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Objective. To observe the effect of adenosine A1 receptor in the hippocampus of mice on GSK-3β phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. Method. The model of middle cerebral artery occlusion (MCAO) was established and grouped into electroacupuncture pretreatment group (EA group), MCAO group, and sham-operated group (Sham group). The neurobehavioral manifestation, the volume of cerebral infarction, and its related protein changes in mice in each group were observed. Then, adenosine Α1 receptor antagonist and agonist were injected intraperitoneally to observe the effects of A1 receptor on the phosphorylation level of GSK-3β, neurobehavioral changes, and infarction volume. Results. (1) Compared with the MCAO group (24 hours after reperfusion), the infarct size in the EA group decreased significantly, and the Garcia neurological score and phosphorylation level of GSK-3β are increased. (2) Compared with the EA group, the infarct size in the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) group increased significantly, and the Garcia neurological score and phosphorylation level of GSK-3β are decreased. (3) Compared with the MCAO group, the infarct size in the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine (CCPA) group decreased significantly, and the Garcia neurological score and phosphorylation level of GSK-3β are increased. There was no significant difference between the EA group and CCPA group. Conclusions. Electroacupuncture pretreatment can increase GSK-3β phosphorylation level via activating A1 receptor, to protect neurons in ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

• Electroacupuncture(EA) pretreatment can alleviate the cerebral ischemia /reperfusion injury. • The mechanism of EA preventing cerebral ischemia /reperfusion injury relies on the inhibition of autophagy. • EA inhibits autophagy through the Wnt/GSK3β signaling. Electroacupuncture (EA), a traditional Chinese replacement therapy, is widely accepted to treat ischemic stroke. Increasing evidence show that autophagy is involved in the process of cerebral ischemia injury and the Wnt/GSK3β pathway, playing an important role in protecting central nervous system. In this study, rats were treated with EA prior to focal ischemia by middle cerebral artery occlusion (MCAO). Deficit score, infarct volumes and levels of autophagy markers, such as LC3I, LC3II and p62, were assessed with either PI3K inhibitor wortmannin or a GSK-3β inhibitor LiCl. Oxygen-glucose deprivation/re-oxygenation (OGD/R) was made in the primitive neuron in vitro , and was respectively treated with autophagy inhibitors 3-MA, LiCl, GSK3β siRNA, or mTOR inhibitor rapamycin. The results indicated that EA pretreatment increased the levels of autophagy marker LC3-II and reduced the levels of p62. Meanwhile, deficit outcome was improved, and infarct volumes were reduced by EA pretreatment. Furthermore, the beneficial effects of EA pretreatment were reversed by wortmannin. LiCl and GSK3β siRNA can mimic the neuroprotective effects of EA pretreatment by downregulating autophagy, and increasing protein levels of p-mTOR, p-GSK3β and β-catenin in OGD/R neurons. However, the protective effects of GSK3β siRNA were blocked by rapamycin. These results suggest that EA pretreatment induces tolerance to cerebral ischemia by inhibiting autophagy via the Wnt pathway through the inhibition of GSK3β. [ABSTRACT FROM AUTHOR]