α-Asarone promotes tendon-bone healing through regulating Dmp1 transcription via targeting transcription factor PPARG

Background: The tendon-bone interface (TBI) frequently proves refractory to restoration following surgical reconstruction or even surgical failure, due to the intricate nature of its structure. α-Asarone (αASA), an active ingredient derived from the traditional Chinese medicinal plant Calamus, has been demonstrated to possess beneficial effects in the treatment of inflammatory and osteoporotic conditions. However, its impact on TBI has yet to be investigated. Objective: This study aimed to elucidate the therapeutic effects of αASA on TBI in vivo and in vitro, gain insight into the underlying mechanisms involved, and assess its potential medicinal value in humans. Methods: A mouse model and a cellular model of tendon-bone interface (TBI) healing were developed. The impact of αASA on TBI was investigated at both the in vitro and vivo levels through a range of techniques, including protein gene assays, cell staining, biomechanics, imaging, and histology. Concurrently, network pharmacology, transcriptomics, molecular docking, transcription factor prediction, and experimental validation were employed to conduct a comprehensive investigation of its specific effects and mechanism of action. Subsequently, In addition, the clinical medicinal value of αASA was validated by human BMSCs (hBMSCs). Results: In vivo, αASA treatment enhanced the biomechanical properties and osseointegration of tendon-bone samples in mice. In vitro, αASA promoted osteogenic differentiation of BMSCs, as evidenced by staining and the expression of osteogenic marker genes. Using network pharmacology, we identified 29 core co-target genes of αASA in the treatment of TBI. The top 20 differentially expressed genes (DEGs) underwent GO, KEGG, and GSEA enrichment analyses, revealing their involvement in tissue mineralization and ossification processes. Additionally, 207 transcription factors (TFs) were predicted for these DEGs, with 9 identified as core co-target genes. Surface plasmon resonance (SPR) confirmed the strong affinity of αASA for the transcription factor PPARG, while luciferase assays demonstrated PPARG binding to the Dmp1 promoter to regulate transcription. αASA also enhanced osteogenic differentiation in human BMSCs, supporting its potential for clinical application. Conclusion: αASA promotes osteogenic differentiation and improves TBI healing in mBMSCs by targeting and down-regulating the transcription factor PPARG to inhibit its binding to the Dmp1 promoter, while exerting the same effect in hBMSCs.