The impact of catalpol on osteogenic differentiation of the jaw bone marrow mesenchymal stem cells on the basis of network pharmacology, molecular docking and experimental validation.

Abstract

Objective: The research aims to explore the effects of catalpol on the osteogenic differentiation of Jaw Bone Marrow Mesenchymal Stem Cells(JBMMSCs).

Methods: The study employed network pharmacology, molecular docking, and in vitro experiments.Potential drug targets were identified through databases, and a protein-protein interaction network was constructed.Gene Ontology and KEGG analyses were conducted on the overlapping targets. Molecular docking was used to confirm the binding of catalpol to core targets. In vitro, JBMMSCs were utilized to investigate the effects of catalpol on the osteogenesis of the cells via CCK-8 assays, ALP staining, Alizarin Red S staining, WB and RT-PCR.

Results: A total of 99 common therapeutic targets of catalpol for bone defect regeneration were identified. Protein-protein interaction (PPI) Network analysis screened 10 core target proteins: ALB, EGFR, ESR1, HSP90AA1, PPARG, SRC, RHOA, GSK3B, SERPINE1, and KDR. Gene Ontology (GO) and Functional analyses revealed 161 biological processes (BP), 38 cellular components (CC), and 59 molecular functions (MF) associated with Catalpol-mediated bone defect repair. KEGG pathway enrichment analysis demonstrated that the target genes were primarily enriched in 16 signaling pathways, notably including osteoclast differentiation, apoptosis, and metabolic pathways. Molecular docking results indicated that catalpol exhibited binding energies < -5 kcal·mol⁻¹ with key target proteins (ALB, EGFR, ESR1, HSP90AA1, and PPARG). The vivo experiments demonstrated that catalpol (0.1µM、1µM、10µM、20µM concentration range) promoted the proliferation of JBMMSCs. Catalpol significantly enhanced alkaline phosphatase (ALP) activity, ALP expression, and osteogenic Mineralization in JBMMSCs, with 1 µM exhibiting the most pronounced effect (p < 0.05). Furthermore, 1 µM catalpol upregulated the expression of osteogenesis-related proteins (BMP-2, RUNX2, and OCN) and their Corresponding mRNA transcripts in JBMMSCs. The most significant regulatory Effects were observed at 72 h post-treatment (p < 0.05).

Conclusion: Catalpol enhances alveolar bone defect regeneration by regulating multiple genes and pathways. Studies have shown that catalpol boosts the proliferation and osteogenic differentiation/mineralization of JBMMSCs and increases the expression of osteogenesis-related genes and proteins. The ideal concentration for ex vivo induction was identified as 1 μM in this investigation. These results establish a scientific foundation for the potential use of catalpol in osteogenesis therapy, although additional research is necessary to clarify its clinical effectiveness and mechanisms of action.