plate는오스코텍 ( 천안, 한국 ) 사의제품을사용하였다. 2. 파골세포분화에미치는녹용의효과골수세포를얻기위해 ICR 5주령생쥐의대퇴골과경골을분리하고골속질공간을 1cc 주사기로수세하여골수세포를얻었다. 또한생후 1일령생쥐의두개골을적출하고 0.1% collagenase와 0.2% dispase를이용하여두개골에서조골세포를추출하였다. 조골세포 (2 X 10 4 /well) 와골수세포 (2 X 10 5 /well) 는 48-well plate에첨가하고 LPS와녹용추출물을투여하여 6일간배양하였다. 6일배양후, 배양한세포는 TRAP 용액으로염색하고붉은색으로염색된세포를파골세포로간주하였다. 3. RANKL 발현에대한녹용의효과녹용추출물을전처리한실험군과대조군조골세포에 LPS 를각각 1일과 2일처리하였다. 실험에사용한세포는 TRIzol (Invitrogen) 용액으로용해하여제조사의방법에따라 RNA를분리했다. 분리한 RNA 1 μg은 oligo dt primer, dntp, buffer, dithiothreitol, RNase inhibitor와 Superscript II reverse transcriptase를이용해 cdna를합성했다. 합성된 cdna는다음과같은 primer를이용하여 PCR 증폭을했다. RANKL sense, 5'-CAGGTTTGCAGGACTCGAC-3' ; RANKL antisense 5'-AGCAGGGAAGGGTTGGACA-3 '; GAPDH sense, 5'-ACCACAGTCCATGCCATCAC-3' ; GAPDH antisense, 5'-TCCACCACCCTGTTGCTGTA-3' ; PCR 후, PCR 산물은 1% agarose gel에서전기영동하고 Et-Br로염색하여 U.V. 상에서관찰했다. 4. 골흡수에대한녹용의효과성숙파골세포를얻기위해 collagen을 90-mm 배양접시에코팅하고골수세포와조골세포를첨가하여 VitD3와 PGE2를넣고배양하였다. 6일후, collagenase로성숙파골세포를떼어내고 hydroxyapatite-coated 48-well plate에첨가하였다. 골흡수에녹용의효과를관찰하기위해녹용추출물을농도별로처리하고 24시간배양하였다. 배양후세포는제거하고골흡수정도를분석하였다. 5. 생체내골흡수에미치는녹용의효과 ICR 6주령생쥐는마취하고녹용추출물 (1 mg/ 생쥐 ) 과 LPS(30 μg/ 생쥐 ) 를머리에피하주사하였다. 생쥐는 SPF(specifie pathogen free) 조건에서 5일간생육하였다. 5 일후생쥐는경추탈골법으로희생하고두개골을얻었다. 두개골은 4% paraformaldehyde로 1일간고정하고 12% EDTA로탈회하여 paraffin 포매후조직절편은 hematoxylin & eosin (H&E) 으로염색하였다. 6. 조골세포의분화에대한녹용의효과조골세포는 (2 X 10 4 /well) 48-well plate에첨가하고 ascorbic acid (50 μg/ml) 와 β-glycerophosphate (10 mm) 를처리하고녹용추출물을투여하여 7일간배양하였다. 7일간배양후 alkaline phosphatase (ALP) 용액으로염색하고파란색으로염색된세포를조골세포로간주하였다. 7. 통계분석정량적인결과는평균값과표준편차로표시하였다. 통계적인차이는 Student's t-test를이용하여분석하였고 p 값이 0.05 이하인경우통계적으로유의한것으로간주하여별표 (*) 로표시하였다. 결과 1. 파골세포의분화에미치는녹용의효과저자는최근에파골세포의분화에녹용의효과를보고하였다 19). 그러나생체내에서파골세포의분화와골흡수에녹용의효과를검증하기위해생체내에서파골세포의분화에중요한역할을하는조골세포와파골세포의분화에녹용의효과를규명하고자하였다. 조골세포와골수세포를공배양하고파골세포의분화를유도하여골흡수를촉진하는 LPS와녹용추출물을처리하였다. 녹용추출물 10 μg/ml로처리한실험군에서는억제효 - 1300 -
파골세포의골흡수에미치는녹용의억제효과 과가없었지만녹용추출물농도의증가에따라파골세포의수가유의하게억제되었다 (Fig. 1A). 또한파골세포의수가농도증가에따라감소되는것을확인하였다 (Fig. 1B). 이결과로녹용추출물이조골세포에의해촉진되는파골세포의분화를억제한다고할수있다. 2. 조골세포에서파골세포유도인자발현에미치는녹용의효과비록조골세포는골형성에중요한세포이지만, 생체내에서파골세포의분화에중요한 RANKL를발현하여파골세포의형성을조절한다 1). 녹용추출물이조골세포에의한파골세포의분화를억제하였기때문에조골세포에의한 RANKL발현에녹용추출물의효과를검증하였다. 조골세포에녹용추출물을처리하고 LPS를처리하였다. LPS는조골세포에서 RANKL의발현을촉진하였고녹용추출물을전처리한실험군에서도 LPS는 RANKL 의발현을촉진하였다 (Fig. 2). 이결과로녹용추출물은조골세포의기능을억제하지않고파골세포에직접적으로작용한다고할수있다. 3. 파골세포의골흡수에대한녹용의효과다음으로파골세포의골흡수에녹용추출물의효과를연구하였다. 골수세포와조골세포의공배양으로얻은성숙파골세포를 hydroxyapatite plate에첨가하고녹용추출물을농도별로처리하였다. 대조군에서는파골세포에의해 hydroxyapatite가흡수되었지만녹용추출물을처리한실험군에서는농도증감에따라 hydroxyapatite 흡수가억제되는것을확인하였다 (Fig. 3). 