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To evaluate the mechanism of governor vessel electroacupuncture in rats with post-stroke limb spasm by observing the changes of glutathione antioxidant system-related factors. Methods A total of 60 SD rats were randomly divided into the normal group (n=12), sham operation group (n=12) and modeling group (n=36). The middle cerebral artery obstruction model was prepared by thread approach method in the modeling group, and 24 rats with successful modeling were randomly divided into the model group and the electroacupuncture group, with 12 rats in each group. At the 3rd day after modeling, the electroacupuncture group was treated with electroacupuncture at three acupoints of the governor vessel, namely, "Dazhui" (GV14), "Jizhong" (GV6) and " Houhui" (anteromedial of the transverse process of the sixth lumbar vertebra), for 30 min each time, once a day for 7 days. The neurological function of rats was assessed by Zea Longa neurological deficit score. The muscle tension of rats was detected by modified Ashworth dystonia rating and electrophysiological tracing method. The brain tissue water content was measured by the dry-wet weight method. The volume of cerebral infarction of rats was measured by the TTC staining method. The contents of glutathione(GSH), catalase(CAT), oxidized glutathione(GSSG), superoxide dismutase(SOD), and malondialdehyde(MDA) in the cortex of rats were detected by colorimetry. The protein and mRNA expressions of glutathione reductase(GR), glutamate cysteine ligase(GCL)C, GCLM, and glutathione peroxidase 4(GPX4) in the cortex of rats were measured by Western blotting and real-time PCR, respectively. Results Compared with rats in the normal and sham operation groups, the Zea Longa neurological deficit score, modified Ashworth dystonia rating, the volume of cerebral infarction, brain tissue water content, and GSSG and MDA contents in cortex were increased in the model group, the tension signal value and the proteins and mRNA expressions of GR, GCLC, GCLM, and GPX4 in cortex were decreased, and the contents of GSH, CAT, and SOD in cortex were decreased(P<0.05). Compared with the model group, the Zea Longa neurological deficit score, modified Ashworth dystonia rating, the volume of cerebral infarction, brain tissue water content, and GSSG and MDA contents in cortex were decreased in the electroacupuncture group, the tension signal value and the proteins and mRNA expressions of GR, GCLC, GCLM, and GPX4 in cortex were increased, and the contents of GSH, CAT, and SOD in cortex were increased(P<0.05). Conclusion Governor vessel electroacupuncture can improve the severity of post-stroke limb spasm in rats, and its mechanism may be related to the regulation of glutathione antioxidant system in cerebral cortex. [ABSTRACT FROM AUTHOR]