Abstract: BACKGROUND: Electroacupuncture, a therapy that integrates traditional and modern elements, has been shown to improve the prognosis of stroke. However, whether it can intervene in neuroinflammatory injury by regulating mitochondrial copper homeostasis remains unclear.  
OBJECTIVE: To observe the therapeutic effects of electroacupuncture stimulation at “Baihui” and “Dazhui” on cerebral ischemia-reperfusion injury, and to explore the possible mechanisms by which it improves copper homeostasis imbalance in cerebral ischemia-reperfusion injury.  
METHODS: Male Sprague-Dawley rats were randomly divided into a sham-operated group, a model group, and an electroacupuncture group, with 12 rats in each group. The latter two groups were used to establish a rat model of cerebral ischemia-reperfusion injury using the modified middle cerebral artery occlusion method. The electroacupuncture group received acupuncture at “Baihui” and “Dazhui” acupoints, once daily for 30 minutes each time, for 14 consecutive days. Zea-Longa scores were used to assess the degree of neurological deficits. Triphenyltetrazolium chloride staining was used to measure cerebral infarct volume. Hematoxylin-eosin staining and Nissl staining were employed to observe pathological structural changes in brain tissue, and rhodanine staining was used to observe copper ion deposition. Transmission electron microscopy was used to observe the ultrastructure of rat cortical neurons. Colorimetric assays were used to detect the levels of reactive oxygen species, iron-sulfur clusters, and cuprous ions in the infarcted brain tissue. Immunohistochemical staining and western blot assay were used to observe the protein expression levels of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), ATPase copper transporting beta (ATP7B), and dihydrolipoamide S-acetyltransferase (DLAT).  
RESULTS AND CONCLUSION: (1) Compared with the sham-operated group, the model group showed significantly increased Zea-Longa scores (P < 0.001) and a significantly increased percentage of cerebral infarct volume (P < 0.001); in the infarcted area, the number of neurons was reduced, their arrangement was loose, neurons showed obvious pathological changes, and the number of organelles was decreased; the contents of reactive oxygen species and cuprous ions in brain tissue were significantly increased (P < 0.001), while the content of iron-sulfur clusters was significantly decreased (P < 0.001); in the cortex, the protein expressions of Nrf2, ATP7B, and DLAT were significantly decreased (P < 0.05, P < 0.001, P < 0.01, respectively), and the protein expression of Keap1 was significantly increased (P < 0.001). (2) Compared with the model group, the electroacupuncture group showed significantly decreased Zea-Longa scores (P < 0.001) and a decreased percentage of cerebral infarct volume (P < 0.05); in the infarcted area, the number of neurons was increased, their arrangement was relatively regular, the nuclear membrane of neurons was relatively intact, and the number of organelles such as mitochondria and endoplasmic reticulum was increased; the contents of reactive oxygen species and cuprous ions in brain tissue were significantly decreased (P < 0.05, P < 0.001), and the content of iron-sulfur clusters was increased (P < 0.01); in the cortex, the protein expressions of Nrf2, ATP7B, and DLAT were significantly increased (P < 0.05, P < 0.001, P < 0.05, respectively), and the protein expression of Keap1 was significantly decreased (P < 0.05). To conclude, these findings suggest that electroacupuncture can protect mitochondrial structural and functional integrity by regulating the Keap1-Nrf2 signaling pathway, promoting ATP7B-mediated mitochondrial copper transport, and maintaining mitochondrial copper homeostasis and normal energy metabolism, ultimately improving neurological function impairment after ischemia-reperfusion and promoting brain tissue repair.  

Key words: electroacupuncture, ischemic stroke, copper homeostasis, Kelch-like ECH-associated protein 1, Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 signaling pathway, cerebral ischemia-reperfusion injury