Formononetin Protects Osteoblasts Against Oxidative Stress and Attenuates Ovariectomy-Induced Bone Loss in Association with MAPK/AP-1 and NRF2 Signaling

Postmenopausal osteoporosis (PMOP) is associated with estrogen deficiency and oxidative stress (OS), which impair osteoblast survival and osteogenic function. Formononetin (FMN) is a phytoestrogen with antioxidant properties, but its protective effects on osteoblasts under OS and the signaling pathways associated with these effects remain incompletely understood. Here, we evaluated the effects of FMN in ovariectomized (OVX) mice and in a tert-butyl hydroperoxide (t-BOOH)-induced oxidative injury model in MC3T3-E1 cells. Bone microarchitecture, histology, serum OS markers, cell viability, reactive oxygen species (ROS) production, apoptosis, mitochondrial morphology, and osteogenic differentiation were assessed. Network pharmacology, molecular docking, molecular dynamics simulations, RNA sequencing, Western blotting, and pharmacological pathway-modulation experiments were used to investigate signaling changes associated with FMN treatment. FMN attenuated trabecular bone deterioration and systemic oxidative imbalance in OVX mice. In MC3T3-E1 cells, FMN improved cell viability, reduced ROS accumulation and apoptosis, alleviated mitochondrial damage, and preserved osteogenic differentiation following t-BOOH exposure. FMN treatment was accompanied by reduced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), together with reduced expression of activator protein 1 (AP-1)-related proteins, including c-Fos, c-Jun, and phosphorylated c-Jun. Pharmacological reactivation of p38/JNK signaling with anisomycin attenuated several antioxidant, anti-apoptotic, and pro-osteogenic effects associated with FMN treatment, whereas nuclear factor erythroid 2-related factor 2 (NRF2) inhibition partially diminished FMN-mediated protection. Collectively, these findings support an association between the protective effects of FMN and modulation of mitogen-activated protein kinase (MAPK)/AP-1 and NRF2-associated signaling.