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Journal of Korean Medicine Rehabilitation Vol. 29 No. 2, April 2019 pissn 1229-1854 eissn 2288-4114 https://doi.org/10.18325/jkmr.2019.29.2.135 Original Article 박정식 임형호가천대학교한의과대학 Effects of Pyrola japonica Extracts on Osteoclast Differentiation and Bone Resorption Jung-Sik Park, K.M.D., Hyung-Ho Lim, K.M.D. College of Korean Medicine, Gachon University RECEIVED March, 28, 2019 REVISED April, 8, 2019 ACCEPTED April, 12, 2019 CORRESPONDING TO Hyung-Ho Lim, College of Korean Medicine, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam 13120, Korea TEL +82-31-750-8599 FAX +82-31-750-5416 E-mail omdlimhh@gachon.ac.kr Copyright 2019 The Society of Korean Medicine Rehabilitation Objectives This study was performed to evaluate the effect of Pyrola japonica extract (NJ) and its principal constituent, homoarbutin (HA) on osteoclast differentiation and gene expression and bone resorption. The osteoclastogenesis and gene expression were determined in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated RAW264.7 cell. Methods In order to evaluate the effect of HA extracted from NJ on bone resorption, osteoclasts were used to be differentiated and formed by stimulating RAW264.7 cells with RANKL. Tartarate-resistant acid phosphatase (TRAP) (+) polynuclear osteoclast formation ability was evaluated, and differentiation control genes including cathepsin K, matrix metalloproteinases-9 (MMP-9), and TRAP in osteoclast differentiation were analyzed by real-time polymerase chain reaction (PCR). Immunoblotting was performed to measure the effect of mitogen-activated protein kinase (MAPK) factors on bone resorption, and the effect of osteoclasts on osteoclast differentiation was measured. Results Both NJ and high concentration of HA blocked RANKL-stimulated differentiation from RAW264.7 cell to TRAP-positive multinucleated cells. NJ reduced RANKL-induced expression of TRAP, cathepsin K. Both NJ and high concentration of HA inhibited RANKLmediated expression of MMP-9, nuclear factor of activated T-cells, cytoplasmic 1, and cellular Jun-fos. NJ suppressed RANKL-stimulated expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase, tumor necrosis factor-alpha, and levels of interleukins. Both NJ and HA decreased bone resorption in osteoclast-induced bone pit formation model. Conclusions These results suggest that NJ and HA blocked bone resorption by decreasing RANKL-mediated osteoclastogenesis through down-regulation of genes for osteoclast differentiation. (J Korean Med Rehabil 2019;29(2):135-147) Key words Osteoclast, Poncirin, Receptor activator of nuclear factor kappa B ligand (RANKL), Tartarate-resistant acid phosphatase (TRAP), Japonica extract, Homoarbutin 서론»»» 골다공증은미세구조이상과골량감소를특징으로하며골을형성하는조골세포보다골을파괴하는파골세포의기능이우세하여나타나는전신적인골격계질병이다. 특히여성갱년기이후에에스트로겐부족으로많이발생하고있다. 골다공증의치료는조골세포의기능을활성화시켜골형성을촉진하거나파골세포의활성을억제하여골흡수를방해하는방법이시도되고있다 1,2). 골수에존재하는골세포전구세포는여러분화촉진인 www.e-jkmr.org 135

