Ju-Young Kim 1,2,3, Jong Min Baek 4, Sung-Jun Ahn 4, Yoon-Hee Cheon 1,4, Sun-Hyang Park 4, Miyoung Yang 3,4, Min Kyu Choi 3,4 and Jaemin Oh 1,2,4*

Transcription

1 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 DOI /s RESEARCH ARTICLE Ethnolic extrct of Schizonepet tenuifoli ttenutes osteoclst formtion nd ctivtion in vitro nd protects ginst lipopolyscchride-induced one loss in vivo Open Access Ju-Young Kim 1,2,3, Jong Min Bek 4, Sung-Jun Ahn 4, Yoon-Hee Cheon 1,4, Sun-Hyng Prk 4, Miyoung Yng 3,4, Min Kyu Choi 3,4 nd Jemin Oh 1,2,4* Astrct Bckground: Excessive osteoclst ctivity is mjor cuse of metolic one disorders, such s osteopeni, rheumtoid rthritis, nd osteoporosis. Thus, discovery of gents trgeting osteoclst differentition nd one resorption is importnt for development of novel tretments for one diseses. It hs een demonstrted tht ethnolic extrct of schizonepet tenuifoli (EEST) hs potent nti-oxidnt nd nti-inflmmtory ctivities. However, the eneficil effects of EEST on one metolism hve not een studied. Therefore, we intend to investigte the effects of EEST on osteoclst differentition. Methods: We exmined the effects nd mechnisms of ction of the EEST on osteoclstogenesis in vitro in one mrrow mcrophges (BMMs) stimulted with receptor ctivtor of nucler fctor kpp-b lignd (RANKL) nd in vivo using mouse model of lipopolyscchride (LPS)-induced one destruction. Results: We found tht EEST inhiited phosphoryltion of Akt nd IkB t erly stges of RANKL-induced osteoclstogenesis. Furthermore, EEST negtively controlled the trnscription nd trnsltion levels of nucler fctor of ctivted T cells c1 (NFATc1) nd the trnsltion level of c-fos t the finl stge of osteoclst differentition. Reflecting these effects, EEST locked oth filmentous ctin (F-ctin) ring formtion nd one resoring ctivity of mture osteoclsts in vitro. The inhiitory effects of EEST on osteoclst formtion nd ctivity were oserved in n LPS-medited one erosion mouse model using micro-ct nd histologicl nlysis. Conclusions: EEST is potentil gent tht is le to tret osteoclst-relted one diseses, such s osteoporosis. (Continued on next pge) * Correspondence: jmoh@wku.c.kr Equl contriutors 1 Imging Science-sed Lung nd Bone Diseses Reserch Center, Wonkwng University, Iksn, Jeonuk , Repulic of Kore 2 Institute for Skeletl Disese, Wonkwng University, Iksn, Jeonuk , Repulic of Kore Full list of uthor informtion is ville t the end of the rticle 2016 The Author(s). Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 2 of 9 (Continued from previous pge) Keywords: Schizonepet tenuifoli, Osteoclst differentition, Bone resorption, Osteoporosis Arevitions: BMCs, Bone mrrow cells; BMMs, Bone mrrow mcrophges; BV/TV, Bone volume per tissue volume; DC-STAMP, Dendritic cell-specific trnsmemrne protein; EEST, Ethnolic extrct of Schizonepet tenuifoli; ELISA, Enzyme-linked immunosorent ssy; ERK, Extrcellulr signl-regulted kinse; F-ctin, Filmentous ctin; FBS, Fetl ovine serum; GAPDH, Glycerldehyde 3-phosphte dehydrogense; H&E, Hemtoxylin nd eosin; IKK, Inhiitor of IkB kinse; JNK, c-jun N-terminl kinse; LPS, lipopolyscchride; MAPKs, Mitogen-ctivted protein kinses; M-CSF, Mcrophge colony-stimulting fctor; NFATc1, Nucler fctor of ctivted T cells c1; NFκB, Nucler fctor kpp-b; PGE 2, Prostglndin E 2 ; RANKL, Receptor ctivtor of nucler fctor kpp-b lignd; T.N, Treculr numer; T.Sp, Treculr seprtion; T.Th, Treculr thickness; TRAF6, Tumor necrosis fctor receptor-ssocited fctor6; TRAP + MNCs, TRAP-positive multinucleted cells; Vitmin D 3, Dihydroxyvitmin D 3 ;XTT,Sodium3ʹ-[1-(phenylminocronyl)-3,4-tetrzolium]-is(4-methoxy-6-nitro); α-mem, α-minimum essentil medium Bckground Skeletl tissue continuously undergoes remodeling. This is defined y three physiologicl processes. First, in the resorption phse, osteoclsts dissolve the old one. Next, in the reversl phse, mononucler cells rrive on the one surfce to complete resorption stge. Finlly, in the formtion phse, osteolsts initite formtion of new one mtrix in response to signls of the mononucler cells [1]. Osteoclst over-ctivity, cused y such risk fctors s n inflmmtory response nd estrogen hormone deficiency, expedites perturtion of stedy-stte one remodeling. This leds to severe one diseses, including osteoporosis nd osteopeni, which re directly ssocited with excessive one destruction nd impired one qulity [2, 3]. The initition of osteoclst differentition from hemtopoietic stem cell of the monocyte/mcrophge linege is dependent on stimultion y two importnt cytokines: mcrophge colony-stimulting fctor (M-CSF), which is required for osteoclst prolifertion nd survivl, nd receptor ctivtor of nucler fctor kpp-b lignd (RANKL), which triggers vrious signls for osteoclstogenesis y inding to the RANK receptor, the surfce mrker of osteoclst precursors [4, 5]. In response to M-CSF nd RANKL stimultion, tumor necrosis fctor receptorssocited fctor6 (TRAF6) is recruited, which leds to susequent ctivtion of downstrem trnsducers of the RANKL-dependent pthwy. The downstrem trnsducers include mitogen-ctivted protein kinses (MAPKs), such s p38, c-jun N-terminl kinse (JNK), nd extrcellulr signl-regulted kinse (ERK); Akt; nd nucler fctor kpp-b (NFκB). Activtion of this signling pthwys leds to the nucler trnsloction of c-fos nd nucler fctor of ctivted T cell c1 (NFATc1), which re recognized s two mster osteoclst regultors. This results increse expression of vrious osteoclst-specific mrker genes tht re crucil for development nd function of mture osteoclsts, such s β3-integrin, dendritic cell-specific trnsmemrne protein (DC-STAMP), nd Cthepsin K [6 10]. Lipopolyscchride (LPS) leds to the intrcellulr induction of p38, JNK, nd NFκB in mcrophges nd monocytes, nd promotes the differentition nd survivl of osteoclsts through the production of other fctors such s PGE 2, interleukin 1, RANKL, nd TNF [11 13]. Therefore, LPS is n importnt meditor of pthologicl one destruction ssocited with inflmmtion. In this study, we screened severl plnt-derived extrcts y trtrte-resistnt cid phosphte (TRAP) stining nd confirmed tht ethnolic extrct of Schizonepet tenuifoli (EEST) cn suppress osteoclst ctivity. Although previous reports demonstrted tht EEST exerts vrious phrmcologicl effects, including nti-inflmmtory, nti-oxidnt, nd hemosttic ctivity, the effects of EEST on one metolism hve not een studied [14 16]. Therefore, we investigted the effects of EEST on RANKL-induced osteoclst differentition nd its underlying intrcellulr mechnisms in vitro. Furthermore, we performed in vivo experiments using LPS-medited one erosion mouse model in order to verify the therpeutic vlue of EEST for tretment of osteoporosis. Methods Plnt mterils nd EEST preprtion The 95 % EEST (sle numer: CA03-094) of the Koren Plnt Extrct Bnk (KPEB) t the Kore Reserch Institute of Bioscience nd Biotechnology (KRIBB) (Dejeon, Kore) ws cquired from the plnt smples purchsed from n Orientl medicine mrket in Kore nd then uthenticted y three txonomic experts t Chunguk, Chungnm, nd Pusn Ntionl University. Also, ll forms of extrction from the KPEB were produced through stndrdiztion procedure. The KPEB extrction protocol consists of 5 stges: extrction, filtrtion nd yield testing, concentrtion, drying, nd storge. First, extrction of ST ws performed using 95 % ethnol with sonictor (SDN- 900H, SD Ultrsonic Clener, Seoul, Kore) t 45 C for 3 dys (15 min soniction followed y 2 h stnding; repeted 10 times per dy). Next, The EEST ws filtered

