基于EPO介导的JAK2/STAT5信号通路研究百会、大椎刺络促缺血性脑卒中后血管新生的调控机制
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安徽省自然科学基金项目(1908085QH371)


Angiogenesis After Ischemic Stroke: A Study Based on the Erythropoietin-mediated JAK2/STAT5 Signaling Pathway
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    摘要:

    目的 基于促红细胞生成素(erythropoietin,EPO)介导的JAK2/STAT5信号通路探讨百会、大椎刺络促缺血性脑卒中后血管新生的调控机制。方法 选取健康SD雄性大鼠150只,随机分为正常对照组、假手术组、模型对照组、刺络治疗组、刺络+EPO拮抗组,每组30只;正常对照组不进行手术,假手术组仅分离血管而不插线栓,模型对照组、刺络治疗组、刺络+EPO拮抗组采用改良的Zea-Longa法制备大脑中动脉局灶性脑缺血再灌注模型(middle cerebral artery occlusion/reperfusion model,MCAO/R)大鼠。模型制备后刺络治疗组、刺络+EPO拮抗组采用“百会”“大椎”穴刺络进行干预,刺络+EPO拮抗组在针刺前将脂质体-siEPO2复合物按5 mg/kg剂量对大鼠进行腹腔注射,使之进入合成EPO的靶细胞,在细胞内引起针对EPO的RNA干扰效应。采用苏木精-伊红染色观察各组大鼠脑皮质病理形态,RT-PCR法检测各组大鼠脑皮质中EPO、血管内皮生长因子(vascular endothelial growth factor,VEGF) mRNA表达水平,Western Blot法检测各组大鼠脑皮质中P-JAK2、P-STAT5、VEGF蛋白表达水平。结果 与正常对照组比较,模型对照组及刺络+EPO拮抗组大鼠脑皮质病理损伤严重;与模型对照组比较,刺络治疗组大鼠脑皮质病理损伤明显改善。与正常对照组比较,模型对照组大鼠脑皮质中EPO、VEGF mRNA表达水平显著升高(P<0.05),P-JAK2、P-STAT5、VEGF蛋白表达水平显著升高(P<0.05),刺络治疗会进一步增强这种趋势;与刺络治疗组比较,刺络+EPO拮抗组大鼠脑皮质中EPO、VEGF mRNA表达水平降低(P<0.05),P-JAK2、P-STAT5、VEGF蛋白表达水平降低(P<0.05)。结论 通过百会、大椎刺络能上调MCAO/R模型大鼠EPO的表达水平,激活JAK2/STAT5信号通路,促进下游VEGF的表达而促进缺血性脑卒中后血管的新生。

    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.

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王保国,曹 奕,陈倩倩,张静波,高 纺,张 璨,施婧婧,周文婷.基于EPO介导的JAK2/STAT5信号通路研究百会、大椎刺络促缺血性脑卒中后血管新生的调控机制[J].安徽中医药大学学报,2022,41(5):67-72

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  • 在线发布日期: 2022-10-11