Inhibitory Effects of Water Extracts of Eucommiae Cortex ...骨萎 ), Golgeuk (骨極 ),...

Inhibitory Effects of Water Extracts of Eucommiae

Cortex and Psoraleae Semen Alone and in

Combination on Osteoclast Differentiation and Bone※

Jin Soo Park1, Ga Young Park1, Han Gyul Choi1, Seong Joung Kim1, June Hyun Kim1,

Min Cheol park2,3, Yun Kyung Kim4,5, Sang Yong Han4,5 and Eun Heui Jo1,3,*

1Department of Acupuncture & Moxibustion Medicine, College of Oriental

Medicine, Wonkwang University2Department of Oriental Medical Ophthalmology & Otolaryngology & Der-

matology, College of Oriental Medicine, Wonkwang University3Research Center of Traditional Korean Medicine, Wonkwang University4Department of Herbal Medicine, College of Pharmacy, Wonkwang Uni-

versity5Wonkwang oriental Medicines Research Institute, Wonkwang University

[Abstract]

Objectives :The�purpose�of�this�study�was�to�evaluate�the�effects�of�water�extracts�of�Eucommiaecortex�(EC),�Psoraleae�semen�(PS),�and�their�combination�on�receptor�activator�of�nuclear�fac-tor-kappa-B�ligand�(RANKL)-induced�osteoclast�differentiation.

Methods : We�assayed�the�protein�expression�levels�of�nuclear�factor�of�activated�T-cells,�cy-toplasmic�1�(NFATc1),�c-Fos,�mitogen-activated�protein�kinases�(MAPKs),�and�β-actin�in�celllysates�using�western�blotting.�Similarly,�mRNA�expression�levels�of�NFATc1,�c-Fos,�tartrate-resistant�acid�phosphate�(TRAP),�and�glyceraldehyde-3-phosphate�dehydrogenase,�sper-matogeni�(GAPDHS)�from�bone�marrow�macrophages�(BMMs)�were�analyzed�using�reversetranscription-polymerase�chain�reaction�(RT-PCR).�Furthermore,�we�determined�the�anti-os-teoporotic�effects�of�the�water�extracts�of�EC,�PS,�and�their�combination�in�a�lipopolysaccharide(LPS)-induced�bone-loss�mouse�model.�

Results : The�in�vitro data�revealed�showed�that�the�combination�of�EC�and�PS�extract�showeda�more�remarkable�inhibition�of�osteoclast�differentiation�than�each�herb�did�alone.�The�com-bination�downregulated�the�induction�of�c-Fos,�NFATc1,�and�TRAP�by�suppressing�the�phos-phorylation�of�p38�and�c-Jun�N-terminal� kinases� (JNKs)�and� inhibiting�nuclear� factorkappa-light-chain-enhancer�of�activated�B�cells�(NF-κB).�Lastly,�the�in�vivo data�showedthat�PS�reduced�the�LPS-induced�bone�erosion.�

Conclusion : The�result�of�this�study�suggests�that�EC�and�PS�could�be�potential�therapeuticagents�for�bone�loss�diseases�such�as�osteoporosis.

※This�study�was�supported�by�academic�research�grant�of�Wonkwang�University�in�2017✱Corresponding�author�:�Department�of�Acupuncture�&�Moxibustion�Medicine,�College�of�Oriental

Medicine,�Wonkwang�University,�460,�Iksandae-ro,�Iksan-si,�Jeollabuk-do,�54538,�Republic�ofKoreaTel�:�＋82－63-270-1022 E－mail�:�[email protected]

Original�ArticleThe Acupuncture Vol. 34 No. 2 May 2017 : 1-18

pISSN 1229-1137 eISSN 2287-7797

http://dx.doi.org/10.13045/acupunct.2017079

Key words : Eucommiae�cortex;�Psoraleae�semen;�Osteoporosis;�Osteoclast�differentiation;�

Bone�resorption

Received : 2016. 11. 28.Revised : 2017. 03. 16.

Accepted : 2017. 05. 02.On－line : 2017. 05. 20.

This�is�an�Open-Access�article�distributed�under�the�terms�of�the�Creative�Commons�Attribution�Non-Commercial�License�(http://creativecommons.org/licenses/by-nc/3.0)�which�permits�unrestricted�non-commercial�use,�distribution,�and�reproduction�in�any�medium,�provided�the�original�work�is�properly�cited.

The�Acupuncture is�the�Journal�of�Korean�Acupuncture�&�Moxibustion�Medicine�Society.�(http://www.TheAcupuncture.org)Copyright��2017�KAMMS.�Korean�Acupuncture�&�Moxibustion�Medicine�Society.�All�rights�reserved.

http://dx.doi.org/10.13045/acupunct.2017079 1

I. Introduction

Boneundergoes a continuous remodelingprocessof regeneration and degradation. Osteoblasts areinvolved in bone-forming while osteoclasts are as-sociatedwiththeboneresorption, andanimbalancebetween these two cell types can lead to the devel-opment of bone-related disorders such as osteo-porosis1). Medications available for osteoporosis inthe modern field of medicine include boneresorption inhibitors and bone-forming agents.Most clinical treatments for osteoporosis involvetheuseofboneresorptioninhibitorsthathavecom-mon side effects including gastrointestinal distur-bance mediated by cells in the T-cell lineage andmonocytes, 3-day flu-like symptoms2), increasedbone mass during the early treatment period, jawbone necrosis with long-term treatment, atypicalsubtrochanteric fracture, and delayed fracture re-covery3). These side effects haveprompted researchon alternative osteoporosis medications derivedfrom natural substances, such as herbal medicinesthathave fewside effects. Furthermore, apreviousstudy has reported that various kinds of herbalmedicines can increase the bone mass of the spinalcolumn more significantly than existingmedications forosteoporosis4).

Although there has been no direct report on os-teoporosis in the oriental medicine literature, os-teoporosis appears to fall under the category ofGolwi (骨萎), Golgeuk(骨極), Golbi (骨痺), andGolgo(骨枯) basedon itsclinical symptomsandpatterns5).In addition, EC and PS have function of Bosin (補腎) according to theories of physiology in orientalmedicine, Sinjugol(腎主骨). Thus, in this study, wepostulated thatECandPSwouldhave therapeuticeffects onosteoporosis6).

Previous studies investigating the therapeuticeffects of EC extracts on osteoporosis using scien-tific methods7-11) have only used ovariectomizedwhite (albino) mice. Furthermore, a studyreportedthat EC extracts inhibit the expression of nuclearfactor of activatedT-cells, cytoplasmic 1 (NFATc1)

by inhibiting the activation of p38, c-Jun N-ter-minal kinases (JNKs), extracellular signal-regu-lated kinases (ERKs), and nuclear factorkappa-light-chain-enhancer of activated B cells(NF-κB)12). However, the experiment conducted inthat study used relatively high intracellularextract concentrations of 50–300 μg/mL12).