3 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 3 of 9 through Whtmn filter pper No.2 (Advntec, Tokyo, Jpn). The filtrtes were comined, evported under vcuum, nd then lyophilized with ClenVc 12 vcuum freeze dryer (Biotron; Gngneung, Kore) t -70 C for 24 h under reduced pressure (<20 P). A 50 mg/ml stock solution of EEST ws prepred in dimethyl sulfoxide (DMSO) nd stored t -20 C. Regents A TRAP stining solution ws otined from Sigm Aldrich (St. Louis, MO, USA) nd sodium 3ʹ-[1-(phenylminocronyl)-3,4-tetrzolium]-is(4-methoxy-6-nitro) (XTT) ssy kit ws purchsed from Roche (Indinpolis, IN, USA). The α-minimum essentil medium (α-mem), fetl ovine serum (FBS), nd penicillin-streptomycin were purchsed from Gico-BRL (Grnd Islnd, NY, USA), nd solule humn recominnt M-CSF nd RANKL were purchsed from Peprotech (London, UK). Specific ntiodies ginst c-fos nd NFATc1 were otined from Snt Cruz Biotechnology (Snt Cruz, CA, USA). Specific primry ntiodies ginst phospho-p38, p38, phospho-akt, Akt, phospho-erk, ERK, phospho-jnk, JNK, phospho-iκb, nd IκB were purchsed from Cell Signling Technology (Beverly, MA, USA), nd tht ginst the house-keeping gene GAPDH ws purchsed from Snt Cruz Biotechnology. Osteoclst differentition from mouse one mrrow mcrophges (BMMs) To otin osteoclst precursors, we prepred mouse BMMs s descried previously [17] nd BMMs were incuted with M-CSF (30 ng/ml) nd RANKL (50 ng/ml) in the sence nd presence of EEST (1 50 μg/ml). In this experiment, the control group ws treted with 0.1 % DMSO, nd the other 5 groups were treted with EEST t concentrtions of 1, 5, 10, 25, nd 50 μg/ml. After 3 dys, the culture medium ws replced with fresh medium with the sme composition. After n dditionl dy, cells were stined with TRAP solution nd TRAPpositive multinucleted cells (TRAP + MNCs) contining more thn 5 nuclei were oserved nd counted s descried previously [17]. Evlution of cytotoxicity, nlysis of western lotting nd quntittive rel-time reverse trnscriptse polymerse chin rection (RT-PCR), retrovirl gene trnsfection, nd ssy of one resorption in co-culture system of BMCs nd primry osteolsts The experiments were performed s descried previously [17]. The primer sets used for the rel-time PCR were listed in Tle 1. The retrovirl vectors used for gene trnsfection were pmx-ires-egfp, pmx-akt-ires-egfp, nd pmxconstitutively ctive (CA)-IKKβ-IRES-EGFP pckging. Tle 1 Primer sequences used for rel-time PCR nlysis Gene nme Primer sequence (5 3 ) GAPDH Forwrd 5 -TCA AGA AGG TGG TGA AGC AG-3 Reverse 5 -AGT GGG AGT TGC TGT TGA AGT-3 c-fos Forwrd 5 -GGT GAA GAC CGT GTC AGG AG-3 Reverse 5 -TAT TCC GTT CCC TTC GGA TT-3 NFATc1 Forwrd 5 -GAG TAC ACC TTC CAG CAC CTT-3 Reverse 5 -TAT GAT GTC GGG GAA AGA GA-3 Cthepsin K Forwrd 5 -CCA GTG GGA GCT ATG GAA GA-3 Reverse 5 -CTC CAG GTT ATG GGC AGA GA-3 β3-integrin Forwrd 5 -GGA GTG GCT GAT CCA GAT GT-3 Reverse 5 -TCT GAC CAT CTT CCC TGT CC-3 Atp6v0d2 Forwrd 5ʹ-GAC CCT GTG GCA CTT TTT GT-3ʹ Reverse 5ʹ-GTG TTT GAG CTT GGG GAG AA-3ʹ DC-STAMP Forwrd 5 -TCC TCC ATG AAC AAA CAG TTC CA-3 Reverse 5 -AGA CGT GGT TTA GGA ATG CAG CTC-3 Immunofluorescence stining nd confocl microscopy BMMs were incuted with M-CSF (30 ng/ml) nd RANKL (50 ng/ml) in the presence nd sence of EEST (50 