Formononetin alleviates hypoxic-ischemic brain damage by inhibiting neuronal ferroptosis via activation of Nrf2 signaling

Hypoxic-ischemic brain damage (HIBD) represents a serious neurological disorder linked to ferroptosis, posing major clinical difficulties. This study investigates the neuroprotective role of formononetin (FMN), a natural antioxidant isoflavone, through its regulation of ferroptosis mediated by the Nrf2 pathway. In vivo, a neonatal mouse HIBD model is used, and FMN is administered alone or with the Nrf2 inhibitor ML385. Brain injury is evaluated via histopathological and functional tests. Ferroptosis markers and Nrf2 expression are measured both molecularly and by imaging. In vitro, HT22 cells undergoing oxygen-glucose deprivation (OGD) are treated with FMN and inhibitors (Fer-1, ML385) to examine ferroptosis and mitochondrial status. The results show that FMN markedly reduces cerebral infarction, brain edema, and neuronal degeneration in HIBD mice. It also lowers oxidative stress (DHE, MDA), iron overload, and pro-ferroptotic markers (TfR1, ACSL4), while raising anti-ferroptotic factors (GSH, GPX4, SLC7A11). FMN specifically increases Nrf2 and its targets (HO-1, NQO1) in the penumbra, downregulates Keap1, and facilitates Nrf2 nuclear translocation. Through network pharmacology, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR), Nrf2 activation is identified as a key mechanism. In OGD-treated HT22 cells, FMN suppresses lipid peroxidation (MDA, C11-BODIPY⁵⁸¹/⁵⁹¹) and iron accumulation (FerroOrange) while restoring GSH and mitochondrial membrane potential, paralleling ferrostatin-1 effects. Critically, FMN’s benefits are counteracted by ML385. These findings demonstrate that FMN protects against HIBD by directly binding Nrf2 and modulating the Keap1/Nrf2 pathway, thereby inhibiting ferroptosis and attenuating neuronal damage.