Introduction: Rehmannioside D (RD) is an iridoid compound of Rehmanniae Radix (RR). Runx2 is known for its role in bone formation; emerging evidence implicates it in osteoclast (OC) activity. This study investigates the in vitro effects of RD on osteoclast differentiation and explores the role of Runx2 in this process. Materials and Methods: OC differentiation was induced in mouse Bone marrow-derived macrophages (BMDMs). The effects of RD were assessed using Tartrateresistant acid phosphatase (TRAP) staining, F-actin ring formation, bone resorption assays, and ELISA for Cathepsin K. Underlying mechanisms were explored via Western Blot assay, immuno-fluorescence, immunoprecipitation, qPCR, and kinase assays. The role of Runx2 was vali-dated using siRNA and lentiviral transfection. Results: RD significantly inhibited OC differentiation, as evidenced by reducing TRAP-positive cells and disrupted F-actin ring. It also suppressed bone resorption and Cathepsin K secretion. Mechanistically, RD inhibited Runx2 phosphorylation, its interaction with Cbf-β, and the expression of downstream targets (bone sialoprotein (bsp) and osteocalcin (ocn)). These effects were mediated through the PI3K/Akt pathway, as RD suppressed Akt kinase activity and phosphorylation. Silencing Runx2 enhanced RD’s inhibitory effects, while Runx2 overexpression reversed them. Furthermore, counteracted the effects of both an Akt activator (SC-79) and inhibitor (MK-2206) on OC differentiation. Discussion: This study demonstrated that RD inhibits OC differentiation and function by suppressing the PI3K/Akt signaling axis. These findings reveal a novel mechanism of RD action and highlight its potential as a therapeutic agent for osteolytic diseases, warranting further in vitro investigation. Conclusion: RD significantly inhibits OC differentiation and activity by down-regulating Runx2 phosphorylation through the PI3K/Akt pathway.
Chen et al. (Wed,) studied this question.