Apreviousstudy, whichreportedthetherapeuticeffects of PS extracts in osteoporosis13), only used awhitemousemodelofosteoporosisinducedbyestro-gen deprivation via ovariectomy. Furthermore, thetherapeutic effectsof thePCextractswereassessedin vivo by assaying related biochemical markers in-cluding serumandurine components aswell as andbonedensity13).

Naturalsubstancesandtheirderivativeswithin-hibitory cellular effects on different stages of os-teoclast differentiation have been investigatedincluding deer antler14) and Taxilli ramulus ex-tracts15-26). There has not been any research on thecellular inhibitoryeffects ofECandPSextracts onosteoclast differentiation, inflammatory bone loss,or their mechanisms of action and combinedeffects. Therefore, the present studywas aimed atinvestigating the inhibitory effects of single orcombined administration ofECandPSextracts onosteoclast differentiation in a white mouse modelof experimentally inducedbone loss.

II. Materials and Methods

1.�Materials

The receptor activator of nuclear factor-kappa-B ligand (RANKL) and macrophage colony-stimu-lating factor (M-CSF) used in our research werepurchased from Peprotech (London, UK). The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay kit waspurchased from Roche (Indianapolis, IN, USA).Phosphorylated-JNK(p-JNK), JNK, p-ERK, ERK,

2 http://dx.doi.org/10.13045/acupunct.2017079

The�Acupuncture�Vol.�34�No.�2�May�2017

p-p38, p38, and inhibitor of NF- κB (I-κB) anti-bodies were purchased from Cell Signaling Tech-nology (Beverly, MA, USA). c-Fos, NFATc1, andactin antibodies were purchased from Santa CruzBiotechnology (SantaCruz, CA, USA).

2.�Methods

1)�Preparation of EC and PS extractsThe EC and PS material used in this study were

from Yeongchun of Gyeongbuk Province andChina, respectively, andwerepurchasedfromOm-niherb Company. The EC and PS samples wereevenly cut into pieces, macerated for 30 min, andboiled in a heating mantle for 2 h. Freeze-driedsamples of the concentrates were used in our ex-periments, and their yields are shown in Table 1.Purifiedwater (1 L) wasaddedto100 gofeachherband heated in a Glas-Col (Terre Haute, IN, USA)heating mantle for 2 h for the extraction. The ob-tained extractswere filtered, concentratedusingarotary vacuum evaporator, freeze-dried, and thenpowdered for use in the experiments. The extractcombination was prepared by mixing the EC andPS extracts in a 1:1 ratio, followed by further ex-traction and processing using the procedure de-scribed above.

2)�Osteoclast culture and differentia-tion inhibition

Bonemarrowcellswere obtainedbyeuthanizing5-week-old male ICR mice by dislocating the cer-vical vertebrae. The tibias and femurs were thenseparated, and a 1× antibiotic solution containingα-minimum essential medium (MEM) wasinjected into the bones using a 1-cc syringe to

flush out the bone marrow inside. Following re-moval of the redblood cells, thebonemarrowcellswere cultured in α-MEM medium containing 10%fetal bovine serum (FBS), antibiotics, and M-CSF(30 ng/mL) for3 days. Acellsuspensioncontaininglymphocytes was removed, and the adherent cellswereusedasbonemarrowmacrophages(BMMs) ofosteoclastprogenitors. TheBMMswereseededintoa48-well plateat 3.5 × 104 cells/well andculturedwith different concentrations of EC or PC extractsor their combination in a growth medium contain-ing M-CSF (30 ng/mL) and RANKL (100 ng/mL).Thegrowthmediumwasreplaced3 days later, andthe cells were stained with tartrate-resistant acidphosphate (TRAP) solution4 days later. Redviolet-coloredTRAP-positivecellswereconsideredosteo-clasts and those with three or more nuclei wererecorded for statistical analysis.

3)�Toxicity test

BMMswereseeded intoa96-well plateat 1 × 104

cells/wellandculturedwithM-CSF(30 ng/mL), fol-lowedbytheextractsatdifferentconcentrationsfor3 days. Then, 50 μL XTT was added to each well,and after a 4-h incubation, the optical density wasmeasured at 450 nm using an enzyme-linked im-munosorbentassay (ELISA) plate reader (MolecularDevices, CA, USA).

4)�Western blot analysis

The cultured cells were washed 2–3 times with1 × phosphate-buffered saline (PBS) to removeany residue and dissolved in a lysis buffer (50 mMTris-chloride [Cl], 150 mM sodium chloride [NaCl],5 mM ethylenediaminetetraacetic acid [EDTA], 1%TritonX-100, 1 mMsodiumfluoride, 1 mMsodiumvanadate, 1% deoxycholate, and protease


Inhibitory�Effects�of�Water�Extracts�of�Eucommiae�Cortex�and�Psoraleae�Semen�Alone�and�in�Combination�on�OsteoclastDifferentiation�and�Bone�

Table 1. Extraction�Yield�of�Medicinal�Herbs�Used�in�the�Study

Korean name Pharmaceutical name Abbreviation Yield (%)

Duchung Eucommiae Cortex EC 11.1

Bogolji Psoraleae Semen PS 16.9

Duchung plus Bogolji Eucommiae Cortex plus Psoraleae Semen EC + PS 14.4

inhibitors). The solution was centrifuged (14,000rpm, 20 min) to obtain the proteins, which werequantified using a DC protein assay kit (Bio-Rad,Hercules, CA, USA), and separated using 10%sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE). The separated proteinswere transferred to a polyvinylidene fluoride(PVDF) membrane, which was blocked with 5%skimmilkinTris-bufferedsalineplusTween(TBS-T) to prevent nonspecific reactions, at 20℃ for 1 h,followedbyincubationwiththeprimaryantibodiesat 4°C. Following the antibody reaction, the PVDFmembranewaswashedthricewithTBS-Tevery10minand then incubatedwith secondary antibodies(1:5000) at room temperature for 1 h. Afterwashing the PVDF membrane thrice with TBS-T,an enhanced chemiluminescence (ECL) kit wasused to develop and visualized the blots to assesstheprotein expression levels.

5)�Reverse transcription-polymerasechain reaction (RT-PCR)�analysis

IntracellularRNAwasseparatedusing1 mL isol-RNAlysis reagent (5PRIME) according to theman-ufacturer’s instructions. Briefly, 0.5 pg–1 μgof theseparatedRNAwasusedtosynthesizecDNAusingthe ReverTra Ace® quantitative polymerase chainreaction(qPCR) reversetranscription(RT) kit (Toy-obo, Japan). The synthesized cDNA was amplified

usingPCRwith theprimers listed inTable2. Then,the amplifiedDNAwas electrophoresedusinga 1%agarose gel, stained with ethidium bromide (Et-Br), and subsequently visualized under ultraviolet(UV) light.

