Electroacupuncture attenuates ferroptosis by promoting Nrf2 nuclear translocation and activating Nrf2/SLC7A11/GPX4 pathway in ischemic stroke

Abstract Objective Electroacupuncture has been shown to play a neuroprotective role following ischemic stroke, but the underlying mechanism remains poorly understood. The ferroptosis has been shown to play a key role in the injury process. In present study we wanted to explore whether electroacupuncture could inhibit ferroptosis by promoting nuclear factor erythroid-2-related factor 2(Nrf2) nuclear translocation. Methods The ischemic stroke model was established by middle cerebral artery occlusion/reperfusion(MCAO/R) in adult rats. These rats have been randomly divided into the EA + MCAO/R group, the MCAO/R group, the EA + MCAO/R + Brusatol group(the inhibitor of Nrf2) and the EA + MCAO/R + DMSO group, and the Sham group. The EA + MCAO/R group, EA + MCAO/R + Brusatol group and the EA + MCAO/R + DMSO group received EA intervention at 24 hours after modeling for consecutive 7 days. The behavioral function was evaluated by Neurologic severity score(NSS), Garcia score, Foot-fault Test and Rotarod Test. The infarct volume were detected by TTC staining..The levels of Fe 2+ , reactive oxygen species (ROS), superoxide dismutase(SOD) and malondialdehyde(MDA) were measured by ELISA. The immunofluorescence and Western blotting was used to detect the expression of Total Nrf2, p-Nrf2, Nuclear Nrf2 and Cytoplasmic Nrf2, and the essential ferroptosis proteins, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11(SLC7A11) and ferritin heavy chain 1(FTH1). The mitochondria were observed by transmission electron microscopy(TEM). Results Electroacupuncture improved neurological deficits in rats model of MCAO/R, decrease the brain infarct volume, and inhibited the Fe 2+ , ROS and MDA accumulation, increased SOD levels, increased the expression of GPX4, SLC7A11 and FTH1, and rescued injured mitochondria. Especially, we found that the electroacupuncture up-regulated the expression of Nrf2, and promote phosphorylation of Nrf2 and nuclear translocation,However, Nrf2 inhibitor Brusatol reversed the neuroprotective effect of electroacupuncture. Conclusion These findings indicate that electroacupuncture confers neuroprotection against ischemic stroke through inhibitting ferroptosis. The underlying mechanism may be that electroacupuncture promote Nrf2 nuclear translocation and activate Nrf2/SLC7A11/GPX4 pathway.