Epimedin B attenuates ovariectomy-induced bone loss by suppressing osteoclastogenesis through decreasing ROS production and targeting ESR1.

Excessive activation of osteoclasts plays a pivotal role in the pathophysiology of osteoporosis, which is predominantly characterized by bone resorption. However, therapeutic strategies targeting osteoclasts remain limited. In this study, we investigated the inhibitory effects of Epimedin B (EB) on osteoclastogenesis and its potential anti-osteoporotic properties. An ex vivo osteoclast differentiation model was established using BMMs. Osteoclastogenesis was assessed via TRAP staining and F-actin ring formation. Hydroxyapatite bone plates were employed to evaluate the impact of EB on bone resorption function. Network pharmacology predictions combined with transcriptomic analysis were employed to untangle the signaling pathways and targets regulated by EB. Cellular thermal shift assays were performed to validate EB-interacting proteins. The expression levels of osteoclast-specific and ROS-related proteins/genes, as well as phosphorylation changes in signaling pathways, were quantified by Western blot and RT-PCR. Protein expression and mitochondrial ROS levels were detected by fluorescence staining, while intracellular ROS generation was measured via flow cytometry. An ovariectomized (OVX) mouse model of osteoporosis was established to evaluate the bone-protective effects and in vivo mechanisms of EB. The results demonstrated that EB significantly suppressed RANKL-induced osteoclast differentiation, particularly during the early phase and cytoskeletal organization stage, while also inhibiting bone resorption activity in mature osteoclasts. Mechanistic studies revealed that EB directly binds to ESR1 and protects it from degradation. Furthermore, EB was found to downregulate the expression of osteoclast-specific genes/proteins and inhibit the phosphorylation of MAPK and PI3K-AKT pathways. Furthermore, EB activated the AMPK pathway, subsequently initiating the Nrf2-mediated antioxidant system, leading to reduced ROS levels. Notably, EB markedly rescued ovariectomy-induced bone loss, with efficacy comparable to estrogen therapy, an effect associated with suppressed osteoclast activity and AMPK pathway activation. These findings provide, for the first time, novel theoretical evidence and a potential therapeutic strategy for EB in the prevention and treatment of osteoporosis.