Rh3R Attenuates RANKL-Induced Osteoclast Differentiation and F-Actin Ring Formation via the Suppression of c-Fos/NFATc1 Signaling in Primary Murine Cells

The homeostatic balance of bone remodeling is governed by the precise coordination between bone-forming osteoblasts and bone-resorbing osteoclasts. In this study, we investigated the anti-resorptive properties of rhamnocitrin-3-rhamnoside (Rh3R), a flavonoid isolated from Loranthus tanakae, using primary bone marrow-derived macrophages (BMMs) and calvaria-derived osteogenic progenitor cells (COCs) to ensure biological relevance. Our findings demonstrate that Rh3R potently inhibits the RANKL-induced differentiation of BMMs into TRAP-positive multinucleated osteoclasts in a dose-dependent manner, without inducing cytotoxicity. Mechanistically, Rh3R effectively attenuates RANKL-induced downstream signaling cascades, as evidenced by the attenuated phosphorylation of MAPKs (ERK1/2, JNK, p38), AKT, and IκB. This signaling blockade subsequently suppresses the induction of the master transcription factors, c-Fos and NFATc1. Furthermore, Rh3R impairs the functional resorptive capacity of mature osteoclasts by destabilizing F-actin-rich ring structures accompanied by decreased integrin β3 expression, thereby preventing the formation of a functional sealing zone. The inhibitory effect of Rh3R on bone-degrading activity was further confirmed by a significant reduction in the total area of resorption pits on bone slices. Notably, Rh3R exhibits a lineage-specific inhibitory effect, showing no adverse influence on osteoblastogenesis or the mineralizing capacity of primary osteogenic cells. Furthermore, the effect of Rh3R was consistently maintained in a co-culture system of primary osteoblasts and BMMs. Collectively, these in vitro findings identify Rh3R as a bioactive modulator of osteoclast differentiation and function via suppression of RANKL-induced downstream signaling, warranting future in vivo and pharmacological studies to evaluate efficacy, exposure, and safety.