Osteoporosis (OP) is characterized by an imbalance in which bone marrow mesenchymal stem cells (BMSCs) preferentially differentiate into adipocytes rather than osteoblasts. The natural polyhydroxyflavone fisetin functions as an antioxidant and multi-target signaling regulator with potential to modulate stem cell fate. However, its role and underlying mechanisms in OP remain unclear. This study investigated whether fisetin ameliorates OP by regulating osteogenic and adipogenic differentiation using integrated in vitro and in vivo models. The effects of fisetin on BMSC differentiation were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, alkaline phosphatase (ALP)/Alizarin Red S staining, Oil Red O and lipid-droplet fluorescence staining, and immunofluorescence. RNA sequencing and network pharmacology were used to explore the underlying mechanisms, followed by validation using pharmacological and genetic approaches. The in vivo effects of fisetin were confirmed in an ovariectomy (OVX)-induced OP mouse model by micro-computed tomography (micro-CT) and bone histomorphometry. Fisetin dose-dependently promoted osteogenic differentiation and inhibited adipogenic differentiation in cultured BMSCs. Mechanistically, fisetin activated the canonical Wnt/β-catenin signaling pathway, which underpinned its pro-osteogenic and anti-adipogenic effects. In OVX-induced OP mice, fisetin treatment significantly attenuated trabecular bone loss and reduced bone marrow adiposity, demonstrating its anti-osteoporotic efficacy as evidenced by micro-CT and histomorphometric analyses. This study demonstrates that fisetin alleviates OVX-induced OP by mitigating trabecular bone loss and suppressing marrow adipogenesis via activation of the Wnt/β-catenin pathway, providing a mechanistic foundation for its development as a therapeutic agent.