이결과는녹용추출물이파골세포의형성뿐만아니라골흡수도억제한다고할수있다. Fig. 1. Effect of water extract of deer antler (WEDA) on LPS-induced osteoclast differentiation. (A) Bone marrow cells and osteoblasts were cocultured for 6 days with LPS (1 μg/ml) in the presence of increasing concentrations of WEDA (μg/ml). Cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained for TRAP. (B) TRAP-positive cells were counted as osteoclasts. Significant difference from control (no treatment) is marked by an asterisk. n.s., not significant. Fig. 3. Effect of WEDA on osteoclastic bone resorption. (A) Bone marrow cells and osteoblasts were co-cultured for 6 days in the presence of VitD3 and PGE2. Mature osteoclasts were seeded on hydroxyapatite-coated 48-well plates and then treated for 24 hours in the presence of the WEDA concentrations. (B) Pit area (indicated by the arrows) was quantified using Image Pro-plus program version. 4.0. *P<0.05, significant differences from no WEDA treatment. Fig. 2. Effect of WEDA on the expression of RANKL induced by LPS. (A) Osteoblasts were pretreated with or without WEDA (100 μg/ml) for 1 hour and then treated with LPS (1 μg/ml) for the indicated time. Total RNA was isolated from the cells and the expression of RANKL and GAPDH mrna was analyzed by RT-PCR. (B) Relative levels of RANKL mrna was quantified using Image Pro-plus program version. 4.0 and normalized to GAPDH. 4. 생체내골흡수에미치는녹용의효과녹용추출물이골수세포에서파골세포로의분화와골흡수를억제하였기때문에생쥐를이용하여생체내골흡수에녹용추출물의효과를검증하였다. LPS는강력한염증인자로생체 - 1301 -
김윤경 최윤홍 송정훈 장성조 김현정 이창훈 안선호 이지은 김정중 최민규 내검증하였다. LP촉진하여골흡수를유도한다. 따라서 LPS를생쥐에투여하고녹용추출물을투여한생쥐에서골흡수였다젮도를확인하였다. LPS는골흡수를촉진하였지만녹용추출물을같이투여한생쥐에서는골흡수가억제되는것을확인할수있다 (Fig. 4). Fig. 4. Effect of WEDA on LPS-induced bone loss. (A) 6-week-old ICR mice were injected subcutaneously at the calvaria with WEDA (1 mg/mouse) and/or LPS (30 μg/mouse). Mice were sacrificed 5 days. Calvarial bones were isolated, fixed in 4% paraformaldehyde for 1 day, decalcified with 12% EDTA, and embedded in paraffin. Sections were stained with H&E. (B) Bone volume/tissue volume (BV/TV) was analyzed using the histomorphometric results. Significant difference between the indicated groups are marked by an asterisk. Fig. 5. Effect of WEDA on osteoblast differentiation. Osteoblasts were cultured for 7 days with ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mm) in the presence of increasing concentrations of WEDA (μg/ml). Cells were fixed in 3.7% formalin and stained for alkaline phosphatase. 5. 조골세포분화에대한녹용의효과 녹용추출물이생체내에서골파괴를억제하는효과를관찰하였다. 