박정식 임형호 자들의작용으로파골세포로분화될수있는데, 분화촉진인자로 macrophage colony-stimulating factor (M-CSF) 나 receptor activator of nuclear factor kappa B ligand (RANKL) 가잘알려져있다 2). 이러한분화과정에는 RANKL 이세포표면에존재하는수용체에결합하는것을시작으로 activator protein-1, cellular Jun-fos (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), cellular-jun (c-jun) 등의활성화와 extracellular regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 등의인산화과정등을통해진행된다 3). 궁극적으로파골세포의골흡수는분화한파골세포에서 tartarate-resistant acid phosphatase (TRAP) 이나, matrix metalloproteinases-9 (MMP-9), cathepsin K 등의작용을통하여나타나게된다 4-6). 한의학적으로골다공증은임상적으로骨萎, 骨痺, 骨枯, 虛勞등에해당하며 素問痿論 에서 腎氣熱, 卽腰脊不擧, 骨枯而髓減, 發爲骨痿. 有遠行勞倦, 逢大熱而渴, 渴卽陽氣內伐, 內伐卽熱舍於腎, 腎者水臟也. 今水不勝火, 卽骨枯而髓虛, 故足不任身, 發爲骨痿. 故下經曰, 骨痿者, 生於大熱也. 라하여발병기전및坐不能起, 腰脊不擧, 肌膚削而形鸁廋, 腰膝疼痛등의골다공증과유사한증상에대하여설명하였다 7). 본연구에사용된녹제초 (Pyrola japonica) 는노루발풀로불리며, 한국을비롯하여난온대지방에서식하는여러해살이풀로꽃피는시기에줄기와잎을채취해서약재로사용한다 8). 최근녹제초는지혈촉진, 이뇨촉진, 항염증작용및면역활성에대한연구결과가보고되었다 9,10). 성분으로는 pirolatin, hyperin, monotropein and chimaphilin, arbutin 과 homoarbutin (HA) 등이보고되었다 11). 한의학적으로녹제초 ( 鹿蹄草 ) 의성미는甘苦하고溫하며祛風濕, 健筋骨, 補肺腎, 止血하는효능이있다 12). 그러나녹제초라는본초가널리사용되지않고연구또한활발히진행되지않았기에근골격계질환과관련된연구를하여새로운가능성을넓히고자健筋骨하는효능에집중하여본연구를시행하였다. 이에녹제초및그주성분의골다공증및파골세포에미치는영향에대해조사하였다. 본연구는녹제초및그주성분인 HA가골다공증에미치는유효성을검증하기위하여 RANKL 유도파골세포분화및관련인자들의발현을측정하고골편을사용한골흡수에미치는영향을측정하여골다공증억제효능을평가하고자하였다. 재료및방법»»» 1. 재료 1) 녹제초추출및 HA 분리녹제초 (Pyrola japonica) 100 g을분말화하여플라스크에투입한후, 30% 에탄올 500 ml를가하고 6시간이상가열과정을거쳐환류추출하였다. 여과지를이용하여여과한후, 여액을 evaporator (EYELA, Tokyo, Japan) 를이용하여감압농축한후, 동결건조하여녹제초추출물 (Pyrola japonica extract, NJ) 로실험에사용하였다. 유효성분을분리하기위하여위에서감압농축한추출물에증류수를넣어현탁시켰다. 현탁시킨액을 ethyl Acetate (EtOAc) 로추출하고, 다시나머지액을 butanol (BuOH) 을이용하여추출하여각각의추출액을농축시키고농축액을 Silica gel column을사용하여분리정제하였다. 2) HA 분리동정및시료제조실험에사용한 HA는기존의보고된방법을사용하여분리하였으며 11), 3 hydrogen-nuclear magnetic resonance 을사용하여동정하여확인하였다. 실험시에는 dimethyl sulfoxide를이용하여배지에녹인후, pore size 0.45 μm 의여과지를통과시킨다음사용하였다. 3) 세포구입파골세포전구세포, mouse myeloid 세포주인 RAW 264.7 세포에 RANKL 을처리하여유도한세포모델을실험에사용하였다. 사용한 RAW 264.7은한국세포주은행 (Korean Cell Line Bank, Seoul, Korea) 에서구입하였다. 2. 방법 1) RAW 264.7 Cell 배양 RAW 264.7 세포는 Dulbecco's modified eagle medium/ 10% Fetal bovine serum (FBS)/PC-SM 배지를사용하여 CO 2 세포배양기에서배양하였으며, 세포수는 5 103 cells/well 로 96 well plated에서배양하였다. 24시간배양후배양액을버린다음, 10% FBS, 100 ng/ml RANKL, 1 ng/ml transforming growth factor beta (TGFβ) 를첨가한 α -minimal essential media 로교환한다음세포배양하였다. 136 J Korean Med Rehabil 2019;29(2):135-147.

배양액에여러농도의 NJ 및 HA를첨가해주었다. 2일에 1회씩동일배지로교체해주면서 6일동안배양하였다. 2) 파골세포생성측정 RANKL 로 RAW 264.7 cell을파골세포로유도한후, TRAP 을염색하여 TRAP-positive 세포의분화정도를평가하였다. phosphate buffered saline (PBS) 로분화시킨세포를 2회에걸쳐세척하고, 3.7% formaldehyde-citrate-acetone 용액으로 10분고정시키고증류수로 2회세척하였다. 2% TRAP fast garnet glucobrassicin base 용액과 NaNO 3 용액을 1:1 비율로섞은용액과 4% acetic acid, 2% tartaric acid, 5% naphtha AS-BI phosphoric acid를포함한용액을고정시킨세포에처리한후 30분이상상온에방치하였다. 광학현미경으로관찰하여 3개이상의핵을가진 TRAPpositive한다핵세포 (TRAP[+] multinucleated cells [MNCs]) 를계산하여파골세포의생성지표로지정하였다. 3) 파골세포유전자발현측정 (1) Total RNA 분리 RNA의분리를위해배양세포배지를제거한후, 4 PBS로 3회세척하였다. 세포를모아 750 g에서 10분간원심분리하고 ice cold PBS로 2회세척하였다. 침전된세포에 lysis buffer를가하여용해시킨다음, 12,000 g에서 10분간원심분리하는과정을거쳐상층을취하였다. Trisamine (Tris) buffer에 1M sucrose를함유하여 loading한다음, 36,000 rpm에서원심분리를 160분간시행하였다. Polysome pellet을취하여 sodium dodecyl sulfate (SDS) buffer에녹인다음 30분간 37 에서 incubation하였다. Sodium acetate buffer 를가한후 phenol/cskl 3 용액으로추출하였다. NaCl을 0.2 M이되도록추가하고 ethanol (EtOH) 를가한후 12시간동안 -20 에보관하였다. 75% EtOH로침전물을 2회 washing한후건조하였다. 유전자발현에필요한 RNA 양을대조군과시험군에서 mrna 양이같아지도록조정한후전기영동으로확인하여사용하였다. (2) cdna 제조대조군및시험군에서각기분리한 total RNA액 10 μl (10 μg RNA 함유 ) 에 oligo dt 1 μl (2 μg/μl) 을넣어혼합한후 5분간 90 에서 incubation하였다. Primer 가 annealing 하도록약 10분간실온에서방치한다음 4 온 도에서다음과같이시약을가하였다. 