6)�Animal experiment

To investigate the effects of the herbal extractson bone loss in vivo, five 5-week-old male ICRmicewereassignedtoeachofthefollowinggroups:EC, PS, combination, and PBS groups (0.4 or 0.2mgperbodyweight ing). Themicewereorally ad-ministeredthepreassignedtreatmentsolutionsfor10 days. To induce bone loss, lipopolysaccharide(LPS) was intraperitoneally injected into the miceintheexperimentalgroupsonday4 and7 afterex-tract administration (day 1)while thecontrolgroupmicewere injectedwithPBSto reduceexperimentalerrors. On day 10, the mice were euthanized, theirfemurswereharvested, fixedwith4% paraformalde-hyde, and then three-dimensional (3D) images ofthe interior were captured using micro-CT (Bruker,Kartuizersweg 3B, 2550 Kontich, Belgium, Skyscan1172).

7)�Statistical analysis

The statistical analysis was performed thrice ormore for each experimental group, and the resultsare presented as the means ± standard deviation



Primer Sequence

c-FosForward 5'- CTGGTGCAGCCCACTCTGGTC-3'

Reverse 5'- CTTTCAGCAGATTGGCAATCTC-3'

NFATc1*Forward 5'- CAACGCCCTGACCACCGATAG-3'

Reverse 5'- GGCTGCCTTCCGTCTCATAGT-3'

TRAP†Forward 5'-ACTTCCCCAGCCCTTACTAC-3'

Reverse 5'-TCAGCACATAGCCCACACCG-3'

GAPDH‡Forward 5'-ACCACAGTCCATGCCATCAC-3'

Reverse 5'-TCCACCACCCTGTTGCTGTA-3'

Table 2. Primers�Used�for�Polymerase�Chain�Reaction�(PCR)�Amplification

* NFATc1, nuclear factor of activated T-cells, cytoplasmic1. † TRAP, tartrate-resistant acid phosphate. ‡ GAPDHS, glyceraldehyde-3-phosphate dehydrogenase, spermatogeni.

(SD). All experimentswererepeatedthriceormore,andonlyconsistent resultswereused in thestatis-tical analyses. For quantitative results, the Stu-dent’s t-test was used. The results of the animalexperiments did not meet the assumption of nor-mality since each group contained only fiveanimals. Therefore, nonparametric Kruskal-Wallis, as well as Mann-Whitney tests, were per-formed to analyze results. The level of statisticalsignificancewas set at p < 0.05.

III. Results

1)�InhibitoryeffectsofECandPSwaterextracts in combination on RANKL-induced osteoclast differentiation

The differentiation of osteoclasts, which resorbbone, plays an important role in inducing osteo-porosis. In this study, we investigated the effectsofECandPSextracts incombinationonosteoclastdifferentiation. BMMs were treated with M-CSFand RANKL at concentrations of 5 and 10 μg/mL



Fig. 1.�Eucommiae�cortex�(EC)�plus�Psoraleae�semen�(PS)�water�extracts�inhibit�osteoclast�differentiation

Bone marrow macrophages (BMMs) were cultured for 4 days with macrophage colony-stimulating factor (M-CSF, 30 ng/mL) and recep-tor activator of nuclear factor-kappa-B ligand (RANKL, 100 ng/mL) in the presence or absence of EC and PS water extracts. (A) After 4 days, the cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton x-100, and stained for tartrate-resistant acid phos-phate (TRAP). (B) TRAP-positive cells were counted as osteoclasts. *Statistically different from control (***p < 0.001).

A

B

and cultured. The differentiation of BMMs intoTRAP-positive multinucleated osteoclasts was ob-served in the control group, which was treatedwith M-CSF and RANKL only. However,osteoclast differentiation was inhibited in the ECandPSgroups(Fig. 1A). Thenumberofmultinucle-ated osteoclasts after staining with TRAP wascounted, and the results revealed that differentia-tion into multinucleated osteoclasts was signifi-cantly inhibitedbytheECandPSextracts (Fig. 1B).In the subsequent experiments, the EC and PS ex-tractswereused at a concentration of 10 μg/mL.

2)�Effects of ECandPSextracts aloneon RANKL-induced osteoclast dif-ferentiation

The combination of the EC and PS extracts in-hibited osteoclast differentiation and, therefore,we investigatedtheir individualeffectsonRANKL-inducedosteoclastdifferentiation. BMMswerecul-tured with M-CSF and RANKL and treated withEC or PS extracts at concentrations of 5 or 10μg/mL. In contrast to BMMs in the control group,which were treated with M-CSF and RANKL only,the EC group showed inhibition of the productionof TRAP-positive multinucleated osteoclasts. Fur-



Fig. 2.�Eucommiae�cortex�(EC)�and�Psoraleae�semen�(PS)�inhibit�osteoclast�differentiation

Bone marrow macrophages (BMMs) were cultured for 4 days with macrophage colony-stimulating factor (M-CSF, 30 ng/mL) and recep-tor activator of nuclear factor-kappa-B ligand (RANKL, 100 ng/mL) in the presence or absence of EC or PS.(A) After 4 days, cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton x-100, and stained for tartrate-resistant acid phosphate(TRAP).(B) TRAP-positive cells were counted as osteoclasts. *statistically different from control (***p < 0.001).

A

B

thermore, thedifferentiationofBMMsintomature,multinucleated osteoclasts was also inhibited.However, this inhibition was not significant com-paredwiththatobservedinthecombinationgroup.ThePS extract inhibited osteoclast differentiation;however, the cells treated at a concentration of 10μg/mLexhibited cytotoxicity (Fig. 2).

2.�CytotoxicityofECandPSextracts

To eliminate the involvement of cytotoxicity inthe inhibitory effects of EC and PS extracts aloneand in combination on osteoclast differentiation,an XTT assay was performed using BMMs treatedwith different concentrations. Based on the cellsurvival rate of the untreated control group thatwas considered tobe 100%, the survival rateswere103.2%, 92.7%, 91.6%, and 81.0% in the EC groupmice treated with extract concentrations of 5, 10,20, and 40 μg/mL, respectively. Furthermore, sur-vival rates of 80.6%, 73.5%, 69.9%, and71.4% werefound in the PS group mice treated with extractconcentrations of 5, 10, 20, and 40 μg/mL. Thecombination treatment showed survival rates of80.4%, 86.7%, 81.5%, and 73.5% at extract concen-

trationsof5, 10, 20, and40 μg/mL. Withacell sur-vival rate ≥ 70% as the minimum standard for nocytotoxicity, a slightcytotoxicitywasobserved fol-lowing administration of the PS extract at 20μg/mL while no cytotoxicity was observed in theEC and combination groups. Based on this result,weset theconcentrationof theECandPSextractsat 5 μg/mL and that of the extract combination at10 μg/mL. At this concentration, the extract com-bination exhibited synergistic effects over those ofthe EC and PS extracts while cytotoxicity was notobserved (Fig. 3).