골은골의흡수와형성에의해서양질의골이유지되는데녹용추출물이조골세포의분화에는어떤효과를나타내는지확인하였다. 조골세포는 ascorbic acid와 β-glycerophosphate 를첨가하고녹용추출물을처리하였다. 7일후 ALP 염색으로 조골세포의분화를관찰하였지만녹용추출물은조골세포의분화에효과가없었다 (Fig. 5). 이결과로녹용추출물은파골세포의분화와기능에만관여한다는것을알수있다. 고찰 조골세포는골형성에중요한세포이지만 RANKL의발현을통해파골세포의형성과기능에깊이관여하는세포이다 3). 본연구에서녹용추출물은조골세포에의해유도되는파골세포분화를억제하였다 (Fig. 1). LPS는강력한염증유도인자로수용체인 Toll-like receptor 4와결합하여 myeloid differentiation protein 88 (MyD88) 을활성화시키고여러신호전달물질의활성화유도로조골세포에서 RANKL의발현을촉진하여파골세포의분화를유도한다고보고되었다 8,24). 그러나 LPS를처리한조골세포에서발현되는 RANKL은녹용추출물에의해억제되지않았다 (Fig. 2). 저자는녹용추출물은 RANKL에의해유도되는전사인자 c-fos와 NFATc1의발현을억제하여파골세포분화를억제한다고보고하였다 19). 이들의결과로녹용추출물은파골세포의분화를조절하는조골세포에는영향없이골수세포에서파골세포로의분화에직접적으로관여한다고할수있다. 파골세포는유일하게골을흡수하는세포로여러신호전달경로를통해골흡수가이루어진다. 골을흡수하는파골세포의가장큰특징은세포막이주름지고 clear zone이형성된다. 특히파골세포가골에부착하기위해서 clear zone에 integrin을발현하는데 αvβ3 integrin이골표면에 RGD (arginine-glycineaspartic acid) sequence를인지하여골에부착된다. 골에부착된파골세포는골의흡착부위에 H+, cathepsin K, TRAP등을분비하여골을흡수한다 2). 최근 calcitonin이파골세포의액틴구조를파괴하여골흡수를억제한다고보고되었다 25). 이들의결과로골에파골세포의부착이중요하다할수있다. 본연구에서녹용추출물은분화와골흡수를억제한다는것을확인하였다 (Fig. 1, 3). 비록파골세포의부착과 H+, cathepsin K, TRAP의분비에대한녹용추출물의효과를검증하지못하였지만녹용추출물은골수세포에서파골세포의분화를억제할뿐만아니라골을흡수하는성숙파골세포의기능도억제한다고할수있다. 골항상성은조골세포와파골세포의활성조절에따라유지되지만조골세포와파골세포는수많은사이토카인과호르몬에의해조절된다. 생체내 IL-1, TNF-α등의증가는파골세포의분화를촉진하여골다공증을유도하지만 interferon-γ와에스트로젠등은파골세포의분화를억제한다 11). 비록녹용추출물은조골세포에서발현되는 RANKL에의한파골세포분화에억제효과가나타났지만생체내골의손실에대한녹용추출물의효과는알수없었다. 따라서저자는 LPS를투여한생쥐에서녹용추출물의효과를검증하였다. 녹용추출물은 LPS에의해유도되는골손실을억제하였다 (Fig. 4). 골손실의억제는파골세포의형성및활성의증가로발생되지만최근조골세포의활성을유도하여골손실을억제하는연구가보고되고있다 26). 따라서저자는조골세포의분화에대한녹용추출물의효과를연구하였지 - 1302 -
파골세포의골흡수에미치는녹용의억제효과 만조골세포의분화에녹용추출물은효과가없었다 (Fig. 5). 이들의결과로녹용추출물은조골세포의골형성에는효과없이파골세포의골흡수를억제하여생체내골손실을억제한다고할수있다. 결론 본연구에서생체내골손실에미치는녹용의효과를검증하고자하였다. 먼저조골세포와골수세포를공배양하고 LPS를처리하여파골세포의분화에녹용추출물의효과를확인하였다. 녹용추출물은 LPS를투여한파골세포의분화를억제하였다. 그러나 LPS에의한 RANKL 발현억제에는효과가없었다. 이결과는녹용추출물이파골세포의분화에직접적으로관여한다고할수있다. 또한녹용추출물은파골세포의골흡수를억제하였다. LPS를생쥐에투여했을때골손실이나타났지만녹용추출물을같이투여한생쥐에서는골손실이억제되었으며조골세포의분화에는효과가없었다. 결론으로녹용추출물은조골세포의기능에는관여하지않고파골세포에직접적으로관여하여골다공증과같은질환에녹용이치료제로이용될수있으리라기대한다. 감사의글 이논문은 2007년도원광대학교교비지원에의해수행되었습니다. 참고문헌 1. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M.T., Martin, T.J. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20: 345-357, 1999. 2. Teitelbaum, S.L., Ross, F.P. Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 4: 638-649, 2003. 3. Boyle, W.J., Simonet, W.S., Lacey, D.L. Osteoclast differentiation and activation. 423: 337-342, 2003. 4. Tolar, J., Teitelbaum, S.L., Orchard, P.J. Osteopetrosis. N. Engl. J. Med. 351: 2839-2849, 2004. 