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Background: Animal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear. Purpose: To reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrowderived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts. Study design: Controlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures. Methods: Cell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative realtime PCR; and proteins expression was evaluated by western blot. Results: Nomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways. Conclusion: This study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases. Keywords: Limonoid Nomilin Osteoclastic differentiation, Introduction Bone metabolic balance is maintained by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Bone metabolic diseases, such as osteoporosis, are caused by an imbalance in bone metabolism, so that [...]