3.�Inhibitory effects onRANKL-me-diated expression of osteoclastdifferentiation-inducing factors

1)�Effectsof thecombinationofECandPS extracts on RANKL-inducedgene expression

RANK binds RANKL to induce the expression oftranscription factors such as c-Fos and NFATc1via various intracellular signaling pathways,thereby inducing the expressionofTRAP, which isa marker of osteoclasts. To investigate the effects



Fig. 3.�Effect�of�Eucommiae�cortex�(EC),�Psoraleae�semen�(PS),�and�EC�+�PS�water�extracts�on�cell�viability

Bone marrow macrophages (BMMs) were cultured for 3 days with macrophage colony-stimulating factor (M-CSF, 30 ng/mL) in the pres-ence or absence of EC, PS, and EC + PS water extracts. After 3 days, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) solution was added to each well, followedby incubation for 4–6 h. Reaction solution in plate was measured at 450 nm using a microplate reader.



oftheECandPSextractsaloneandincombinationon the gene expression of c-Fos, NFATc1, andTRAP, weperformedRT-PCR. The results showedincreased mRNA expression of c-Fos, NFATc1,andTRAPinducedbyRANKLonday1–2. However,themRNAexpressionofc-Fos, NFATc1, andTRPAwas significantly inhibited in the combinationgroup (Fig. 4).

2)�Effects of EC and PS alone onRANKL-inducedproteinexpression

The RT-PCR results showed increased mRNAexpression of c-Fos, NFATc1, and TRAP inducedby RANKL on Day 1–2. The mRNA expression ofNFATc1 was significantly inhibited in the EC andPSgroups. ThemRNAexpressionofTRAPwassig-

nificantly inhibited on Day 2 in the EC group, andonDay 1 in thePSgroup (Fig. 5).

4.�Inhibitory effects on RANKL-in-duced expression of c-Fos andNFATC1�proteins

1)�Effectsof thecombinationofECandPS extracts on RANKL-induced ex-pression of c-Fos andNFATc1�pro-teins

RANKL induces C-Fos expression, thusinducingtheexpressionofNFATc1, whichplaysanimportant role in osteoclast differentiation. West-ern blotting was performed to investigate the ef-

Fig. 4.�Eucommiae�cortex�(EC)�plus�Psoraleae�semen�(PS)�water�extracts�suppress�receptor�activator�of�nuclearfactor-kappa-B�ligand�(RANKL)-induced�c-Fos,�nuclear�factor�of�activated�T-cells,�cytoplasmic�1�(NFATc1),�andtartrate-resistant�acid�phosphate�(TRAP)�expression

Bone marrow macrophages (BMMs) were pretreated with or without EC and PS water extracts (10 μg/mL) for 1 h and then stimulatedwith RANKL (100 ng/mL) for indicated times (1 day [24 h] and 2 days [48 h]).(A) mRNA expression was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). (B) Quantitative analysis using ImageJ program. Significance: *indicates significant difference between control and extraction treatmentgroup (***p < 0.001). Error bars indicate standard deviation (SD), n = 3.GAPDHS, glyceraldehyde-3-phosphate dehydrogenase, spermatogeni.

A

B

fects of EC and PS extracts alone and in combina-tion on the expression of c-Fos and NFATc1 pro-

teins. The expression of NFATc1 was increased 12,24, and48 hafterRANKLtreatment ina time-de-



Fig. 5.�Eucommiae�cortex�(EC)�and�Psoraleae�semen�(PS)�suppress�receptor�activator�of�nuclear�factor-kappa-Bligand�(RANKL)-induced�c-Fos,�nuclear�factor�of�activated�T-cells,�cytoplasmic�1�(NFATc1),�and�tartrate-resis-tant�acid�phosphate�(TRAP)�expression

Bone marrow macrophages (BMMs) were pretreated with or without EC and PS (5 μg/mL) for 1 h and then stimulated with RANKL (100ng/mL) for indicated times (1 day [24 h], 2 days [48 h]).(A): mRNA expression was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). (B) Quantitative analysis using ImageJ program. *Significant difference between control and extract treatment groups (*p < 0.05 and ***p < 0.001). Error bars indicate standard deviation (SD); n = 3.GAPDHS, glyceraldehyde-3-phosphate dehydrogenase, spermatogeni.

A

B

Fig. 6.�Eucommiae�cortex�(EC)�plus�Psoraleae�semen�(PS)�water�extracts�suppress�receptor�activator�of�nuclearfactor-kappa-B�ligand�(RANKL)-induced�c-Fos�and�nuclear�factor�of�activated�T-cells,�cytoplasmic�1�(NFATc1)expression

Bone marrow macrophages (BMMs) were pretreated with or without EC and PS water extracts (10 μg/mL) for 1 h and then stimulatedwith RANKL (100 ng/mL) for indicated times. Cell lysates were analyzed using western blotting with antibodies for c-Fos, NFATc1, and β-actin.



pendent manner, and that of c-Fos was increasedat 12 and 24 h. However, the expression of c-FosandNFATc1 proteinswasinhibitedinthecombina-tion treatment group (Fig. 6).

2)�Effects of ECandPSextracts aloneonRANKL-inducedexpressionofC-Fos and NFATc1�proteins

The expression of NFATc1 increased in a time-dependent manner 12, 24, and 48 h after RANKLtreatment while that of c-Fos was increased 12and24 hafterRANKLtreatment. Nochangeintheexpressionlevelofc-FosandNFATc1 proteinswasobserved in the PS group, whereas the expressionof NFATc1 protein was inhibited in the EC group(Fig. 7).

5.�Effects on signaling pathways ofosteoclast differentiation

1)�Effectsof thecombinationofECandPS extracts on signaling pathwaysofRANKL-inducedosteoclastdiffer-entiation

Weinvestigated theeffectsofECandPSextractsalone and in combination on major signaling path-

ways induced by RANKL to identify themechanisms of osteoclast differentiation inhibition.BMMswerepretreatedwith theECandPSextractsaloneor incombination, followedbyRANKLat0, 5,15, and30 min, andMAPKphosphorylationwasas-sessed. While phosphorylation of AKT, p38, andJNK was increased in the EC and PS groups, phos-phorylationofp38 andJNKwassimultaneously in-hibitedinthecombinationgroup. TheproteolysisofI-κBwasalso inhibitedbyRANKL(Fig. 8).