5 xcyscript buffer 4 μl, deoxyuridine triphosphate (dutp) nucleotide mix 1 μl, dutp Cydye-labelled nucleotide 0.1 μl, 0.1M dithiothreitol 2 μl, Cyscript reverse transcriptase 1 μl, H 2 O 0.9 μl를첨가, 20 μl로한다음신중하게수작업으로 tipping 혼합하였다. 그다음 42 에서 90분간 incubation하고얼음에방치하였다. 2.5M NaOH를 2 μl 씩가한다음 15분간 37 에서 incubation하였고 2M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (free acid) 를 10 μl 가하여중화시켰다. 각각의시약은 Amersham Bioscience (Cambridge, UK) 에서구입하였다. (3) Real time reverse transcription polymerase chain reaction (RT-PCR) Optical tube (MicroAmp R Optical 96-Well Reaction Plate with Barcode and Optical Adhesive Films, Applied Biosystems, Cat.No. 4314320) 에각각에 3배의 SybrGreen Mix 2.5 μl (Sigma-Aldrich, Cat.No. S9430) 위에서합성한 10 pmol/μl primer pair mix 1 μl, cdna 1 μl, 각각 2.5 mm의 dntp 2 μl, Tag Polymerase 0.3 μl, 10 Tag polymerase buffer 2.5 μl와 14.7 μl H 2 O를가한후 95 5 min 1 cycle, 95 30 sec, 45 30 sec, 72 60 sec 40 cycles, 95 20 min 1 cycle로증폭시켰다. Primer 는 RT-PCR 에이용한것을사용하였다. PCR이끝난다음 tube를꺼내반응액 5 μl를사용하여 3% agarose gel에서 PCR specificity를측정한다음 ABI PRISM R 7000 Sequence Detection System (Applied Biosystems, Cat.No. 4349157) 를이용하여 real time PCR 결과를분석하였다. 4) Western Blotting 파골세포에 radioimmunoprecipitation assay buffer (ph8, 150 mm NaCl 2, 10 mm NaPO 4, 20 mm Tris-HCl, 100 M Na 3 VO 4, 100 M ammonium molybdate, 10% glycerol, 0.1% SDS, 1% NP-40) 1 ml를첨가하여초음파파쇄기를이용, 5초동안세포벽을파괴시킨후균질액을 20분간 13,000 rpm에서원심분리한상층액을단백질추출액으로하였다. 10% polyacrylamide/sds gel에서정량된단백질을전기영동시킨다음, polyvinylidene difluoride membrane 에흡착시켰다. JNK, p-38 kinase, ERK의 1차항체를 5% skim milk, 0.1% Tween-20 을함유한 PBS with www.e-jkmr.org 137

박정식 임형호 tween-20 (PBST) 에희석시켜서 4 에서 16시간동안반응시킨다음 0.01% Tween-20을함유한 PBST 로 3차례에걸쳐 15분씩세척하였다. Blocking solution 으로 1:10,000배로희석시킨 peroxidase-conjugated anti-immunoglobulin g 이차항체와 1시간동안실온에서반응시킨다음 0.1% Tween-20을함유한 PBS로 3차례에걸쳐세척하고발색은 enhanced chemiluminescence system 으로확인하였다. 5) 골흡수억제실험 NJ 및 HA의골흡수억제효능을평가하기위하여골편에서골흡수에미치는영향을관찰하는방법을사용하였다. 골미네랄흡수에대한연구는 Bone resorption assay kit (Gibco, Los Angeles, CA, USA) 를사용하여수행하였다. 우선 calcium phosphate-coated 골편에 RAW264.7 cells를옮겨심고, ethanol extract of Pyrola japonica와 HA를적절한농도로전처리한다음 RANKL 을처리하였다. 위의세포가심어진골편을이산화탄소배양기에서 12일간배양하였다. 12일째에배지를제거하고골흡수측정용배지를첨가한다음이산화탄소배양기에서배양한후 extension 485 nm and emission 535 nm 파장에서현광광도를측정하였다. 이후에형성된 pit area를측정하기위하여 plate에 5% sodium hypochlorite solution을처리하여세포를제거하고건조한다음현미경으로관찰하고대조군과비교하여골흡수억제정도를평가하였다. 3. 통계학적분석각결과에대한유의성검증은 student's t-test를이용하였다. 모든통계적유의수준은 p<0.05로설정하였다. 결과»»» 1. 파골세포분화에미치는영향 RAW 264.7 cell을 RANKL 과 TGFβ를첨가한배지에배양한경우다수의 TRAP(+) MNCs 가형성되었다. 이것을파골세포로한다음 NJ 및 HA가파골세포분화에미치는 (A) (B) Fig. 1. Effects of Pyrola japonica extract (NJ) on the formation of tartarate-resistant acid phosphatase positive (TRAP[+] multinucleated cells [MNCs]). (A) The histogram represents the number of TRAP(+) MNCs compared with receptor activator of nuclear factor kappa B ligand (RANKL) treated as a control. (B) RAW264.7 cells were cultured with 35 μg/ml, 75 μg/ml, 100 μg/ml and 150 μg/ml concentration of NJ in the presence of 100 ng/ml RANKL. Cells were fixed and stained for TRAP, and TRAP(+) MNCs were observed using inverted microscope( 100). Each bar represents mean±standard deviation of 5 tests. NC: negative control, C: control. * Represents p<0.01 compared to the control group. 138 J Korean Med Rehabil 2019;29(2):135-147.

