基于HIF-1α-VEGF-EPO信号通路探究电针干预大脑中动脉栓塞大鼠脑血管新生的机制

目的 基于低氧诱导因子（hypoxia inducible factor，HIF）-1α-血管内皮生长因子（vascular endothelial growth factor，VEGF）-促红细胞生成素（erythropoietin，EPO）通路探究电针“百会”“水沟”“足三里”“曲池”治疗脑缺血的作用机制。方法 将108只SD雄性大鼠随机分为假手术组、模型组、电针组。采用线栓法复制右侧大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）大鼠模型。电针组于模型复制后4 h电针“百会”“水沟”和左侧“后三里”（“足三里”）、“曲池”，疏密波，频率5～100 Hz，每次20 min，每日1次，治疗14 d。假手术组、模型组同等抓取固定，不进行治疗。采用改良神经功能缺损体征评分（modified neurological severity scores，mNSS）评价大鼠神经功能损害情况，多普勒超声检测缺血半暗带区局部脑血流量（regional cerebral blood flow，rCBF）变化，HE染色观察脑缺血半暗带病理学改变，免疫组织化学法检测缺血半暗带区皮质CD34阳性细胞数，Western blot 法和RT-PCR法分别检测缺血半暗带区皮质HIF-1α、VEGF、EPO蛋白及其mRNA表达水平。结果 与同时点假手术组比较，模型组大鼠mNSS、CD34阳性细胞数、HIF-1α、VEGF、EPO蛋白和mRNA表达水平均显著增加（P＜0.05），rCBF显著减少（P＜0.05），HE染色可见大量神经元坏死；与同时点模型组比较，电针组大鼠mNSS评分显著减少（P＜0.05），rCBF、CD34阳性细胞数及HIF-1α、VEGF、EPO蛋白和mRNA表达水平均显著增加（P＜0.05），脑缺血半暗带区病理学改变均有所好转。结论 电针“百会”“水沟”“足三里”“曲池”能激活HIF-1α-VEGF-EPO通路蛋白和基因表达，增加CD34阳性细胞数量，介导内皮细胞血管新生，从而提高MCAO大鼠rCBF，改善脑组织结构和功能，且治疗效果具有时间累积效应。

Mechanism of Electroacupuncture Intervention on Angiogenesis in Rats with Middle Cerebral Artery Occlusion:A Study Based on the HIF-1α-VEGF-EPO Signaling Pathway

Objective To investigate the mechanism of action of electroacupuncture (EA)at Baihui,Shuigou,Zusanli,and Quchi points in the treatment of cerebral ischemia based on the hypoxia-inducible factor-1α (HIF-1α)-vascular endothelial growth factor(VEGF)-erythropoietin (EPO) signaling pathway.Methods A total of 108 male Sprague-Dawleyrats were randomly divided into sham-operation group,model group,and EA group.The suture method was used to establish a rat model of right middle cerebral artery occlusion (MCAO).At 4 hours after modeling,the rats in the EA group were treated with EA at Baihui,Shuigou,left Housanli (Zusanli),and Quchi points with dilatational wave at a frequency of 5-100 Hz for 20 minutes each time,once a day for 14 days,and those in the sham-operation group and the model group were caught and fixed without intervention.The modified Neurological Severity Score (mNSS) was used to evaluate neurological deficit;Doppler ultrasound was used to measure the change in regional cerebral blood flow(rCBF) in the ischemic penumbra;HE staining was used to observe the pathological changes of the ischemic penumbra;immunohistochemistry was used to measure the number of CD34-positive cells in the ischemic penumbra;Western blot and RT-PCR were used to measure the protein and mRNA expression levels of HIF-1α,VEGF,and EPO in the ischemic penumbra.Results Compared with the sham-operation group at each time point,the model group had significant increases in mNSS score,the number of CD34-positive cells,and the protein and mRNA expression levels of HIF-1α,VEGF,and EPO (P<0.05) and a significant reduction in rCBF (P<0.05),with massive neuronal necrosis shown by HE staining.Compared with the model group at each time,the EA group had a significant reduction in mNSS score (P<0.05) and significant increases in rCBF,the number of CD34-positive cells,and the protein and mRNA expression levels of HIF-1α,VEGF,and EPO (P<0.05),with certain improvements in the pathological changes of the ischemic penumbra.Conclusion EA at Baihui,Shuigou,Zusanli,and Quchi points can activate the protein and mRNA expression levels of the HIF-1α-VEGF-EPO pathway,increase the number of CD34-positive cells,and mediate endothelial angiogenesis,thereby improving rCBF and brain structure and function in MCAO rats,and its therapeutic efficacy has a time cumulative effect.