2)�Effects of ECandPSextracts aloneonsignalingpathwaysofRANKL-in-duced osteoclast differentiation

BMMs were pretreated with EC and PS extracts,followed by RANKL at 0, 5, 15, and 30 min, andthen MAPK phosphorylation was assessed. Thephosphorylation of AKT and p38 was inhibited inthe EC group, and that of p38 and JNK was inhib-ited in thePSgroup (Fig. 9).

6.�Effects on LPS-induced inflam-matory bone loss

A mouse model of inflammatory bone loss wasusedto investigate the invivoeffectsofECandPS

Fig. 7.�Eucommiae�cortex�(EC)�and�Psoraleae�semen�(PS)�suppresses�receptor�activator�of�nuclear�factor-kappa-B�ligand�(RANKL)-induced�c-Fos�and�nuclear�factor�of�activated�T-cells,�cytoplasmic�1�(NFATc1)�expression

Bone marrow macrophages (BMMs) were pretreated with or without EC and PS (5 μg/mL) for 1 h and then stimulated with RANKL (100ng/mL) for indicated times. Cell lysates were analyzed using western blotting with antibodies for c-Fos, NFATc1, and β-actin.



Fig. 8.�Eucommiae�cortex�(EC)�plus�Psoraleae�semen�(PS)�water�extracts�inhibit�receptor�activator�of�nuclear�fac-tor-kappa-B�ligand�RANKL�signaling�pathways

Bone marrow macrophages (BMMs) were pretreated with or without EC and PS water extracts (10 μg/mL) and further stimulated withRANKL(100 ng/mL) for indicated times. Cells were lysed, proteins were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and sub-jected to western blot analysis.p-JNK, phosphorylated-c-Jun N-terminal kinase; inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells, I-κB.

Fig. 9.�Eucommiae�cortex�(EC)�and�Psoraleae�semen�(PS)�inhibit�receptor�activator�of�nuclear�factor-kappa-B�lig-and�RANKL�signaling�pathways

Bone marrow macrophages (BMMs) were pretreated with or without EC and PS (5 μg/mL) and further stimulated with RANKL (100 ng/mL)for indicated times. Cells were lysed, proteins were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and sub-jected to western blot analysis.p-JNK, phosphorylated-c-Jun N-terminal kinase; inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells, I-κB.

extracts alone and in combination. LPS (5 μg/g)was intraperitoneally injected into the mice onceevery 3 days, and then each extract was orally ad-ministered once daily at various concentrations.Theextent ofLPS-inducedbone losswasassessedbased on 3D images of the femur specimens ob-tained using the Skyscan BE/skyscan 1172 (Table3).

The mean bone loss was 16.9% in the LPS-treated group compared to that in the controlgroup. No bone loss was observed in groupstreated with the single EC and PS extracts alone.The bone volume/tissue volume (BV/TV) was sig-nificantly increased in the group treated with thePS extract and LPS-stimulated, indicating a sig-nificant recovery of bone loss. No bone lossrecovery was observed in the group treated withthe extract combination (Fig. 10).

IV. Discussion

Osteoporosis is a systemic musculoskeletal dis-order characterized by reduced bonemass andmi-crostructureabnormalities and is amajorgeriatricdisease that causes the bones to weaken and be-come brittle27). The aging population has led to an

increase in thenumberofpatientswithosteoporo-sis.28 Bone diseases such as osteoporosis are be-lieved to be caused by excessive proliferation andactivation of osteoclasts, which degenerate bone.Therefore, medications that can inhibit osteoclastactivitieshavebeenwidelyusedtotreatosteoporo-sis. These medications include bisphosphonates,which mechanically inhibit bone resorption by os-teoclasts and induce osteoclasts to undergo apop-tosis.29 While bisphosphonateshavebeen shown tobe effective in the treatment of osteoporosis, theycanresult inserioussideeffectssuchasnecrosisofthe jaw bone in patients with periodontaldiseases.30 For this reason, much attention hasbeen focused on the development of osteoporosismedications from natural substances that tend tohave few side effects. Natural substances derivedfromplantshavebeenwidelyused to treatvariousdiseases from ancient times. Identifying theirmechanisms of action using modern researchmethods as a strategy to assess their values as os-teoporosismedicationswould contribute to thede-velopment of osteoporosis medications fromnatural substances.

EC is the dried bark of Eucommia ulmoidesOLIV.), whichbelongstotheEucommiaceaefamily.The bark is peeled off the trees in April and May,cut, stacked, and dried until the inner side turnspurplishbrown. ECismild, non-toxic (溫無毒), and



CTR* EC†200 PS‡200LPS§+

- EC200 PS200 EC+PS200 EC+PS200

BV/TV (%) 30.9±0.0 28.6±0.4 31.5±0.9 25.7±0.7 26.5±1.4 30.6±1.9 26.5±2.5 26.3±2.8

Trabecularthickness(㎛)

15.6±0.0 15.1±0.7 16.3±0.5 14.8±1.0 15.2±1.4 16.0±0.2 15.1±6.9 15.4±0.1

Trabecularseparation(㎛)

35.0±0.0 37.7±1.2 35.4±0.2 42.8±1.4 42.2±6.2 36.5±2.8 42.1±6.9 43.7±6.5

Trabecularnumber(1/㎛)

0.020±0.0 0.019±0.0 0.019±0.0 0.017±0.0 0.018±0.0 0.019±0.0 0.018±0.0 0.017±0.0

Table 3. Skyscan�Analysis�of�Lipopolysaccharide�(LPS)-induced�Bone�Loss�in�Mice

* CTR, control group † EC, Eucommiae Cortex‡ PS, Psoraleae Semen asdasd§ LPS, lipopolysaccharideBV/TV, bone volume/trabecular volume

has a sweet and slightly spicy taste (甘微辛). It ef-fects on (歸經) the kidney and liver meridian (肝腎經) and strengthens muscle and bone (强筋骨) and,therefore is used to treat numbness and weakness

of feet and knees (足膝痿弱) and other diseases6). Itis combined with Psoraleae semen to treat chillsand pain in the waist and knees (腰膝酸痛) andmusculoskeletal weakness (筋骨無力) caused by

liver andkidney yangdeficiencies (肝腎不足)31).Psoraleae fructus is the dried ripe fruit of Pso-

ralea corylifolia L., which is an annual herb thatbelongs to theLeguminosae family. It is harvestedin September and then dried. Psoraleae fructus ismild andnon-toxic (溫無毒) withabitter and spicytaste (辛苦). It effects on (歸經) the kidney andspleen meridian (腎脾經), and adding Yang energy



Fig. 10.�Skyscan�analysis�of�lipopolysaccharide�(LPS)-induced�bone�loss�in�mice

(A) Mice were orally treated with Eucommiae cortex (EC), Psoraleae semen (PS), and EC + PS extracts (200 or 400 mg·kg-1·day-1) or1× phosphate-buffered saline (PBS) daily for 10 days. LPS (5 µg/g body weight) or 1× PBS was administered intraperitoneally on day 4and 7, and the femurs were harvested on day 10 after EC, PS, and EC + PS extract treatments. Radiographs were captured using Sky-scan. (B) Bone volume/tissue volume (BV/TV), trabecular thickness, separation, and number were analyzed using histomorphometric results.*Statistically different from LPS group. (*p < 0.05).