(A) (B) Fig. 2. Effects of homoarbutin (HA) on the formation of tartarate-resistant acid phosphatase (TRAP[+] multinucleated cells [MNCs]). (A) The histogram represents the number of TRAP[+] MNCs compared with receptor activator of nuclear factor kappa B ligand (RANKL) treated as a control. (B) RAW264.7 cells were cultured with 35 μg/ml, 75 μg/ml, 100 μg/ml and 150 μg/ml concentration of HA in the presence of 100 ng/ml RANKL. Cells were fixed and stained for TRAP, and TRAP[+] MNCs were observed using inverted microscope(x100). Each bar represents mean±standard deviation of 5 tests. NC: negative control, C: control. * Represents p<0.01 compared to the control group. 영향을평가하였다. 측정결과, NJ는모든군에서파골세포형성을유의성있게억제하였고 HA는 150 μg/ml 처리시파골세포형성을유의성있게억제하였다 (Fig. 1, 2). 2. 파골세포생존율에미치는영향 NJ 및 HA가세포사멸을통해분화를억제하는지평가하기위하여미분화된세포에추출물을첨가한다음 3일간배양하여 MTT 방법으로세포생존율을평가하였다. 실험결과, NJ 및 HA는실험에사용한모든군에서세포생존율에영향을미치지않았다 (Fig. 3, 4). 3. 파골세포유전자발현에미치는영향 1) Cathepsin K 발현에미치는영향 Cathepsin K는 RANKL 이나 M-CSF 등을처리하면 TRAP, MMP-9 등과같은단백질이발현되어골흡수에관여하고있다. 그러므로 cathepsin K는 MMP-9, TRAP과함께파 Fig. 3. Effects of Pyrola japonica extract (NJ) on cell viability of osteoclast. CON: vehicle. NJ25: 25 μg/ml of NJ. NJ50: 50 μg/ml of NJ. NJ75: 75 μg/ml of NJ. NJ150: 150 μg/ml of NJ. NJ300: 300 μg/ml of NJ. 골세포활성화지표로이용할수있다. 본실험에서는파골세포기능과관련된유전자들에 NJ 및 HA가미치는영향을평가하는지표로 cathepsin K 발현에주는영향성을 real time PCR system 을이용하여평가하였다. 실험결과, NJ는모든군에서 cathepsin K 유전자발현을유의성있 www.e-jkmr.org 139

박정식 임형호 게억제하였고 HA는실험결과유의성있는감소효과가없었다 (Fig. 5, 6). 2) MMP-9 발현에미치는영향 Fig. 4. Effects of homoarbutin (HA) on cell viability of osteoclast. CON: vehicle. HA25: 25 μg/ml of HA. HA50: 50 μg/ml of HA. HA75: 75 μg/ml of HA. HA150: 150 μg/ml of HA. MMP-9는파골세포에서분비되는 collagen 을파괴하는단백질파괴물질이다. RANKL 로자극된파골전구세포는파골세포로분화하여발생하는 osteoprotegerin (OPG), TRAP 단백질등과더불어 MMP-9 의작용으로골흡수를유도한다. MMP-9 활성을 NJ 및 HA를처리한파골세포로부터측정한결과, NJ는모든군에서, HA는 150 μg/ml 농도처리시 RANKL 처리된파골세포에서증가한 MMP-9 발현을유의하게억제하였다 (Fig. 7, 8). 3) TRAP 발현에미치는영향골수에서유래된전구세포들이파골전구세포로분화되면파골세포표식유전자인 TRAP 유전자를발현하게된다. 최종적으로 TRAP 양성세포들은다핵파골세포 (multinucleated osteoclasts) 로분화하여골흡수작용을한다. 실험결과, NJ는모든군에서 RANKL 처리하여증가된분화한파골세포의 TRAP 유전자발현을유의하게억제하였고 HA는실험결과유의성있는결과가없었다 (Fig. 9, 10). Fig. 5. Effects of Pyrola japonica extract (NJ) on the expression of cathepsin K in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated Osteoclast. Negative control (NC): vehicle, 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. Fig. 6. Effects of homoarbutin (HA) on the expression of cathepsin K in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 100: RANKL (100 ng/ml) +100 μg/ml of HA. 150: RANKL (100 ng/ml)+150 μg/ml of HA. Each bar represents mean±standard deviation of 5 tests. Fig. 7. Effects of Pyrola japonica extract (NJ) on the expression of matrix metalloproteinases-9 (MMP-9) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. 140 J Korean Med Rehabil 2019;29(2):135-147.

