Osteoporosis, characterized by excessive osteoclast activity and disrupted bone remodeling, remains a significant health concern. This study investigated the potential of shiitake mushroom (Lentinula edodes) extracts, prepared with various drying methods, to inhibit osteoclast differentiation and promote bone formation. Mushroom stipe extracts, irrespective of the drying method, effectively suppressed osteoclast differentiation by downregulating nuclear factor of activated T-cells, cytoplasmic 1 expression in osteoclast precursor cells treated with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-Β ligand (RANKL), whereas the mushroom cap extracts exhibited minimal inhibitory effects. This disparity highlights the importance of part-specific analyses as the stipes contained higher levels of bioactive molecules, including denatonium, which was identified as a key RANKL signaling disrupter. In contrast, the caps lacked the anti-osteoclastogenic activity observed in the stipes, which were notably consistent across the freeze-drying, air-drying, and hot-air drying methods, indicating the stability of the bioactive compounds under various processing conditions. In vivo studies using an osteoporosis-induced zebrafish model further validated the therapeutic potential of the stipe extracts, which restored bone mineralization and mitigated prednisolone-induced bone loss. These findings emphasize the importance of identifying and optimizing specific mushroom parts for their unique therapeutic properties. Shiitake mushroom stipe extracts are a natural product potentially useful in osteoporosis therapy. This therapy would be a promising alternative to conventional treatments, such as bisphosphonates, which are associated with adverse side effects.

Abstract
INTRODUCTION
MATERIALS AND METHODS
    Preparation of shiitake mushroom extracts by different drying methods
    Osteoclast differentiation and tartrate-resistant acid phosphatase staining
    Cell viability assay
    Western blot analysis
    Quantification of denatonium
    Zebrafish maintenance and drug treatment
    Whole-mount skeletal staining
    Statistical analysis
RESULTS
    Effect of different shiitake mushroom parts and drying methods on osteoclast differentiation
    Effect of different shiitake mushroom parts and drying methods on osteoclast precursorcell proliferation
    Denatonium-rich shiitake mushroom stipe extracts suppress nuclear factor of activatedT-cells, cytoplasmic 1 expression and osteoclast differentiation
    Mitigation of prednisolone-induced bone mineralization loss by shiitake mushroomextracts on bone formation in an osteoporosis-induced zebrafish model
DISCUSSION
REFERENCES

• Tae Kyung Hyun(Department of Industrial Plant Science & Technology, Chungbuk National University, Cheongju 28644, Korea)
• Hyerim Lee(Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju 28644, Korea)
• Sun-Ju Yi(Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju 28644, Korea)
• Kyunghwan Kim(Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju 28644, Korea) Corresponding author