title: Dual effect of WIN-34B on osteogenesis and osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells.
abstract: BACKGROUND
  As an n-butanol fractionated extracted mixture of Lonicera japonica Thunb, dried flowers and Anemarrhena asphodeloides Bunge, root, WIN-34B has been reported the analgesic, anti-inflammatory, cartilage-repairing and protective effects in previous studies.
  
  
  OBJECTIVE
  To investigate the effect of WIN-34B on osteogenesis and osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells.
  
  
  METHODS
  To examine the effect of WIN-34B on osteogenic differentiation, human mesenchymal stem cells (hMSCs) were treated with WIN-34B (1μg/mL and 10μg/mL). Alkaline phosphatase (ALP) activity was evaluated and Von Kossa staining was conducted. Mice bone marrow macrophages (BMMs) were obtained and treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (m-CSF) to induce osteoclastogenesis. To investigate osteoclast differentiation, tartrate-resistant acid phosphatase (TRAP) staining was conducted after treatment with WIN-34B. Osteoclastogenic conditions were induced by stimulating the cells with interleukin (IL)-1α, IL-17, and tumor necrosis factor (TNF-α) in hMSCs and BMMs co-culture systems. The expression levels of osteoprotegerin (OPG), RANKL, runt-related transcription factor 2 (RUNX2), IL-17, c-Fos, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured by reverse transcription polymerase chain reaction (RT-PCR). The expression levels of nuclear factor-kappaB (NF-κB), inhibitory kappa B-α (IκBα), phospho-NF-κB, phospho-IκBα, β-actin, p38 MAPK, phospho-c-Jun N-terminal kinase (JNK), phospho-extracellular-signal regulated kinase (ERK), phospho-p38 mitogen-activated protein kinase (MAPK), phospho-JNK, and phospho-ERK were measured by western blot analysis.
  
  
  RESULTS
  WIN-34B promoted ALP activity and mineralization of hMSCs. In TRAP-stained BMMs, the number of multinucleated cells decreased after WIN-34B treatment. WIN-34B increased the OPG/RANKL ratio and the expression of RUNX2, and suppressed the expression of IL-17, c-Fos, and TNF-α. It also suppressed the activation of NF-κB, IκBα, p38 MAPK, and JNK in a dose-dependent manner.
  
  
  CONCLUSIONS
  These results demonstrated that WIN-34B increased osteogenesis and decreased osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells via inhibition of the NF-κB, JNK, and p38 MAPK pathways.
authors.affiliation: Department of Clinical Korean Medicine, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea.
authors.forename: Byung-Kwan
authors.initials: BK
authors.lastname: Seo
authors.affiliation: Department of Clinical Korean Medicine, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea.
authors.forename: Hee-Kyoung
authors.initials: HK
authors.lastname: Ryu
authors.affiliation: Department of Clinical Korean Medicine, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea.
authors.forename: Yeon-Cheol
authors.initials: YC
authors.lastname: Park
authors.affiliation: Oriental Medicine Research Center for Bone & Joint Disease, East-West Bone & Joint Research Institute, Kyung Hee University, 892, Dongnam-ro, Gangdong-gu, Seoul 05278, Republic of Korea.
authors.forename: Jeong-Eun
authors.initials: JE
authors.lastname: Huh
authors.affiliation: Department of Clinical Korean Medicine, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea. Electronic address: byhacu@khu.ac.kr.
authors.forename: Yong-Hyeon
authors.initials: YH
authors.lastname: Baek
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects*; D018920:Coculture Techniques; D016207:Cytokines / Q000494:pharmacology*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects*; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects*; D010012:Osteogenesis / Q000187:drug effects*; D010936:Plant Extracts / Q000494:pharmacology*
chemical_list: D016207:Cytokines; D010936:Plant Extracts; C569023:WIN 34B
keywords: Bone marrow cells; Mesenchymal stem cells; Osteoclastogenesis; Osteogenesis; Osteoporosis; WIN-34B