Electroacupuncture Inhibits Neuronal Ferroptosis in MCAO/R Model Mice by Regulating the GSK-3β/NRF2 Signaling Pathway

Background: Neuronal ferroptosis following acute ischemic stroke (AIS) contributes to ischemic brain injury and is regulated by nuclear factor erythroid 2-related factor 2 (NRF2), a key regulator of antioxidant defense and iron homeostasis. Whether electroacupuncture (EA) suppresses neuronal ferroptosis by regulating NRF2 stability and nuclear translocation through upstream glycogen synthase kinase 3 beta (GSK-3β) remains unclear. We investigated whether EA inhibits neuronal ferroptosis and alleviates ischemic brain injury by modulating the GSK-3β/NRF2 axis. Methods: Middle cerebral artery occlusion/reperfusion (MCAO/R) models were established in C57BL/6 and conditional Nrf2-knockout mice. The GSK-3β inhibitor laduviglusib (CHIR-99021; CHIR), the ferroptosis inhibitor ferrostatin-1 (Fer-1), and conditional Nrf2 knockout were used to investigate the anti-ferroptotic mechanism of EA and the regulatory hierarchy between GSK-3β and NRF2. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence were used to examine the GSK-3β/NRF2 pathway and ferroptosis-related molecules. NRF2 nuclear localization, neuronal nuclei (NeuN) /acyl-CoA synthetase long-chain family member 4 (ACSL4) double immunofluorescence, and co-immunoprecipitation were used to assess NRF2 nuclear translocation, neuronal ferroptosis, and the GSK-3β–NRF2 interaction, respectively. Results: EA inhibited GSK-3β activity after MCAO/R, restored NRF2 protein expression, promoted nuclear accumulation, and upregulated NAD(P)H:quinone oxidoreductase 1 (NQO1). EA also downregulated ACSL4 and transferrin receptor 1 (TfR1), upregulated glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), reduced NeuN/ACSL4-colocalized neurons, decreased ferrous iron (Fe2+) and malondialdehyde (MDA) levels, and increased superoxide dismutase (SOD) activity, glutathione (GSH) content, and the GSH/oxidized glutathione (GSSG) ratio. CHIR and Fer-1 produced similar anti-ferroptotic effects, and co-immunoprecipitation confirmed GSK-3β–NRF2 interaction. Conditional Nrf2 knockout did not affect EA- or CHIR-mediated GSK-3β phosphorylation but markedly attenuated their effects on NQO1, SLC7A11, GPX4, and TfR1. EA also alleviated mitochondrial ultrastructural damage, reduced cerebral infarction and Fluoro-Jade B (FJB)-positive neurons, and improved neurological function and motor coordination in MCAO/R mice. Conclusion: These findings identify the GSK-3β/NRF2 axis as an important regulatory mechanism of regulating neuronal ferroptosis after MCAO/R and support EA as a potential therapeutic strategy for alleviating neuronal ferroptosis and cerebral ischemia-reperfusion injury through GSK-3β inhibition and NRF2 activation.