Byung-Kwan Seo1, Hee-Kyoung Ryu1, Yeon-Cheol Park1, Jeong-Eun Huh2, Yong-Hyeon Baek3. 1. Department of Clinical Korean Medicine, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea. 2. 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. 3. 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.
Abstract
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 (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.
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 (1μg/mL and 10μg/mL). Alkaline phosphatase (ALP) activity was evaluated and Von Kossa staining was conducted. Micebone 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, p38MAPK, 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, p38MAPK, 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 p38MAPK pathways.