Ganoderic acid A accelerates osteoporotic fracture healing by promoting osteogenic differentiation via estrogen receptor alpha binding

Objectives Ganoderic acid A (GAA), a natural triterpenoid derived from the fungal species Ganoderma lucidum which is known for its medicinal properties, is well recognized for its anti-inflammatory, anti-aging, kidney-protective, and hepatoprotective properties. However, its role in osteoporotic fracture (OF) healing remains unclear.This study aims to explore the pharmacological effects of GAA on OF healing and to clarify its underlying mechanisms. Methods An ovariectomized femoral fracture model was treated with GAA or estradiol benzoate. Callus formation, mineralization and bone mineral density (BMD) were assessed by micro–computed tomography, radiographic scoring and DXA. Mechanical properties were evaluated by three-point bending tests. Histological analyses and enzyme-linked immunosorbent assay for serum bone formation and resorption markers were performed. RNA sequencing with GO, KEGG and GSEA analyses was used to explore molecular mechanisms. In vitro, MC3T3-E1 cells were used to assess osteogenic differentiation under GAA, estrogen receptor (ER) antagonist (ICI 182780) or selective ERα antagonist (Methyl-piperidino-pyrazole dihydrochloride, MPP) treatment. Biophysical interaction assays and molecular simulations were employed to characterize GAA–ERα interactions. Results GAA enhanced early callus mineralization, bone volume fraction, callus volume and trabecular number, and increased radiographic union, bone mineral density and flexural rigidity in OF mice. Histological analysis showed increased soft callus and mineralized bone with elevated bone formation and resorption markers. Transcriptomic analyses indicated that GAA modulated hormone-related pathways, identifying the estrogen signaling pathway as a key target. In vitro, GAA promoted alkaline phosphatase activity, mineral deposition and expressions of phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2), Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), all of which were abrogated by ICI 182780 and MPP. Notably, molecular interaction studies demonstrated direct and stable binding of GAA to ERα. Conclusion GAA promotes osteogenic differentiation and accelerates OF healing via ERα. The translational potential of this article The work offers a conceptual advance by identifying a fungal-derived small molecule that functions as an ERα ligand to promote fracture repair, supporting its potential as a therapeutic candidate for OF.