5,7-Dihydroxycoumarin counteracts osteoporosis by inhibition of PI3K/Akt signaling to suppress osteoclastogenesis: A study-based network pharmacology and experimental validation.

BACKGROUND: Osteoporosis is characterized by excessive osteoclast mediated bone resorption, resulting in trabecular deterioration and increased fracture risk. Natural small molecules with multi-target regulatory properties have attracted increasing attention as potential candidates for anti-osteoporotic therapy. 5,7-Dihydroxycoumarin (5,7-DHC), a coumarin derivative with reported anti-inflammatory potential, has not yet been fully investigated for its therapeutic effects on osteoporosis.

METHODS: Network pharmacology and molecular docking were applied to identify potential 5,7-DHC targets and signaling pathways associated with osteoporosis. The binding stability of the 5,7-DHC-Akt1 complex was further evaluated using molecular docking and 100-ns molecular dynamics simulations. In vitro, bone marrow derived macrophages were used to assess osteoclast differentiation, F-actin ring formation, acidified vesicle secretion, bone resorption, and expression of osteoclast related genes and proteins using TRAP staining, phalloidin/AO staining, bone resorption assays, RT-PCR, and western blotting). In vivo, the bone protective effects of 5,7-DHC were evaluated in ovariectomized (OVX) mice using micro-CT and histological analyses.

RESULTS: Network pharmacology identified 47 overlapping genes between 5,7-DHC targets and osteoporosis-related genes, enriched mainly in the PI3K/Akt, MAPK, and EGFR pathways. Molecular docking showed strong affinity between 5,7-DHC and Akt1, with a binding energy of -7.4 kcal/mol. This predicted interaction was further supported by molecular dynamics simulations, which yielded a favorable binding free energy of -63.38 ± 3.38 kJ/mol. In vitro, 5,7-DHC (75-200 μM) significantly inhibited RANKL induced osteoclast differentiation, disrupted F-actin ring formation, reduced acidified vesicle secretion, bone resorption, and downregulated osteoclast-related markers including NFATc1, c-Fos, DC-STAMP, OC-STAMP, CTSK, and MMP9. Mechanistically, 5,7-DHC suppressed phosphorylation of PI3K, Akt, and GSK3β during early osteoclastogenesis. In OVX mice, 5,7-DHC markedly attenuated estrogen deficiency-induced bone loss and improved trabecular microarchitecture, as reflected by increased BV/TV, BMD, BS/TV, reduced Tb.Sp, and improved histological morphology.

CONCLUSION: These findings suggest that 5,7-DHC exerts bone-protective effects by suppressing osteoclast differentiation and function, at least partly through inhibition of the PI3K/Akt/GSK3β signaling axis. This study provides preliminary experimental evidence supporting 5,7-DHC as a potential natural compound for the prevention or treatment of osteoclast mediated bone loss.