4) NFATc1 발현에미치는영향 NFATc1는파구전골세포에서파골세포로분화를촉진하는단백질로신호전달과정은 RANKL/receptor activator of nuclear factor kappa (RANK) 복합체가형성되면서시작되어 tumor necrosis factor receptor-associated factor (TRAF) 가활성화되고 mitogen-activated protein kinase (MAPK) 의 pathway 활성화 (JNK, ERK, p38) 과정을거쳐 transcription factor 의활성화에 NFATc1, nuclear factor kappa light chain enhancer of activated B cells (NF-kB), Akt, Src 등이포함되어있다. 본연구에서는신호전달과정의일부인 NFATc1의발현에 NJ 및 HA가미치는영향을측정하였다. 실험결과 NJ는모든군에서, HA 는 150 μg/ml 농도처리시에서, RANKL 처리된파골세포분화과정에서증가하는 NFATc1 의발현을유의성있게억제하였다 (Fig. Fig. 8. Effects of homoarbutin (HA) on the expression of matrix metalloproteinases-9 (MMP-9) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 100: RANKL (100 ng/ml)+100 μg/ml of HA. 150: RANKL (100 ng/ml)+150 μg/ml of HA. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.05 compared to the control group. Fig. 10. Effects of homoarbutin (HA) on the expression of tartarate-resistant acid phosphatase (TRAP) in osteoclast. Negative control (NC): vehicle. 0 (control group): receptor activator of nuclear factor kappa B ligand (RANKL) (100 ng/ml). 100: RANKL (100 ng/ml)+100 μg/ml of HA. 150: RANKL (100 ng/ml)+150 μg/ml of HA. Each bar represents mean±standard deviation of 5 tests. Fig. 9. Effects of Pyrola japonica extract (NJ) on the expression of tartarate-resistant acid phosphatase (TRAP) in osteoclast. Negative control (NC): vehicle. 0 (control group): receptor activator of nuclear factor kappa B ligand (RANKL) (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. Fig. 11. Effects of Pyrola japonica extract (NJ) on the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean± standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. www.e-jkmr.org 141

박정식 임형호 11, 12). 5) c-fos 발현에미치는영향 RANKL은 Rac1을통해서 c-fos 발현을촉진하고그결과 prostaglandine2 (PGE2) 발현이증가한다. Cyclooxygenase-2 (COX-2) 가활성화되면생성된 PGE2 는 RANKL 과 RANK 의결합을방해하는 OPG 유리를조골세포로부터분리되는것을방해한다. 즉 PGE2 생성이증가하면파골세포의분화가촉진된다. 따라서 c-fos 발현은파골세포분화를촉진할수있다. 실험결과, NJ는모든군에서, HA는 150 μg/ml 농도처리후 RANKL 처리시증가되는 c-fos 발현을유의성있게억제하였다 (Fig. 13, 14). 7) TNF-α 발현에미치는영향 TNF family 계열의수용체는 RANK/OPG 등이있다. 이들의활성화는파골세포의분화를촉진한다. RANKL 과 OPG는조골세포에서유리되어파골세포의분화에관여하는데, OPG 는주로 RANKL 이 RANK 에결합하는것을방해하는작용을한다. 이들은작용시에서로결합되어있어밀접한작용을하는데 TNF-α 는파골세포분화를촉진 6) Inducible neuronal nitric oxide synthase (inos) 발현에미치는영향 Nitric oxide (NO) 는 endothelial 또는 neuronal nitric oxide synthase (NOS) 에의해생성되거나또는 inducible NOS (inos) 에의해생성되는물질로 NOS 에의해생성된다. 파골전구세포에서파골세포로분화하는과정에서 ino는분화조절에관여한다. 실험결과, NJ는모든농도군의분화한파골세포에서 RANKL 처리로증가된 inos 발현을유의성있게억제하였다 (Fig. 15). Fig. 13. Effects of Pyrola japonica extract (NJ) on the expression of cellular Jun-fos (c-fos) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. Fig. 12. Effects of homoarbutin (HA) on the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 100: RANKL (100 ng/ml)+100 μg/ml of HA. 150: RANKL (100 ng/ml)+150 μg/ml of HA. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. Fig. 14. Effects of homoarbutin (HA) on the expression of c-fos in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 100: RANKL (100 ng/ml)+100 μg/ml of HA. 150: RANKL (100 ng/ml)+150 μg/ml of HA. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. 142 J Korean Med Rehabil 2019;29(2):135-147.

