Abstract
Acupuncture (AC) has been demonstrated to improve outcomes in ischemic stroke. However, its precise mechanism of action remains incompletely understood. This study investigated the effects of AC in a rat model of cerebral ischemia-reperfusion injury (CIRI). A middle cerebral artery occlusion/reperfusion (MCAO/R) model was established to create CIRI rats. Rats were randomly assigned to the Sham, MCAO/R, AC + MCAO/R, IRP2((+)) + MCAO/R, IRP2((-)) + MCAO/R and IRP2((-)) + AC + MCAO/R groups. Neurological function was assessed using the Garcia scores. TTC staining was used to observe cerebral infarction volume ratios. Transmission electron microscopy (TEM) examined mitochondrial structure in neurons within the ischemic penumbra. Colorimetric assays measured Fe(2+) content in neurons, along with ferroptosis-related biomarkers Reactive oxygen species (ROS), Glutathione peroxidase 4 (GPX4), Malondialdehyde (MDA) and Glutathione (GSH). Immunofluorescence staining detected Transferrin receptor 1 (TFR1), Ferritin (FER), Ferroportin (FPN) and Iron regulatory protein 2 (IRP2) expressions in the ischemic penumbra. RT-qPCR measured IRP2 mRNA expression, whilst RNA immunoprecipitation (RIP) analysis assessed the impact of each group on IRP2 binding to IRE. Our results showed AC improved neurological deficits, reduced infarct volume, decreased Fe(2+) overload, enhanced antioxidant markers GPX4 and GSH, and lowered lipid peroxidation levels of ROS and MDA. It also attenuated mitochondrial structural damage related to ferroptosis. Immunofluorescence staining and RT-qPCR analyses revealed that AC downregulated TFR1 and FER expression whilst upregulating FPN and IRP2 expression. Furthermore, RNA immunoprecipitation assays revealed enhanced binding affinity between IRP2 and IRE after AC. These findings indicate that AC inhibits neuronal ferroptosis after CIRI and protects brain tissue, possibly by promoting IRP2-IRE binding, modulating neuronal iron metabolism, and reducing lipid peroxidation.