Keap1-independent GSK-3β/Nrf2 signaling mediates electroacupuncture inhibition of oxidative stress to induce cerebral ischemia-reperfusion tolerance

Purpose: Cerebral ischemia-reperfusion (CIR) injury is a devastating consequence of stroke characterized by oxidative stress-induced neuronal damage. Electroacupuncture (EA) has emerged as a potential therapeutic intervention for ischemic stroke, but its underlying mechanisms remain incompletely understood. This study aimed to elucidate whether EA exerts anti-oxidative stress effects against CIR injury by modulating the GSK-3β/Nrf2 pathway. Methods: CIR mouse models were established using the suture-occluded method and underwent EA pretreatment. Cognitive and neurologic function, cerebral infarct volume, and neuronal damage were assessed in mice. Oxidative stress levels and the expression of components of the GSK-3β/Nrf2 pathway in the cerebral cortex were measured. The regulatory effect of GSK-3β on Nrf2 and its role in electroacupuncture to alleviate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury were investigated by modulating GSK-3β expression in HT22 hippocampal neuronal cells and electroacupuncture serum intervention. Ultimately, Nrf2 knockout mice, GSK-3β knockout mice, and wild-type mice treated with TBHQ (an Nrf2 activator) were utilized for further validation. Results: EA pretreatment improved cognitive impairment and neuronal damage induced by CIR injury. Mechanistically, EA inhibited oxidative stress in the cerebral cortex, manifested by reduced levels of reactive oxygen species and malondialdehyde, along with increased superoxide dismutase activity. Furthermore, EA upregulated the expression of Nrf2 and its downstream antioxidant enzymes HO-1 and NQO1, while Keap1 expression remained unaffected. In vitro, GSK-3β overexpression inhibited the protective effects of EA serum on OGD/R-induced neuronal damage. In vivo, knockout of either Nrf2 or Gsk-3β genes abolished the neuroprotective effects of EA, and TBHQ exerted effects similar to EA, confirming the significant role of GSK-3β/Nrf2 in mediating EA antioxidative effects. Conclusion: EA exerts antioxidative stress effects against CIR injury by activating the GSK-3β/Nrf2 signaling pathway, independent of Keap1 regulation.