A

B

to kidney-Gi with warm medicine (溫腎助陽 ).Therefore, it is used to treat cold feeling and painin the waist and knees (腰膝冷痛)6). It is combinedwith EC to treat kidney yang deficiency and backpain31).

Considering the description of osteoporosis as

“腎主骨髓” in《素問∙陰陽應象大論》, as“腎主骨” in《素問∙宣明五氣》, and as“腎之合骨也” in《素問∙五藏生成論》, Sinheo (腎虛) can be recognized as the maincause of osteoporosis, and medications that Bosin(補腎) may be effective in the treatment of osteo-porosis, a geriatric disease. We hypothesized thatEC and PS, both of which enhance kidney yang,can effectively treat osteoporosis. Therefore, weinvestigatedtheinhibitoryeffectsoftheECandPSextracts alone and in combination on osteoclastdifferentiation andbone resorption.

M-CSF and RANKL are cytokines that are es-sential for the differentiation of stem cells intomultinucleated osteoclasts32). RANK binds withRANKL to promote the expression of c-Fos andNFATc1 via intracellular signaling pathways,thereby inducing the expressionofTRAP, which isa marker of osteoclasts. TRAP is the only bone re-sorption factor expressed by mature osteoclastsand can be used to determine their differentiationstage33). TRAP-positive cellswere stained to inves-tigate the inhibitory effects of EC and PS extractsand their combination on osteoclastdifferentiation, which was found to besignificantly inhibited by the extract combination.While osteoclast differentiation was also inhibitedincells treatedwitheachextractofECorPSalone,this inhibition was not significant compared tothat observed in the combination group. AlthoughEC and PS extracts inhibited osteoclast differenti-ation, their combination had stronger effects thaneach extract did alone. Furthermore, while the PSextract showed cytotoxicity, the EC extract andcombination treatment showed no cytotoxicity,suggesting that EC may mitigate the cytotoxicityof PS. The combination of the EC and PS extractsmay provide important insights that could con-tribute to thedevelopmentofosteoporosismedica-

tions.The c-Fos protein, whose expression is induced

by RANKL, induces the expression of NFATc1 andplays an important role in osteoclastdifferentiation. Westernblottingwasperformedtoinvestigate the effects of EC and PS extracts andtheir combination on the expression of c-Fos andNFATc1 proteins. The expression of NFATc1 wasincreased ina time-dependentmanner 12, 24, and48 h after RANKL treatment. The expression of c-Fos was increased at 12 and 24 h after RANKLtreatment; however, the expression of c-Fos andNFATc1 was inhibited in the combination group(Fig. 4). On the other hand, no change in the ex-pression level of c-Fos and NFATc1 was observedin the PS group while the expression of NFATc1was inhibited in this group. The extract combina-tion inhibited osteoclast differentiation more ef-fectively than the single ES or PS extracts didalone, possibly because the EC extract likely en-hanced the inhibitory effects of the PS extract onthe expression ofNFATc1 proteins.

C-Fos is a transcription factor that is expressedwithin 30 min after RANKL stimulation. C-Fos in-creases the expression of NFATc1, which subse-quently induces the expression of theosteoclast-specificmarkergenessuchasTRAPandcathepsinKto induceosteoclastdifferentiation. In-hibiting the expression of c-Fos reduces that ofNFATc1, whichleadstoreducedexpressionofTRAP,an osteoclast-specific marker34-35). We performedRT-PCRtoassess theexpression levelof thec-Fos,NFATc1, and TRAP genes and observed increasedmRNA expression of c-Fos, NFATc1, and TRAP in-duced by RANKL on day 1–2. However, the mRNAexpressionofc-Fos, NFATc1, andTRAPwassignif-icantly inhibited in the combination group.Although the EC and PS extracts significantly in-hibitedtheexpressionofc-Fos, NFATc1, andTRAP,which are the key genes involved in the process ofosteoclast differentiation induced by RANKL, theinhibition of NFATc1 expression by EC and PS ex-tracts was not significant compared to that by theextract combination. The expression of c-Fos was



not inhibited by EC and PS extracts. This explainswhy the inhibition of osteoclast differentiation wasmore evident in the combination group than it wasin the EC and PS groups, and indicates that thecombination of EC and PS extracts inhibits the keymechanismofosteoclastdifferentiation.

Inosteoclast precursor cells, the cytoplasmicdo-main of RANK binds TRAFs following exposure toan RANKL-induced stimulus to activate proteinsinvarioussignalingpathways. P38, JNK, andERK,whichareMAPKs, areknowntobe involved in im-portant signaling pathways of osteoclast differen-tiation36). MAPKs play an important role in theexpression of c-Fos. SB203580 and SP600125,which inhibit p38 and JNK, respectively, also in-hibit RANKL-induced expression of cFos and,thus, the activation of p38 and JNK could be con-sidered an important intermediate process in os-teoclast differentiation. The inhibition of differentstages of the RANKL signaling pathway to inhibitosteoclast production could facilitate the develop-ment of a therapeutic approach for osteoporosis.We assessed the effects of EC and PS extracts andtheircombinationontheactivationofsignaltrans-ducerstoinvestigatetheirmechanismofinhibitionof NFATc1. The combination ofECandPS extractsinhibitedtheactivationofp38 andJNK, suggestingthat the inhibitionofNFATc1 expressionby the ex-tract combination may be associated with the acti-vationofp38 andJNK. PhosphorylationofAKTandp38 was inhibited in theECgroupwhile thatofp38and JNK was inhibited in the PS group. This sug-gests that the effects of the PS extract on the rele-vantsignalingpathwaysweregreater thanthoseofEC in theextract combination.

NF-κB is a transcription factor involved in dif-ferentiation, survival, and activation of variouscells and is known tobe involved in the expressionof NFATc1 in osteoclasts37). I-κB binds NF-κB inthe cytoplasm to prevent it from traveling to thenucleuswhileRANKLdestroysI-κBtoenableNF-κBtotranslocate thenucleus. Continuousexpres-sion of I-κB inhibits NF-κB activity andultimately inhibits differentiation into

osteoclasts38). WeinvestigatedtheeffectsofECandPS extracts and their combination on theactivation of NF-κB. The level of I-κB was re-duced at 5 and 10 min as I-κB was degraded byRANKL. However, in the combination group, thelevelof I-κBwasunchangedover time. Therefore,the inhibition of NFATc1 expression by the combi-nationoftheECandPSextractsmaybeassociatedwith inhibition ofNF-κBactivation.