μg/ml). After 3 dys, the culture medium ws replced with fresh medium contining the sme constituents. After n dditionl dy, cells were stined with phlloidin nd DAPI solution for visuliztion of filmentous ctin (Fctin) nd nuclei s descried previously [17]. The fluorescence signl ws oserved using lser scnning confocl microscope (Olympus FV1200; Olympus, Shinjuku, Jpn), nd imges representtive of 5 experiments were nlyzed using Imge-Pro Plus softwre (Medi Cyernetics Inc., Rockville, MD, USA). LPS-medited one erosion mouse model nd micro-ct nd histologicl nlysis Five-week-old mle ICR mice were purchsed from Smtko Inc. (Osn, Kore). The mice were kept under controlled temperture (22 24 C) nd humidity (55 60 %) with 12 h light/drk cycle. The use of experimentl nimls ws reviewed y the institutionl niml cre nd use committee (IACUC) nd pproved under WKU To exmine the effect of EEST on LPS-induced one destruction, 5-week-old mle ICR mice were rndomly divided into 3 groups (5 mice per group): Control (treted with PBS), LPS (treted only with LPS), nd LPS + ST (treted with oth LPS nd EEST). EEST (200 mg/kg) or PBS ws dministered orlly 1 dy efore LPS injection (5 mg/kg), nd then every other 8 dys. LPS ws injected intrperitonelly on dy 2 nd 6. All mice were scrificed fter 8 dys nd femur of ech mouse were exmined y high-resolution micro-ct nlysis nd histologicl nlysis including hemtoxylin nd eosin (H&E) nd TRAP stining s descried previously [17]. Briefly, micro-ct

4 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 4 of 9 nlysis ws performed using one-relted prmeters, including one volume frction (BV/TV), treculr thickness (T.Th), treculr seprtion (T.Sp), nd treculr numer (T.N) which re miniml set of vriles tht should e investigted for treculr one regions [18]. Nomenclture, symols, nd units used in this study were recommended y the Americn Society for Bone Minerl Reserch (ASBMR) Nomenclture Committee. Sttisticl nlysis Ech experiment ws conducted t lest 3 times, nd dt were expressed s men ± stndrd devition (SD). All sttisticl nlyses were performed using Sttisticl Pckge for the Socil Sciences Softwre (SPSS; Koren version 14.0). Student s t-test ws used to compre prmeters etween 2 groups, while nlysis of vrince followed y Tukey post-hoc test ws used to compre prmeters mong 3 groups. P < 0.05 ws considered sttisticlly significnt. Results nd discussion EEST exerts inhiitory effects on TRAP-positive osteoclst formtion induced y RANKL tretment without cytotoxicity To screen the effects of EEST on osteoclst formtion, we treted mouse BMMs with the indicted concentrtions of EEST in culture medium (α-mem contining 30 ng/ml M-CSF nd 50 ng/ml RANKL). We oserved tht EEST suppressed the numer of TRAP + MNCs with more thn 5 nuclei in dose-dependent mnner compred to DMSO-treted control group (Fig. 1 nd ). In ddition, EEST did not exert ny cytotoxic effects during the differentition of BMMs into osteoclsts (Fig. 1c). Our results indicted tht EEST effectively suppressed c Fig. 1 EEST ttenutes RANKL-induced osteoclst differentition in dose-dependent mnner with no cytotoxicity. BMMs were cultured for 4 dys in the presence of M-CSF (30 ng/ml) nd RANKL (50 ng/ml) with the indicted concentrtions of EEST. Cells were fixed in 3.7 % formlin, permeilized with 0.1 % Triton X-100, nd stined with TRAP solution. TRAP + MNCs were photogrphed under light microscope. TRAP + MNCs with more thn 5 nuclei were counted. ***P < 0.001, **P < 0.01 vs. control. c BMMs were seeded into 96-well plte nd cultured for 3 dys in the presence of M-CSF (30 ng/ml) nd the indicted concentrtions of EEST. After 3 dys, the sornce t 450 nm ws determined using n ELISA reder