하는것으로알려져있다. 실험결과, NJ는 160 μg/ml 농도처리시분화한파골세포에서 RANKL 처리에의해증가한 TNF-α 발현을유의하게억제하였다 (Fig. 16). 8) Interleukin 6 (IL-6) 발현에미치는영향 IL-6는파골세포의 PGs 합성과정을촉진하는사이토카인으로알려져있다. 이는 RANKL 처리된파골세포에서 COX-2 발현과 PGE2 합성증가로나타난다. COX-2 발현으로활성이증가되는 PGE2 는 RANK에 RANKL 이결합하는작용을억제하는 OPG 유리를조골세포로부터떨어지지못하게하는작용을통해 RANKL 에의한파골세포분화를촉진할수있다. 따라서 IL-6의발현증가는 PGE2 생성을증가시키는 COX-2 발현을촉진할수있어파골세포분화를촉진할수있다. 실험결과 NJ는모든농도군의분화한파골세포에서 RANKL 처리에의해증가한 IL-6 발현을유의하게억제하였다 (Fig. 17). 9) COX-2 발현에미치는영향 Fig. 15. Effects of Pyrola japonica extract (NJ) on the expression of inducible neuronal nitric oxide synthase (inos) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml) +40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.05 compared to the control group. Represents p<0.01 compared to the control group. RANKL은 Rac1을통해 COX-2 발현을향상시키며그결과 PGE2 발현이증가한다. COX-2 가활성화되어생성된 PGE2 는 RANK 에 RANKL 이결합하지못하도록하는 OPG 유리를조골세포로부터떨어지는것을방해한다. 그러므로 PGE2 생성이증가하면파골세포의분화가촉진될수있다. 연구결과, NJ는 80 μg/ml, 160 μg/ml 농도처리시분화한파골세포에서 RANKL 처리로증가된 COX-2 발현을유의성있게억제하였다 (Fig. 18). Fig. 16. Effects of Pyrola japonica extract (NJ) on the expression of tumor necrosis factor-alpha (TNF-α) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml) +40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. Fig. 17. Effects of Pyrola japonica extract (NJ) on the expression of interleukin 6 (IL-6) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. www.e-jkmr.org 143

박정식 임형호 Fig. 18. Effects of Pyrola japonica extract (NJ) on the expression of cyclooxygenase-2 (COX-2) in receptor activator of nuclear factor kappa B ligand (RANKL) stimulated osteoclast. Negative control (NC): vehicle. 0 (control group): RANKL (100 ng/ml). 40: RANKL (100 ng/ml)+40 μg/ml of NJ. 80: RANKL (100 ng/ml)+80 μg/ml of NJ. 160: RANKL (100 ng/ml)+160 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to NC group. Represents p<0.01 compared to the control group. Fig. 20. Effect of Pyrola japonica extract (NJ) on osteoclastinduced bone resorption. Negative control (NC): vehicle. Pit formation control (PC): receptor activator of nuclear factor kappa B ligand (RANKL) (100 ng/ml). 35: RANKL (100 ng/ml)+ 35 μg/ml of NJ. 75: RANKL (100 ng/ml)+75 μg/ml of NJ. 150: RANKL (100 ng/ml)+150 μg/ml of NJ. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to PC group. Represents p<0.01 compared to NC group. Represents p<0.05 compared to PC group. Fig. 19. Microscopic image of Pyrola japonica extract (NJ) and homoarbutin (HA)-treated bone pit formation in osteoclast induced bone resorption model. NJ and HA treatment showed significant reduction of bone resorption in osteoclast-loaded bone slice. Pit area was subjected to visualization with inverted microscope(x100). RANKL: receptor activator of nuclear factor kappa B ligand, NC: negative control. 4. 골흡수에미치는영향파골세포에의한골흡수에미치는영향을평가하기위하여파골세포를 loading 한골편에서 pit formation 을측정하였다. 현미경관찰하여사진을판독하였으며, 형광측정기를이용하여측정하였다. 실험결과, NJ 및 HA는골편에서파골세포에의한골흡수를억제하였다 (Fig.19~21). 1 HNMR (CD3OD, 400 MHz):6.87 (d,j=2.8 Hz, 1 H), 6.78 (dd, J=2.8, 8.4 Hz, 1 H), 6.64 (d, J=8.8 Hz, 1 H), 4.72 (d, J=7.2 Hz, 1 H), 3.88 (d, J=11.2 Hz, 1 H), 3.70~ 3.66 (m, 1 H), 3.43~3.30 (m, 4 H), 2.15 (d, J=1.6 Hz, 3 H). 이상의결과를토대로분리된물질은 HA로확인하였다 (Fig. 22). 5. 분자구조결정 녹제초로분리한물질을 NMRfh 분석한결과는다음과같다. 고찰»»» 뼈는끊임없이생성되는흡수되는과정을거치는조직 144 J Korean Med Rehabil 2019;29(2):135-147.

