a-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. a-Asarone (aASA), 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 aASA 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 aASA 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 aASA was validated by human BMSCs (hBMSCs). Results: In vivo, aASA treatment enhanced the biomechanical properties and osseointegration of tendon-bone samples in mice. In vitro, aASA 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 aASA 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 aASA for the transcription factor PPARG, while luciferase assays demonstrated PPARG binding to the Dmp1 promoter to regulate transcription. aASA also enhanced osteogenic differentiation in human BMSCs, supporting its potential for clinical application. Conclusion: aASA 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. Overall design: To explore transcriptional regulation of the therapeutic effect of a-ASA on TBI, we used mouse BMSCs to induce osteogenic differentiation in osteogenic medium and divided them into two groups with or without a-ASA, each with 6 biological replicates, for high-throughput sequencing.We used the RNA-seq data to perform differential gene analysis, and GO, KEGG, and GSEA enrichment analyses were performed for the top 20 differential genes.Subsequently, the specific mechanism of action was deeply explored through the interactive analysis of RNA-seq data and network pharmacology data