5 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 5 of 9 RANKL-dependent osteoclst formtion without cytotoxicity t vrious concentrtions (1 50 μg/ml). EEST ffects erly signling events of osteoclstogenesis vi dephosphoryltion of Akt nd IkB Next, we performed western lotting to confirm whether EEST ws relted with RANKL-dependent signl trnsducers, such s MAPKs, including p38, ERK, nd JNK; Akt; nd IκB. As shown in Fig. 2, EEST strongly reduced the phosphoryltion of Akt nd wekly suppressed the phosphoryltion of IkB. In ddition, we reffirmed the role of EEST on the ctivtion of Akt nd IκB using retrovirl vectors. Overexpression of Akt nd CA form of IKKβ, ctlytic suunit of IκB kinse complex, ws sufficient to reverse the inhiitory effect of EEST on osteoclst formtion. Previously, it hs een shown tht Akt plys criticl role in osteoclst survivl y regulting its downstrem trget, GSK3β, nd the signling cscde of NFATc1, mster trnscription fctor for osteoclstogenesis. An Akt inhiitor, LY294002, significntly suppresses osteoclst formtion nd NFATc1 expression in vitro, nd systemic injection of LY ttenutes multiple myelominduced norml osteoclst formtion nd osteolysis in vivo [19, 20]. The other erly signling molecule, IκB, is lso essentil for the regultion of osteoclst ctivity. The inhiitor of IκB kinse (IKK), which induces phosphoryltion of IkB, suppresses RANKL-induced osteoclst formtion nd ctivity in vitro, nd ovriectomymedited one erosion in vivo y trgeting osteoclstic one resorption [21]. Therefore, our results suggested tht EEST suppresses osteoclst formtion y trgeting two signl trnsducers, Akt nd IκB t the erly stge of osteoclst differentition. EEST suppresses mrna nd protein expressions of NFATc1 nd protein expression of c-fos Through the verifiction of the role of EEST on the erly stge of osteoclstogenesis, we ssumed tht EEST ws lso ssocited with the finl stge of RANKL-medited osteoclst differentition. At this stge, the trnscription fctors c-fos nd NFATc1 re ctivted in response to the ctivtion of erly signl trnsducers [22, 23]. It hs previously een shown tht emryonic stem cells with NFATc1 deficiency re not cple of differentiting into functionl osteoclsts, nd the retrovirus-medited overexpression of NFATc1 induces norml osteoclst differentition even in the sence of RANKL [22]. Ectopic expression of c-fos reverses the osteoclst dysfunctioninduced symptom of osteopetrosis, which is oserved in c-fos knockout mice [23]. In this study, we found tht EEST locked the expression of NFATc1 gene t oth the trnscription nd trnsltion levels nd c-fos gene t Fig. 2 EEST down-regultes RANKL-medited phosphoryltion of Akt nd IkB. BMMs were cultured for 1 dy in the presence of M-CSF (10 ng/ml). Next, BMMs were strved for 3 h, pretreted with EEST (50 μg/ml) for 1 h nd then stimulted with RANKL (50 ng/ml) for the indicted times. Cell lystes were nlyzed y western lotting with ntiodies ginst p-p38, p38, p-erk, ERK, p-jnk, JNK, p-akt, Akt, p-iκb, IκB, nd GAPDH. BMMs were infected with retroviruses expressing pmx-ires-egfp (pmx), pmx- CA-IKKβ-EGFP, nd pmx-akt-egfp. Infected BMMs were cultured with or without EEST (50 μg/ml) in the presence of M-CSF (30 ng/ml) nd RANKL (50 ng/ml) for 4 dys. After culturing, the cells were fixed nd stined for TRAP (left). The TRAP+ MNCs with more thn 5 nuclei were counted (right). *P <0.05,**P <0.01vs. the indicted group

