MAFLD enhances BMP-2-induced heterotopic ossification via the LRP6/Wnt/β-catenin axis

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a systemic metabolic disorder, but whether it aggravates BMP-2-induced heterotopic ossification (HO) remains unclear. This study investigated whether MAFLD enhances HO through the LRP6/Wnt/β-catenin axis and explored a natural compound candidate for further mechanistic validation. Methods A BMP-2-induced rat HO model was established in rats fed either a normal diet or a high-fat diet to compare HO development under control and MAFLD-like metabolic conditions. HO tissues were analyzed by TRAP staining, western blotting and RT-qPCR. In vitro, rBMSCs were treated with control serum or MAFLD serum, with or without BMP-2, followed by LRP6 silencing and CHIR99021 rescue experiments to define the role of LRP6/Wnt/β-catenin signaling. Dual-target virtual screening, molecular docking and molecular dynamics simulations were then performed to identify C1 as a candidate compound targeting LRP6 and PORCN. Results MAFLD enhanced BMP-2-induced ectopic ossification and increased TRAP-positive remodeling. HO tissues and serum-treated cells showed increased LRP6 phosphorylation, β-catenin activation and osteogenic marker expression. LRP6 knockdown suppressed Wnt/β-catenin signaling and osteogenic gene expression, whereas CHIR99021 partially restored downstream β-catenin activation. In vitro, C1 treatment reduced MAFLD-associated Wnt/β-catenin activation and osteogenic marker expression, supporting its role as a candidate compound for further validation. Virtual screening identified C1, 2″-O-rhamnosylicariside II, as a dual LRP6/PORCN-binding candidate with acceptable predicted ADMET properties and stable binding behavior. Conclusion MAFLD promotes BMP-2-induced HO by activating LRP6/Wnt/β-catenin signaling. These findings nominate C1 as a candidate compound for further target validation and in vivo evaluation, rather than establishing it as a therapeutic strategy for MAFLD-associated pathological ossification.