Abstract:Objective To investigate the regulatory mechanism of blood-letting puncture at Baihui and Dazhui in promoting angiogenesis after ischemic stroke based on the erythropoietin (EPO)-mediated JAK2/STAT5 signaling pathway. Methods A total of 150 healthy male Sprague-Dawley rats were selected and randomly divided into normal control group, sham-operation group, model control group, blood-letting puncture group, and blood-letting puncture+erythropoietin antagonism group, with 30 rats in each group. The rats in the normal control group were not given surgery, those in the sham-operation group were treated with vessel isolation without suture, and those in the model control group, the blood-letting puncture group, and the blood-letting puncture+erythropoietin antagonism group were used to establish a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) by the modified Zea-Longa method. After successful modeling, the rats in the blood-letting puncture group and the blood-letting puncture+erythropoietin antagonism group were given blood-letting puncture at Baihui and Dazhui; for the rats in the blood-letting puncture+erythropoietin antagonism group, liposome-siEPO2 complex was intraperitoneally injected at a dose of 5 mg/kg before puncture to enter the target cells synthesizing EPO and induce RNA interference targeting EPO. HE staining was used to observe the pathomorphological changes of the cerebral cortex; RT-PCR was used to measure the mRNA expression levels of EPO and vascular endothelial growth factor (VEGF) in the cerebral cortex, and Western blot was used to measure the protein expression levels of P-JAK2,P-STAT5, and VEGF in the cerebral cortex. Results Compared with the normal control group, the model control group and the blood-letting puncture+erythropoietin antagonism group had more severe pathological injury of the cerebral cortex, and compared with the model control group, the blood-letting puncture group had a significant improvement in the pathological injury of the cerebral cortex. Compared with the normal control group, the model control group had significant increases in the mRNA expression levels of EPO and VEGF and the protein expression levels of P-JAK2,P-STAT5, and VEGF in the cerebral cortex (P<0.05), and this trend of increase was further enhanced by blood-letting puncture; compared with the blood-letting puncture group, the blood-letting puncture+erythropoietin antagonism group had significant reductions in the mRNA expression levels of EPO and VEGF(P<0.05) and the protein expression levels of P-JAK2,P-STAT5, and VEGF in the cerebral cortex (P<0.05). Conclusion Blood-letting puncture at Baihui and Dazhui can upregulate the expression of EPO, activate the JAK2/STAT5 signaling pathway, and promote the expression of downstream VEGF in a rat model of MCAO/R, thereby promoting angiogenesis after ischemic stroke.