<i>β</i> ‐Sitosterol Suppresses Osteoclastogenesis and Alleviates Bone Loss by Targeting Tmed10 to Inhibit <scp>RANKL</scp> ‐Induced Hedgehog/ <scp>MAPK</scp> Signaling and <scp>ROS</scp>

β-sitosterol, a phytosterol widely distributed in Chinese herbal medicines, exhibits significant anti-inflammatory and antioxidant activities. This study aimed to investigate the potential role of β-sitosterol in osteoclastogenesis and bone loss. Primary BMDM were used to induce osteoclastogenesis and evaluate the effect of β-sitosterol on osteoclastogenesis. Meanwhile, an ovariectomy-induced osteoporosis mouse model was employed to examine the effect of β-sitosterol on bone mass in vivo. The results indicated that the MAPK and Hedgehog signaling pathways were identified as key signaling pathways, with Tmed10 recognized as the drug target protein. In vitro experiments demonstrated that β-sitosterol suppressed RANKL-induced osteoclastogenesis and cytoskeleton formation in a concentration-dependent manner, downregulated the expression of osteoclast-related genes and proteins, and inhibited the nuclear translocation of NFATc1. Mechanistically, β-sitosterol inhibited the phosphorylation of ERK in the MAPK pathway and the nuclear translocation of p-ERK. Concurrently, β-sitosterol suppressed cellular and mitochondrial ROS generation and upregulated the expression of antioxidant enzymes. CETSA demonstrated that β-sitosterol protects Tmed10 from thermal degradation, while the DARTS assay confirmed that β-sitosterol binds to Tmed10 and prevents its enzymatic digestion. These results verified the direct binding between Tmed10 and β-sitosterol. Co-immunoprecipitation indicated that Tmed10 directly interacts with HHIP, thereby inhibiting the activation of the downstream Hedgehog signaling pathway. In vivo studies revealed that administration of β-sitosterol activated Tmed10 expression, attenuated osteoclastogenesis, improved bone microstructure and trabecular parameters, and prevented ovariectomy-induced bone loss. In conclusion, β-sitosterol effectively suppresses osteoclastogenesis and rescues bone loss by targeting Tmed10 to regulate the key MAPK/Hedgehog signaling pathways and reduce ROS generation. The significant protective effect of β-sitosterol against bone loss in vivo suggests its potential as a candidate therapeutic agent for osteoporosis.