Fig. 21. Effect of homoarbutin (HA) on osteoclast-induced bone resorption. Negative control (NC): vehicle. Pit form control (PC): receptor activator of nuclear factor kappa B ligand (RANKL) (100 ng/ml). 50: RANKL (100 ng/ml)+50 μg/ml of HA. 75: RANKL (100 ng/ml)+75 μg/ml of HA. 150: RANKL (100 ng/ml)+150 μg/ml of HA. Each bar represents mean±standard deviation of 5 tests. * Represents p<0.01 compared to PC group. Represents p<0.05 compared to PC group. Represents p<0.01 compared to NC group. Fig. 22. Chemical Structure of homoarbutin. [4-Hydroxy-3- methylphenyl beta-d-glucopyranoside]. 으로, 골흡수를유도하는파골세포와골생성작용을하는조골세포간의평형이중요하다 13). 정상적인상태에서는조골세포와파골세포의기능이균형을유지하고있지만, 병적인상태가되면파골세포기능이우세하여골다공증과파젯병, 골화석증, 류마토이드관절염등대부분의대사성골질환을유발한다 14). 대사성골질환중골다공증은주로폐경기이후여성에서많이발생하고있으며염증반응에의해서나스테로이드계열의약물처치의경우에발생한다. 폐경기의여성은여성호르몬인에스트로겐이감소되며그결과칼시토닌활성이저하되어 parathyroid hormone (PTH) 의파골세포에대한활성이높아져골흡수가증가한다 15). 염증반응은 inos와 COX의활성증가로인한파골세포에서 MMP-9, cathepsin K 발현을유도하며, 스테로이드계열 약물은조골세포의기능을저하시켜골형성을방해함으로써골다공증을유발한다 16). 녹제초 (Pyrola japonica) 는노루발풀로불리며보허, 익신, 거풍, 제습, 지혈효능이있어, 주로폐허에의한해수, 피로회복, 풍습관절통, 붕루, 백대하, 외상출혈, 옹종창독등에응용이가능하다고알려져있다 8). 본연구에서는녹제초추출물 (NJ) 및그성분인 HA를사용하여, RANKL 자극에의해대식세포인 RAW 264.7이 TRAP 발현이증가하는세포로분화정도를평가하여, NJ 및 HA가강력한파골세포생성과정억제물질인것을발견하였다. RAW 264.7 세포를 RANKL 로자극하여분화시키는방법은파골세포형성을측정하는모델로널리이용되는연구방법이다 17). TRAP은성숙된파골세포에서발현되며 TRAP 양성세포는파골세포로의분화를판단함에사용될수있다. 연구결과, NJ는독성이없는농도에서 RANKL 로유도한파골세포의분화를억제하였고 HA 는유의성있는결과가없었다. 또한 NJ는파골세포특이적인유전자인 TRAP, MMP-9, cathepsin K의발현을억제하였고, 파골세포분화의핵심적인조절인자인 c-fos, NFATc1의발현도억제하였다. 특징적으로는세포분화의신호전달인자인 JNK의인산화를억제하는효과를나타내었다. HA는 150 μg/ml 농도처리시조절인자의발현만유의하게억제하였다. 파골세포표면에존재하는 TNF receptor family 단백질인 RANK에 RANKL이결합하면파골전구세포의분화가촉진된다 18). RANKL 로자극된파골전구세포로부터분화된파골세포는골흡수에관여하는 cathepsin K, OPG, TRAP 등단백질의발현을증가시킨다. NJ 및 HA를처리한파골세포로부터 TRAP 발현을측정한결과, NJ는 TRAP 유전자발현을모든농도군에서유의성있게억제시켰으므로, TRAP의발현을조절하는전사조절인자들의발현을 NJ가저하시킬것으로예상하였다. 파골세포에서는 RANKL 자극으로인한 TRAF 활성화, MAPK pathway 활성화및 Src나 Akt 등의활성화를통해 transcription factor들인 NF-κB, NFATc1 이활성화된다 19). NFATc1은파골전구세포에서파골세포로분화를촉진하는 TRAP과 osteoclast-associated receptor (OSCAR) 의발현을조절하는중요한전사인자이다. 따라서 TRAP의발현이억제되는것은 NJ가 NFATc1 의발현을억제시키기때문인것으로추정되었고, 실험결과에는없지만 OSCAR www.e-jkmr.org 145