A study using an LPS-induced rat model of in-flammation, which is commonly used in researchon bone loss and destruction in chronic inflamma-toryenvironments, reported thatosteoclastdiffer-entiation is promoted by LPS39). Furthermore, theauthors reported that their observations were at-tributable to theLPS-inducedexpressionof tumornecrosis factor (TNF)-α, which subsequently in-creased the expression of the proto-oncogene ty-rosine-protein kinase Src (c-src)39). Bone lossresults from the effects of inflammatory cytokineexpression, whichisincreasedbyLPSaswellasin-flammatory mediators such as prostaglandin E2

(PGE2)40). Results in this studies have suggestedthat single extracts of EC and PS and their combi-nation inhibitosteoclastdifferentiationtopromotea recovery from bone loss. Therefore, we investi-gated the effects of single EC and PS extracts andtheir combination in an animal model of LPS-in-duced inflammation.

EC has been reported to increase bone densityand bone strength in white rat models of inducedosteoporosis when coadministered with PS andtreadmill exercise and, therefore, could beeffective in thetreatmentofosteoporosis41). ECad-ministered to ovariectomized white rats has beenobserved to increase the trabecular bone volume10).PS has been observed to reduce the serum level ofosteocalcin and increase the level of phosphorusand calcium in bones in ovariectomized white ratsand, thus, has been reported to be effective in theprevention and treatment of osteoporosis42).Basedon these observations, we investigated the effectsof EC and PS extracts and their combination onLPS-induced bone loss. The results showed that





singleextractsofECandPSandtheircombinationdid not promote recovery of LPS-induced boneloss. TheBV/TVwas increased in thePSgroup, in-dicating a significant recovery of bone loss com-pared to the group treated with LPS alone. Nosignificant changes in the trabecular thickness,separation, numberwere observed although slightimprovements were evident. This finding may beassociated with the previous observation whereNFATc1 expression and phosphorylation of p38and JNK were inhibited in the PS group. Since EChaspreviouslybeenreported to increaseboneden-sity inanovariectomizedmodel, itappearsthatECaffectsbonemetabolismviapathways thatarenotassociatedwith inflammation.

In summary, EC and PS inhibited osteoclast dif-ferentiation, and their inhibitory effects were sig-nificantly stronger when the extracts werecombined. ThemechanismsofactionoftheECandPS extracts were associated with the inhibition ofNFATc1 expression by the inhibition of p38, JNK,and NF-κB activation. However, in the in vivoLPS-induced bone loss model, a more significantrecovery of bone loss was observed in animalstreated with PS, which inhibited p38 and JNKphosphorylation and the expression of NFATc1proteins, than in those treated with the extractcombination. This study is meaningful because tothebestofourknowledge, it isthefirststudytore-porttheinhibitoryeffectsofthecombinationofECand PS extracts on osteoclast differentiation andbone resorption. Further investigation on the syn-ergistic effects of ES and PC extracts, their com-bined ratio, and whether the signaling pathwaysthey affect are independent ornot is necessary.

V. Conclusion

We investigated whether single extracts of EC

and PS and their combination had inhibitoryeffects on RANKL-induced osteoclast differentia-tionaswell as bone resorption.

1. Based on the TRAP assay results, RANKL-in-ducedosteoclastdifferentiationwasmoresig-nificantly inhibited by the combination of ECand PS extracts than by each of the extractsalone.

2. TheECandPSextractsataconcentrationof5μg/mL showed no cytotoxicity and, therefore,were used in the experiment. The extractcombination was prepared by mixing the ECandPS extracts in a 1:1 ratio andwasused ataconcentrationof 10 μg/mLsincenocytotox-icitywas observed at this concentration.

3. The EC extract inhibited the phosphorylationofAKTandp38 inRANKL-inducedpathwaysto inhibit the expression of the NFATc1 gene,thereby inhibiting osteoclast differentiation.

4. The PS extract inhibited the phosphorylationofAKTandp38 inRANKL-inducedpathwaysto inhibit the gene expression of NFATc1,thereby inhibiting osteoclast differentiation.

5. The EC and PS extract combination inhibitedthe phosphorylation of p38 and JNK inRANKL-inducedpathwaysandtheactivationofNK-κBto inhibit thegeneandproteinex-pression of c-Fos and NFATc1, thereby in-hibiting osteoclast differentiation.

In the invivo experiment, theECandPSextractcombination had no effects on recovery from LPS-induced bone loss and the BV/TV was increased inthe PS- and LPS-treated group, indicating a sig-nificantrecoveryofbonemasscomparedto there-covery in thegroup treatedwithLPSonly.

In conclusion, the EC and PS extracts inhibitedosteoclast differentiation through different routes.The inhibitionofosteoclastdifferentiationwasen-hanced when the extracts were coadministered.Only the single extract of PSwas effective againstbone loss causedby inflammation.

VI. References

1. WJ Boyle, WS Simonet, DL Lacey. Osteoclastdifferentiation and activation. Nature. 2002;423(6937):337-42.

2. Welton JL, Morgan MP, Marti S et al.Monocyte and γδ T cells control the acute-phaseresponse to intravenouszoledronate: in-sights from a phase IV safety trial. J BoneMinerRes. 2013;28(3):464-71.

3. Rachner TD, Khosla S, Hofbauer LC. Osteo-porosis: now and the future. Lancet. 2011;377(9773):1276-87.

4. Wang ZQ, Li JL, Sun YL et al. Chinese herbalmedicineforosteoporosis: asystematicreviewofrandomized controlled trails. Evid Based Com-plementaryAlternatMed. 2013;2013:356260.

5. TheSociety ofKoreanMedicineRehabilitation.Korea Rehabilitation Medicine. Seoul: KoonjaPublishing INC. 2003:105-7.

6. SinMG. Clinicalherbalmedicine. Seoul: Younglimsa.2000:556-9.

7. OhHS, KimHC, LeeSI, AhnDK. EffectsofEu-commiae cortex and Folium on the ovariec-tomized rat as the model of postmenopausalosteoporosis. Kor. J Herbology. 1995;10(1):59-68.

8. Lee JA, Noh SH, Ann DK, Choi HY. Effect ofEucommiae cortex and Chanenomelis fructuson the aged ovariectomized rat of post-menopausal osteoporosis. Kor. J Herbology.2001;16(1):201-6.

9. Kim MS, Seo BI, Kwak MA, Jee SY. Effect ofChungajihwangtangonosteoporosis inovariec-tomized rats. Kor. J Herbology. 2003;18(2):49-58.

10. LeeDS, ByunSY. Effectsofthedietarymixtureof Eucommia ulmoides oliver on osteoporosis.KoreanJ. Biotechnol. Bioeng. 2001;16(6):614-9.

