电针调节外泌体miR-210对脑缺血后HIF-1α/VEGF/Notch信号通路的影响  

Effect of Electroacupuncture-Guided Exosome miR-210 on Regulation of HIF-1α/VEGF/Notch Signaling Pathway in Angiogenesis after Cerebral Ischemia

作　　者：袁之鹏 吕鹤群 曾春利 冯瑶婷[1] 彭拥军[1] YUAN Zhipeng;LYU Hequn;ZENG Chunlin;FENG Yaoting;PENG Yongjun(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,Jiangsu,China)

机构地区：[1]南京中医药大学附属医院,江苏南京210029

出　　处：《中华中医药学刊》2025年第2期158-164,I0031-I0035,共12页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82174484,81973932);江苏省第六期“333高层次人才培养工程”项目;江苏省中医院专项(Y2022ZR20);江苏省中医院高峰学术人才项目(k2021rc24);南京中医药大学自然科学基金项目(XZR2023096)。

摘　　要：目的观察电针对缺血性脑卒中大鼠分泌外泌体miR-210调控缺氧诱导因子-1α(HIF-1α)/血管内皮生长因子(VEGF)/Notch信号通路的机制。方法前期实验将SD大鼠随机分为假手术组、模型组、电针组,每组6只。后期实验将SD大鼠随机分为假手术组、模型组、外泌体组(造模后注射电针治疗后提取的血浆外泌体组),miR-210siRNA组(造模后注射电针治疗后提取的血浆外泌体和miR-210siRNA组),HIF-1α抑制剂组(造模后注射电针治疗后提取的血浆外泌体和HIF-1α抑制剂组),每组20只。前期实验建立改良后改良后大脑中动脉栓塞/再灌注(MCAO)大鼠模型,缺血1h后进行再灌注,电针组进行电针治疗,取穴“水沟”“百会”,电针30 min,共治疗3 d,提取电针组大鼠血浆外泌体用于后期实验外泌体组、miR-210siRNA组、HIF-1α抑制剂组注射。完成实验后进行取材。神经功能缺损评分评估大鼠实验前后神经功能,二辛可酸(BCA)法测定外泌体浓度及透射电镜观察外泌体形态大小,蛋白质印迹(Western blot)法检测外泌体标志性蛋白CD63、CD9、CD81表达,四氮唑比色法(TTC)显示脑梗死灶分布并检查脑梗死体积,苏木精-伊红(HE)染色法观察大鼠脑缺血组织病理形态,透射电镜观察大鼠脑缺血侧纹状体区超微形态学变化实时荧光定量PCR法检测前期实验大鼠各组外泌体中miR-210mRNA表达和后期实验大鼠各组纹状体脑组织miR-210mRNA、CD34、HIF-1α、VEGF、Notch mRNA表达。结果前期实验中电针组大鼠在干预后神经功能缺损评分显著降低(P<0.001)。在干预后,与模型组相比较,电针组大鼠神经功能缺损评分显著降低(P<0.005)。与假手术组相比,模型组、电针组外泌体浓度升高(P<0.001),外泌体标志性蛋白CD63、CD9、CD81存在属实,即前期实验中提取电针血浆中存在外泌体。后期实验中与模型组相比较,外泌体组、miR-210siRNA组、HIF-1α抑制剂组大鼠脑梗死体�Objective To observe the mechanism of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)/Notch signaling pathway regulated by the secretion of exosome miR-210 in ischemic stroke rats.Methods SD rats were randomly divided into sham operation group,model group and electroacupuncture group,with 6 rats in each group.In the later stage experiment,SD rats were randomly divided into sham operation group,model group,exosome group(plasma exosome extracted after injection of electroacupuncture after modeling)and miR-210siRNA group(plasma exosome and miR-210siRNA extracted after injection of electroacupuncture after modeling).HIF-1αinhibitor group(plasma exosomes and HIF-1αinhibitor group extracted after electroacupuncture treatment after modeling),20 in each group.The modified middle cerebral artery occlusion(MCAO)rat model was established in the preliminary experiment,and was reperfused after ischemia for 1 h.The electroacupuncture group was treated with electroacupuncture,and“Shuigou(DU26)”and“Baihui(DU20)”were selected for 30 min for a total of 3 days.The plasma exosomes of the rats in the electroacupuncture group were extracted for injection in the later experimental exosome group,miR-210siRNA group and HIIF-1αinhibitor group.After the experiment was completed,the samples were collected.The nerve function of rats was evaluated by Longa nerve function deficit score before and after the experiment.The concentration of exosomes was measured by BCA and the morphology and size of exosomes were observed by transmission electron microscopy.The expressions of exosome signature proteins CD63,CD9 and CD81 were detected by Western blot.HE staining was used to observe the pathological morphology of cerebral ischemic tissue in rats.The expressions of miR-210mRNA,CD34,HIF-1α,VEGF and Notch mRNA in the exosomes of rats in the early experimental period and those in the striatal brain of rats in the late experimental period were detected by real-time fluorescence quantitative PCR.Results Compared

关 键 词：缺血性脑卒中 外泌体 MIR-210 HIF-1Α VEGF Notch 

分 类 号：R259.416[医药卫生—中西医结合]