5. Yagi, M., Miyamoto, T., Toyama, Y., Suda, T. Role of DC-STAMP in cellular fusion of osteoclasts and macrophage giant cells. J. Bone Miner. Metab. 24: 355-358, 2006. 6. Fuller, K., Wong, B., Fox, S., Choi, Y., Chambers, T.J. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J. Exp. Med. 188: 997-1001, 1998. 7. Kong, Y.Y., Yoshida, H., Sarosi, I., Tan, H.L., Timms, E., Capparelli, C., Morony, S., Oliveira-dos-Santos, A.J., Van, G., Itie, A., Khoo, W., Wakeham, A., Dunstan, C.R., Lacey, D.L., Mak, T.W., Boyle, W.J., Penninger, J.M. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 397: 315-323, 1999. 8. Sato, N., Takahashi, N., Suda, K., Nakamura, M., Yamaki, M., Ninomiya, T., Kobayashi, Y., Takada, H., Shibata, K., Yamamoto, M., Takeda, K., Akira, S., Noguchi, T., Udagawa, N. MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha. J. Exp. Med. 200: 601-611, 2004. 9. Kanematsu, M., Sato, T., Takai, H., Watanabe, K., Ikeda, K., Yamada, Y. Prostaglandin E2 induces expression of receptor activator of nuclear factor-kappa B ligand/osteoprotegrin ligand on pre-b cells: implications for accelerated osteoclastogenesis in estrogen deficiency. J. Bone Miner. Res. 15: 1321-1389, 2000. 10. Tsukii, K., Shima, N., Mochizuki, S., Yamaguchi, K., Kinosaki, M., Yano, K., Shibata, O., Udagawa, N., Yasuda, H., Suda, T., Higashio, K. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone. Biochem. Biophys. Res. Commun. 246: 337-341, 1998. 11. Takayanagi, H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat. Rev. Immunol. 7: 292-304, 2007. 12. Takayanagi, H., Kim, S., Koga, T., Nishina, H., Isshiki, M., Yoshida, H., Saiura, A., Isobe, M., Yokochi, T., Inoue, J., Wagner, E.F., Mak, T.W., Kodama, T., Taniguchi, T. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev. Cell. 3: 889-901, 2002. 13. Kim, K., Kim, J.H., Lee, J., Jin, H.M., Lee, S.H., Fisher, D.E., Kook, H., Kim, K.K., Choi, Y., Kim, N. Nuclear factor of activated T cells c1 induces osteoclast-associated receptor gene expression during tumor necrosis factor-related activation-induced cytokine-mediated osteoclastogenesis. J. Biol. Chem. 280: 35209-35216, 2005. 14. Lloyd, M. Treatment of postmenopausal osteoporosis. N. Engl. J. Med. 338: 736-746, 1998. 15. Rodan, G.A., Martin, T.J. Therapeutic approaches to bone disease. Science. 289: 1508-1514, 2000. 16. Kwak, H.B., Yang, D., Ha, H., Lee, J.H., Kim, H.N., Woo, E.R., Lee, S., Kim, H.H., Lee, Z.H. Tanshinone IIA inhibits - 1303 -
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