Epimedium Active Component Icaritin Enhances Bone Mass through Regulating the Coupling of Bone Formation and Resorption Mediated by Distinct Signaling Target

Osteoporosis is a skeletal disease characterized by low bone mass and increased risk of fracture. There has been a long term interest in searching novel natural products for the prevention and treatment of osteoporosis. Using in vitro and in vivo systems, we investigated the therapeutic efficacy and molecular mechanisms of Icaritin, an active component from Epimedium (Berberidaceae), on the treatment of osteoporosis. Our results showed that Icaritin promoted osteoblast differentiation indexed by increased alkaline phosphatase (ALP) activity, mineralized nodule formation and upregulated osteogenic maker genes expression. Icaritin enhanced bone acquisition in the calvaria organ culture compared with the controls. Molecular examination showed that Icaritin elevated Smad1/5/8 phosphorylation in osteoblast, while this effect was suppressed by Noggin and siRNA mediated BMPR-II knockdown. Immunofluorescence staining indicated that Icaritin induced BMPR-II endocytosis in association with clathrin. We next identified the ligand-protein binding of Icaritin and BMPR-II using a fluorescence-based thermal shift assay. The result showed that Icaritin affected the BMPR-II thermal stability in a concentration dependent manner, suggests a direct interaction between Icaritin and BMPR-II. These observations suggest that Icaritin may activate the BMP/Smad1 signaling by binding to BMPR-II and increasing its endocytosis in osteoblast. In the osteoclast differentiation model, Icaritin was found to inhibit osteoclast by significantly decreased numbers of tartrate-resistant acid phosphatase (TRAP) positive multi-nucleated cells, disruption of actin-ring, and eliminated bone resorptive activity compared with the controls. Icaritin performed these effects by inducing mature osteoclasts apoptosis without affect their early differentiation. The Icaritin-induced mature osteoclasts apoptosis was mediated by the c-src/Akt signaling but not through the activation of Smad1/5/8, indicating that Icaritin exerts a differential mode of signaling mechanism in osteoclast compared to that observed in osteoblast. In the in vivo system, we found that Icaritin treatment prevented bone loss and maintained the biomechanical properties of the femurs in ovariectomy (OVX) induced mouse osteoporosis model. Osteogenic marker genes were upregulated by Icaritin treatment compared with that in the sham and the OVX groups. Our results suggest that Icaritin promotes osteoblast differentiation and bone formation mediated by the BMP/Smad1 signaling, while inhibits osteoclast bone resorption function by inducing mature osteoclasts apoptosis via the c-src/Akt signaling. Icaritin may serve as a potential therapeutic agent for the treatment of osteoporosis through enhancing the osteoblast anabolic effect and inhibiting the osteoclast functions mediated by distinct signaling targets.