Rosmarinic acid suppresses ferroptosis and confers neuroprotection in cerebral ischemia-reperfusion via direct KEAP1 inhibition and NRF2 activation

Cerebral ischemia–reperfusion injury exacerbates neuronal loss following acute ischemic stroke through oxidative stress and regulated cell-death pathways, notably ferroptosis. Here, we investigated whether rosmarinic acid (RA), a natural polyphenol, confers neuroprotection by inhibiting ferroptosis via activation of the KEAP1–NRF2 axis. In vivo , rats pretreated with RA (30 or 60 mg/kg, i.p.) for 14 days before middle cerebral artery occlusion/reperfusion exhibited dose-dependent improvements in neurological scores, reduced infarct volumes, preserved cerebral blood flow, and attenuated histopathological damage. In vitro , RA (25–50 μM) restored the viability of HT22 and PC12 cells subjected to oxygen-glucose deprivation/reperfusion, suppressed lipid reactive oxygen species accumulation, iron overload, and mitochondrial ultrastructural collapse. Mechanistically, RA promoted nuclear factor erythroid 2-related factor 2 (NRF2) nuclear translocation, enhanced antioxidant response element -driven luciferase activity, and upregulated ferroptosis-defense genes ( Gpx4 , Slc7a11 , and Gclm ) at both mRNA and protein levels. Cycloheximide chase and ubiquitination assays, supported by molecular docking, molecular dynamics simulations, and surface plasmon resonance, demonstrated that RA directly binds Kelch-like ECH-associated protein 1 (KEAP1) to stabilize NRF2. A KEAP1 knockdown abolished RA's induction of NRF2 targets and its anti-ferroptotic effects. These findings establish RA as a targeted NRF2 activator and ferroptosis inhibitor, offering a promising therapeutic strategy for limiting ischemia–reperfusion-induced neuronal injury.