Dual effect of WIN-34B on osteogenesis and osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells

Ethnopharmacological relevance: 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. Aim of the study: To investigate the effect of WIN-34B on osteogenesis and osteoclastogenesis in cytokine-induced mesenchymal stem cells and bone marrow cells. Materials and methods: To examine the effect of WIN-34B on osteogenic differentiation, human mesenchymal stem cells (hMSCs) were treated with WIN-34B (1 mu g/mL and 10 mu g/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-kappa B 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)-1 alpha, IL-17, and tumor necrosis factor (TNF-alpha) 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-alpha, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured by reverse transcription polymerase chain reaction (RT-PCR). The expression levels of nuclear factor-kappaB (NF-KB), inhibitory kappa B-alpha (I kappa B alpha), phospho-NF-kappa B, phospho-I kappa B alpha, beta-actin, p38 MAPK, phospho-c-Jun N-terminal kinase UNK), 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/RAN-KL ratio and the expression of RUNX2, and suppressed the expression of IL-17, c-Fos, and TNF-alpha. It also suppressed the activation of NF-kappa B, I kappa B alpha, 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-kappa B, JNK, and p38 MAPK pathways. (C) 2016 Elsevier Ireland Ltd. All rights reserved.