The therapeutic effect of genistein on microfracture based on network pharmacology, immune cell abundance analysis and experimental verification

Abstract

Postmenopausal osteoporosis is a metabolic bone disease mainly attributed to estrogen deficiency. Genistein, a major phytoestrogen, has been reported to exert a direct suppressive effect on osteoclasts. The aim of this study was to explore the possible impact of genistein on osteoclastic activity and osteogenesis during the microfracture healing process, and to determine its possible mechanisms of action in an ovariectomized rat model. A predictive network depicting the relationship between genistein and microfracture was constructed based on data collected from several databases. Integrated network pharmacology approaches were used to identify potential signaling pathways. The predicted targets were validated by molecular docking, and the effects were further investigated using immunoinfiltration analysis and verified in an ovariectomized rat model. In total, 64 overlapping targets of genistein that were also related to microfractures were identified. These targets were enriched in multiple pathways, including osteoclast differentiation, and pathways relate to monocyte activation and migration. Molecular docking revealed that genistein exhibited high affinity to the Proto-oncogene tyrosine-protein kinase Src and extracellular signal-regulated kinase (ERK), both of which are association with monocyte activation. MoreoverFurthermore, genistein and microfractures were found to increase the abundance of monocyte or M2 macrophage and could elevate subsequent osteoclast function. Immunohistochemical and morphological analyses confirmed that genistein upregulates the expression of key regulatory factors for osteoclast and osteogenic differentiation, which was associated with an increasing trend in resorptive cavities and osteogenic area in fractured ovariectomized rats. These results demonstrated that genistein exerts regulatory effect in bone osteoclastic metabolism and subsequent bone remodeling specifically during the fracture healing phase in OVX rats in vivo, rather than exerting a general anti-osteoporotic effect.