Phillygenin Improves the Focal Adhesion Kinase/Glycogen Synthase Kinase 3β/β‐Catenin Axis to Promote Mesenchymal Stem Cell Osteogenic Differentiation

Forsythiae Fructus, a traditional herb known for its anti-inflammatory and antioxidant properties, has demonstrated inconclusive effects on bone metabolism in prior studies. The aim of this study is to investigate whether phillygenin (PHI), an active component of Forsythiae Fructus, promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by modulating the FAK/GSK-3β/β-catenin signaling axis. Experimental procedure: In vivo, an ovariectomized (OVX) mouse model was employed to mimic postmenopausal bone loss. In vitro, osteoclastogenesis was evaluated using tartarate-resistant acid phosphatase (TRAP) enzymatic assays, while osteogenic differentiation of BMSCs was assessed through alkaline phosphatase (ALP) and alizarin red S (ARS) staining. Cell proliferation and viability were examined using the CCK-8 assay. Osteogenic markers, including ALP, RUNX2, osteopontin (OPN), as well as signaling proteins, such as phosphorylated FAK at Y397 (pFAKY397), phosphorylated glycogen synthase kinase-3 beta (pGSK-3β), and β-catenin, were analyzed by western blot and immunofluorescence analyses. Specific inhibitors of the FAK and β-catenin (Y15 and XAV-939) were employed for mechanistic validation. PHI treatment significantly decreased the number and size of osteoclasts in a dose-dependent way. In OVX mice, PHI preserved trabecular microarchitecture, increased bone mineral density, and inhibited osteoclast formation dose-dependently. Low doses of PHI significantly promoted osteogenic differentiation and mineralization of BMSCs without affecting cell proliferation and viability. Mechanistically, PHI upregulated the expression of pFAKY397, pGSK-3β, and β-catenin. Inhibition of the pathway, achieved through the use of Y15 and XAV-939, attenuated PHI-induced osteogenesis. These findings indicate that PHI enhances BMSCs osteogenesis via the FAK/GSK-3β/β-catenin axis, thereby restoring bone metabolic balance. The results highlight PHI's dual regulatory potential in the management of bone disorders, particularly, estrogen deficiency-related osteoporosis, and suggest its potential applications in the therapeutic intervention of bone diseases.