6 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 6 of 9 the trnsltion level (Fig. 3). Collectively, EEST showed suppression effects on the expression of two mjor trnscription fctors in response to downregultion of Akt nd IκB during osteoclst differentition. EEST inhiits formtion of F-ctin structure nd one resorption ctivity of mture osteoclsts During the development of functionl osteoclsts, the orgniztion of F-ctin structure is required for one resorption ctivity. Once the multinucleted osteoclst ttches to the one surfce, its memrne ecomes polrized nd secretes oth hydrogen ions nd lytic enzymes into the resorption lcun in order to dissolve the one mtrix. This region is surrounded y tight seling zone tht is recognized s physiologicl feture of mture osteoclsts nd composed of F-ctin ring-like structures [24, 25]. In this study, we confirmed tht EEST significntly inhiited the formtion F-ctin ring-positive osteoclsts nd susequently reduced the pit re formed s result of the one resorption ctivity of mture osteoclsts (Fig. 4 nd ). Also, this phenomenon ws induced y the decresed expression of vrious osteoclst-mrker genes, including β3-integrin, DC-STAMP, Atp6v0d2,ndCthepsin K,which re required for the cell-to-cell fusion needed to orgnize F-ctin structure nd one resorption [26 29]. Our results demonstrted tht EEST negtively regulted the development of functionl osteoclsts y ttenuting the trnscription of severl osteoclst-specific mrker genes. EEST exerts protective effects in LPS-induced one erosion mice model Finlly, to determine if the in vitro effect of EEST on osteoclst ctivity could e confirmed in vivo, commonly used mouse model of osteoporosis ws pplied. Among the osteoporotic models, in this study, we focused on the therpeutic vlue of EEST on inflmmtion-induced one loss ecuse EEST hs een proved to exert significnt nti-inflmmtory effect [14]. LPS is n efficient tool for the induction of inflmmtory osteoporosis in mice [30]. Thus, we selected one loss model induced y LPS injection to suggest the therpeutic vlue of EEST on inflmmtory osteoporosis. Mice were injected with LPS intrperitonelly on dy 1 nd 4 to induce systemic inflmmtory response nd susequent one loss nd orlly dministered with EEST or PBS every 8 dys. After, the left femor of scrificed mice were nlyzed y micro-ct nd the right femor were stined with H&E nd TRAP solution. While the one mss in the femor of LPS-treted mice ws lower thn tht of controls, there ws prtil recovery of one density in mice treted with oth LPS nd EEST (Fig. 5). Morphometric nlysis of the femor of LPS-treted mice showed decresed BV/TV nd T.N nd incresed T.Sp, while restortion of these prmeters ws oserved in the LPS + EEST group (Fig. 5). Histologicl nlysis confirmed tht LPS + EEST tretment inhiited LPSinduced erosion of one mtrix nd formtion of TRAP-positive osteoclsts within growth pltes Fig. 3 EEST inhiits protein expression of c-fos nd oth mrna nd protein expression of NFATc1. BMMs were pretreted with or without EEST (50 μg/ml) for 1 h nd then stimulted with M-CSF (30 ng/ml) nd RANKL (50 ng/ml) for the indicted times. The cell lystes were nlyzed y western lotting with ntiodies ginst c-fos, NFATc1, nd GAPDH. BMMs were stimulted with RANKL (50 ng/ml) nd M-CSF (30 ng/ml) in the presence or sence of EEST (50 μg/ml) for the indicted times. Totl RNA ws isolted from cells using the QIAzol regent, nd mrna expression of c-fos nd NFATc1 ws determined using quntittive rel-time RT-PCR. ***P <0.001vs. control in the indicted time

7 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 7 of 9 c Fig. 4 (See legend on next pge.)