11. JeYM, YooDY. EffectsofDokhwalgisaengtang-gamiwaterextractonosteoclastdifferentiationandosteoblastfunctioninRANKL-induedRAW264.7 cell. JKoreanObstetGynecol. 2013;26(2):

1-16.12. Jung YT, Choi YH, Song JH et al. Effect of

water extract ofEucommiae cortex inRANKL-induced osteoclast differentiation. Korean JOriental Physiol Pathol. 2009;23(3):613-8.

13. KwonHT, SeoBI, KimSH, KimMR. Astudyonthe effects of Psoraleae fructus in ovariec-tomized rat model of postmenopausal osteo-porosis. Kor. JHerbology. 1997;12(2):21-38.

14. Kwak HB, Kim JH, Kim DJ, Kwon YM, Oh JM,Kim YK. Effect of water extract of Deer antlerinosteoclastdifferentiation. KoreanJOrientalPhysiol Pathol. 2008;22(4):891-5.

15. Baek JM, Kim JY, Lee MS et al. Inhibitioneffect of Taxilli ramulus extract on osteoclastdifferentiation and bone resorption. Korean JOriental Physiol Pathol. 2013;27(4):431-6.

16. Lee SY, Kim SN, Kim JK. Effects of Asparaguscochinchinensis(Lour.) Merr. onthestimulationof osteoblast differentiation and inhibition ofosteoclast generation. J Korean Soc Food SciNutr. 2008;37(1):16-9.

17. KimJH, Ki JY, AnnJYet al. Inhibitory effectsof Achyranthis bidentatae radix on osteoclastdifferentiationandboneresorption. KorJHer-bology. 2010;25(1):65-74.

18. Yun YJ, Lee JM, Lee CH, Jang JB, Lee KS. Astudy on inhibitory mechanism of Meliafructus extract on osteoclast differentiation. JKoreanObstetGynecol. 2012;25(2):1-11.

19. ParkCK, KimHJ, KwakHBetal. Inhibitoryef-fectsofStewartiakoreanaonosteoclastdiffer-entiation and bone resorption. IntImmunopharmacol. 2007;7(12):1507-16.

20. HanKY, YangD, ChangEJetal. Inhibitionofos-teoclast differentiation and bone resorption bysauchinone. BiochemPharmacol. 2007;74(6):911-23.

21. Jang HJ, Hwang DS, Lee JM, Lee CH, Lee KS,Jang JB. Osteoclast differentiation of polygonicuspidatiradixextractseffectsandmechanismof inhibition studies. J Korean Obstet Gynecol.2014;27(1):17-27.

22. Heo JK, Hwang DS, Lee JM, Lee CH, Jang JB,



Lee KS. Inhibitory effects of ursolic prunellavulgarisonosteoclastdifferentiation. JKoreanObstetGynecol. 2014;27(2):59-70.

23. YoonKH, BaekJM, KimJYet al. Inhibitory ef-fect on RANKL-induced osteoclast differentia-tion by water extract of zizyphus jujuba mill.KoreanJOrientalPhysiolPathol. 2014;28(1):29-34.

24. Cheon YH, Kwack SC, Oh JM et al. Effect ofHoelen inRANKL-inducedosteoclastdifferen-tiation. Korean J Oriental Physiol Pathol.2012;26(3):320-24.

25. Hobson, E.E, Ralsteo, S.H. Role of genetic fac-torsinthepathophysiologyandmanagementofosteoporosis. ClinEndocrinlo. Oxf. 2001;54(1):1-9.

26. Fujiwara S. Epidemiology of osteoporosis andfracture. Clin calcium. 2004;14(11):13-8.

27. Lee Y, Lee HS, Jang SJ, Song JH. Effect ofwaterextractofschisandrachinensisonosteo-clastdifferentiation. KoreanJOrientalPhysiolPathol. 2010;24(5):848-53.

28. KimJH, LeeJH, OhJM, KimYK. Inhibitoryef-fects on bone resorption and osteoblast prolif-eration of Kyungok-go. Korean J OrientalPhysiol Pathol. 2011;19(2):61-71.

29. NishikawaM, AkatsuT, KatayamaYetal. Bis-phosphonates act on osteoblastic cells and in-hibit osteoclast formation in mouse marrowcultures. Bone. 1996;18(1):9-14.

30. Abrahamsen B. Bisphosphonate adverseeffects, lessons from large databases. CurrOpinRheumatol. 2010;22(4):404-9.

31. Ju YS. Ungok Herbology. Soeul: Seolimje.2004:407-13.

32. Roodman G.D. Regulation of osteoclast differ-entiation. AnnNYAcadSci. 2006;1068:100-9.

33. MinkinC. Boneacidphosphatase: tartrate-re-sistant acid phosphatase as amarker of osteo-clast function. Calcif Tissue Int.1982;34(3):285-90.

34. Matsuo K, Galson DL, Zhao C et al. Nuclearfactor of activated T-cells (NFAT) rescues os-teoclastogenesis inprecursors lackingc-Fos. JBiol Chem. 2004;279(25):26475-80.

35. Takayanagi H. Mechanistic insight into osteo-clast differentiation in osteoimmunology. JMolMed. 2005;83(3):170-9.

36. LeeZH, KimHH. Signaltransductionbyrecep-tor activator of nuclear factor kappa B in os-teoclasts. Biochem Biophys Res Commun.2003;305(2):211-4.

37. Jimi E, Aoki K, Saito H et al. SelectiveinhibitionofNF-kappaBblocksosteoclastoge-nesisandpreventsinflammatorybonedestruc-tion in vivo. NatMed. 2004;10(6):617-24.

38. Shimizu H, Nakagami H, Tsukamoto I et al.NFkappaBdecoyoligodeoxynucleotidesamelio-ratesosteoporosisthroughinhibitionofactiva-tion and differentiation of osteoclasts. GeneTher. 2006;13(12):933-41.

39. Abu-Amer Y, Ross FP, Edwards J, TeitelbaumSL. Lipopolysaccharide -stimulated osteoclas-togenesis is mediated by tumor necrosis factorvia its P55 receptor. J Clin Invest. 1997;100(6):1557-65.

40. QureshiST, LariviereL, LevequeGetal. Endo-toxin-tolerantmicehavemutationsinToll-likereceptor4 (Tlr4). JExpMed. 1999;189(4):615-25.

41. YoonSJ, KimKY, KimGYet al. Effects ofEu-commia Ulmoides Oliver and TreadmillExerciseontheOsteoporosisofRatsCausedbyGlucocorticoid Induction. Korean J OrientalPhysiol Pathol. 2008;22(4):884-90.

42. Seo BI, Kim SH. A Study on the effect offructus Psoraleaeon ovariectomy-induced os-teoporosis in rats. Korean J Oriental PhysiolPathol. 1997;5(1):179-83.



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