박정식 임형호 의발현도 NJ 및 HA에의해억제될것으로예상되었다. NJ는 NFATc1 의발현을억제시킴으로써, 하위단계의유전자들인 TRAP, OSCAR, cathepsin K 등의발현을모두억제시키는것으로판단하였다. Cathepsin K는단백질분해효소로서 RANKL을처리하는경우리소좀에서발현되어파골세포의중요한역할을담당한다 20). 따라서 cathepsin K는 MMP-9, TRAP과함께파골세포의유력한지표로사용되는데, 실험결과 NJ는 cathepsin K 유전자발현을모든농도군에서유의성있게억제하였다. TRAP 발현조절에는 NFATc1 전사인자가관여하며, cathepsin K의발현조절은 microphthalmia-associated transcription factor (MITF) 가관여하므로 NJ에의한 cathepsin K의발현억제는 NFATc1 또는 MITF 전사인자가억제될가능성을나타낸다. 그러나 HA는실험결과유의성있는결과가없었다. 파골세포에서는 RANKL, TNF-α, IL-6 등으로인해활성화되는 inducible NOS에의해 NO가생성된다 17). 파골세포에서 NO는파골전구세포의분화를촉진시키는인자로작용하며, 파골세포분화후에는세포의사멸을억제하는생존인자로작용한다 21). 실험결과, NJ는 inos 발현을모든농도군에서유의성있게억제하였으며, 이는 NO 생성을감소시켜파골세포분화를억제하고, 분화된파골세포의사멸은촉진할것으로추정되었다. COX-2 는 interleukin-1β 등의자극으로파골세포에서발현이증가하며결과적으로 PGE2 의합성을증가시킨다. PGE2는파골세포내의 protein kinase C의활성과 MAPK 의활성증가를촉진하는한편, G-protein에연계된 adenylate cyclase와 cyclic adenosine monophosphate (camp)-dependent PKA 의활성을증가시킨다. 또한 camp 에의존적인 RANKL 과 OPG 의작용을증폭시켜 MMP-9의발현을통한 collagen을분해하여골흡수를촉진하는작용을한다 22). 실험결과, NJ는 COX-2 유전자발현을억제하였다. 따라서하위단계유전자인 MMP-9 의발현이억제될것으로예상되었다. 실험결과, MMP-9의발현도 NJ 및고농도의 HA에의해감소되는것이확인되었다. MMP-9 은파골세포단백질인 TRAP, OPG 등과더불어골흡수를유발한다 23). TNF-α는조골세포콜라겐합성과 osteocalcin의발현을억제한다 24). 더불어 IL-6와 M-CSF 를활성화하여파골세포의분화를증가하고골흡수를유도한다 25). TNF-α 는 IL-1β와같이작용하여 TNF-α 자신의합성을유도 하고 IL-6의합성을유도한다 26). IL-6는염증반응을촉진하고, 또한파골세포의분화와활성화를촉진한다고보고되었다 27). 실험결과, NJ는 TNF-α와 IL-6의발현을억제하였다. 이를토대로 NJ 및 HA가골흡수를억제할것으로판단되어골편에파골세포를부착하여골흡수에영향을현미경하에서관찰하고형광을측정하여정량하였다. 실험결과 NJ 및 HA는파골세포작용으로촉발되는골흡수를억제하였다. 이상의연구결과를토대로 NJ 및고농도의 HA는파골세포분화를차단하고, 기능을억제할수있는것으로판명되었다. 또한파골세포의신호전달과정에서관련유전자발현을억제하는것으로판단되었다. ex vivo 실험에서골손실이억제되는것으로확인되어, NJ 및고농도의 HA는골다공증을예방또는치료할수있을것으로판단되었다. 결론»»» 본연구에서녹제초추출물 (NJ) 및 HA가파골세포의활성과분화를저하시켜골의재흡수를억제하는지를알아보기위해 RANKL 로유도된 TRAP(+) MNCs 형성에미치는영향성을실험하였으며골흡수에관련된인자들의유전자발현과이에관련된신호전달과정에끼치는영향을측정하였다. 또한골편을이용한골기질물질의손실에미치는영향을측정하여정리한결과는아래와같다. 1. 녹제초추출물및고농도의 HA 는 RANKL 유도 TRAP(+) MNCs 형성을차단하여파골세포분화를억제하였다. 2. 녹제초추출물은 RANKL 자극파골세포에서 TRAP, Cathepsin K의유전자발현을억제하였다. 3. 녹제초추출물및고농도의 HA는 RANKL 자극파골세포에서 MMP-9, NFATc1, c-fos 유전자발현을억제하였다. 4. 녹제초추출물은 RANKL 자극파골세포에서 COX-2, inos, TNF-α, IL-6의발현을억제하였다. 5. 녹제초추출물및 HA는파골세포를부착시킨골편에서골기질물질의파괴를억제하였다. 146 J Korean Med Rehabil 2019;29(2):135-147.

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