8 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 8 of 9 (See figure on previous pge.) Fig. 4 EEST negtively controls F-ctin structure, one-resoring ctivity, nd expression of osteoclst-specific genes. BMMs were cultured for 4 dys in the presence of M-CSF (30 ng/ml) nd RANKL (50 ng/ml) with or without EEST (50 μg/ml). Cells were fixed with 3.7 % formlin, permeilized with 0.1 % Triton X-100, nd stined with phlloidin nd DAPI. Mture osteoclsts were seeded on hydroxyptite-coted pltes for 24 h with EEST (50 μg/ml) tretment. Adherent cells were removed nd photogrphed under light microscope. The reltive rtio of pit res ws quntified using Imge J. ***P < vs. control. c BMMs were stimulted with RANKL (50 ng/ml) nd M-CSF (30 ng/ml) in the presence or sence of EEST (50 μg/ml) for the indicted times. Totl RNA ws isolted from cells using QIAzol regent, nd mrna expression of β3-integrin, DC-STAMP, Atp6v0d2, ndcthepsin K ws determined using quntittive rel-time RT-PCR. ***P <0.001vs. control in the indicted time (Fig. 5c nd d). The present findings suggested tht EEST exhiited protective effects on osteoclst differentition nd susequent one resorption in vivo. Also, it ws thought tht our further study could e in need of demonstrting the effect of EEST on OVX-medited one loss to more clrify the protective effect of EEST on osteoporosis. Conclusions In the present study, we demonstrted tht EEST ttenuted RANKL-induced osteoclst differentition y downregulting Akt nd IkB phosphoryltion in the erly signling event nd susequently trgeted NFATc1 t the trnscriptionl nd trnsltionl levels nd c-fos t the trnsltionl level. Moreover, EEST exerted suppression c d Fig. 5 EEST recovers LPS-induced inflmmtory one loss in mice. Mice were scrificed 8 dys fter the first LPS injection nd 2D or 3D rdiogrphs of the coronl nd trnsverse plnes of the proximl femor were otined y micro-ct. The BV/TV, T.Sp, T.Th, nd T.N of the femor were determined using the micro-ct dt nd nlyzed y INFINITT-Xelis softwre. ***P <0.001vs. control; ## P <0.01, ### P <0.001vs. LPSgroup.c Dissected femor were fixed, declcified, emedded, nd sectioned. Sections were stined with TRAP nd H&E. d The numer of osteoclsts per field of tissue ws counted y histomorphometric nlysis

9 Kim et l. BMC Complementry nd Alterntive Medicine (2016) 16:301 Pge 9 of 9 effects on F-ctin ring formtion nd one resorption in vitro nd LPS-medited one erosion in vivo. Tken together, our findings supported the potentil vlue of EEST s plnt-derived therpeutic gent to tret onerelted disorders, prticulrly osteoporosis. Funding This study ws supported y grnt from the Wonkwng University in Avilility of dt nd mterils All dt nd mterils re contined nd descried within the mnuscript. Authors contriutions JYK nd JMB: conception nd design, eqully performed most of the experiments in this study, drfted the mnuscript, nd will provide finl pprovl of the version to e pulished; SJA nd SHP: performed the in vivo study; YHC, MY nd MKC: drfted the mnuscript nd revised it criticlly for importnt intellectul content; JO: conception nd design, nd will provide finl pprovl of the version to e pulished. All uthors red nd pproved the finl mnuscript. Competing interests The uthors declre tht they hve no competing interests. Consent for puliction Not pplicle. Ethics pprovl nd consent to prticipte The use of experimentl nimls ws reviewed y the IACUC nd ws pproved under WKU Author detils 1 Imging Science-sed Lung nd Bone Diseses Reserch Center, Wonkwng University, Iksn, Jeonuk , Repulic of Kore. 2 Institute for Skeletl Disese, Wonkwng University, Iksn, Jeonuk , Repulic of Kore. 3 Institute for Environmentl Science, Wonkwng University, Iksn, Jeonuk , Repulic of Kore. 4 Deprtment of Antomy, School of Medicine, Wonkwng University, Sinyong-dong, Iksn, Jeonuk , Repulic of Kore. Received: 27 April 2016 Accepted: 18 August 2016 References 1. Hdjidkis DJ, Androulkis II. Bone remodeling. Ann N Y Acd Sci. 2006;1092: Tnk Y, Nkymd S, Okd Y. Osteolsts nd osteoclsts in one remodeling nd inflmmtion. Curr Drug Trgets Inflmm Allergy. 2005;4(3): Hughes DE, Di A, Tiffee JC, Li HH, Mundy GR, Boyce BF. 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