Electroacupuncture Inhibits Ferroptosis by Modulating Iron Metabolism and Oxidative Stress to Alleviate Cerebral Ischemia–Reperfusion Injury

Ischemic stroke (IS) is one of the leading causes of mortality and long-term disability worldwide. Electroacupuncture (EA) is commonly used in the treatment of IS, meaning that may reduce cerebral ischemia–reperfusion injury (CIRI). The middle cerebral artery occlusion/reperfusion (MCAO/R) rat models were created by the modified Zea Longa suture method. EA treatment was performed for 7 consecutive days at the acupoints Neiguan (PC6), Shuigou (GV26), and Sanyinjiao (SP6). The neurological function was assessed using the Zausinger six-point neurological deficiency score. The cerebral infarct volume was detected by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Hematoxylin and eosin (HE) staining was employed to observe the pathological changes in brain tissues. Prussian blue staining was employed to investigate iron deposition within the brain tissues. Transmission electron microscopy (TEM) was utilized to examine the morphological characteristics of mitochondria. Simultaneously, flow cytometry was utilized to detect the fluorescence intensity of reactive oxygen species (ROS). Assay kits were employed to measure the levels of Fe²⁺ and glutathione (GSH). Additionally, western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR) assays were performed to evaluate the expression levels of proteins associated with ferroptosis. Compared with the MCAO/R group, both the MCAO/R + EA and MCAO/R + DFO groups exhibited significant improvements in neurological function following cerebral ischemia–reperfusion (CIR), attenuated the pathological brain tissue injury, and reduced the cerebral infarct volume and iron deposition in brain tissue. Furthermore, both the MCAO/R + EA and MCAO/R + DFO groups displayed a marked reduction in mitochondrial injury. There was a substantial decrease in Fe²⁺ and ROS levels, accompanied by a notable increase in GSH level and glutathione peroxidase 4 (GPX4) activity. Compared with the MCAO/R group, the levels of ferroportin1 (FPN1) protein and mRNA expression were significantly increased in the MCAO/R + EA and MCAO/R + DFO groups, and the expression levels of transferrin (TF), transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1) protein and mRNA, as well as ferritin (FER) protein, were significantly decreased. EA inhibits ferroptosis by modulating iron metabolism and oxidative stress to alleviate CIRI, exerting neuroprotective effects.

Graphical Abstract