【摘要】 目的 观察补阳还五汤对脑缺血大鼠微小RNA（miRNA）表达谱的影响，探讨其对脑缺血后神经血管单元（NVU）的保护机制。方法 将SD大鼠随机分为对照组、模型组、补阳还五汤组(5 g·kg^-1)及丁苯酞组(54 mg·kg^-1)，灌胃给药。除对照组外，其余各组采用大脑中动脉栓塞法复制脑缺血模型。灌胃给药7 d后，采用神经行为学评分评估神经功能损伤，HE染色评估病理损伤，免疫组化法检测脑组织中神经血管单元相关蛋白微管相关蛋白2（MAP2）、神经胶质纤维酸性蛋白（GFAP）和血管性血友病因子（VWF）的表达，miRNA微阵列筛选差异表达的miRNAs，通过基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路富集分析差异miRNAs靶基因参与的主要生物学过程，实时荧光定量PCR（qRT-PCR）法验证微阵列芯片结果。结果 与对照组比较，模型组大鼠神经行为学评分明显升高（P<0.01）；缺血侧神经元排列不规整，细胞间隙增宽，细胞核固缩；缺血侧脑组织中MAP2及VWF的表达降低（P<0.01）,GFAP表达上升（P<0.01）；共有82个差异表达的miRNAs（FC≥1.5且P<0.05）。与模型组比较，补阳还五汤组大鼠神经行为学评分降低（P<0.01）；缺血侧神经元排列相对规整，细胞间隙减小，核仁较清晰；缺血侧脑组织MAP2及VWF的表达上升（P<0.01）,GFAP表达下降（P<0.01）；并有9个差异表达的miRNAs（FC≥1.5且P<0.05）。生物信息学分析显示核心miRNAs可能主要通过IL-17信号通路、趋化因子信号通路及Hippo信号通路等发挥治疗作用。与对照组比较，qRT-PCR检测发现模型组中rno-miR-532-5p、rno-miR-338-3p及rno-miR-412-5p表达下调（P<0.01,P<0.05）,rno-miR-211-3p及rno-miR-494-3p表达上调（P<0.01,P<0.05）；与模型组比较，补阳还五汤组rno-miR-532-5p、rno-miR-338-3p及rno-miR-412-5p表达上调（P<0.01,P<0.05）,rno-miR-211-3p及rno-miR-494-3p表达下调（P<0.01），与微阵列芯片结果趋势一致。结论 补阳还五汤可能通过影响脑缺血大鼠的miRNA表达谱，保护神经血管单元，发挥抗脑缺血损伤的作用。更多还原

【Abstract】 ObjectiveTo observe the effects of Buyang Huanwu Decoction（BHD）on miRNA expression profiles in rats with cerebral ischemia（CI）and to investigate the mechanisms underlying its protection of the neurovascular unit（NVU）after CI.MethodsSD rats were randomly divided into control group,model group,BHD group(5 g·kg^-1),and buphthalein group(54 mg·kg^-1), which were treated by gavage. Each group except for the control group was modeled by using middle cerebral artery embolization method. Seven days after the intervention, neurobehavioral scores were used to assess the neurological impairment,HE staining was used to assess the pathological damage,and immunohistochemistry was performed to detect microtubule-associated protein 2（MAP2） related to NVU proteins,glial fibrillary acidic protein（GFAP）and von Willebrand factor（VWF）in brain tissue. The differentially expressed miRNAs were screened by Agilent miRNA microarray,and the main biological processes involved in the target genes of differential miRNAs were analyzed by gene ontology（GO）function and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment, quantitative real-time PCR was used to verify the microarray results.ResultsCompared with the control group,the neurobehavioral scores of the model rats increased significantly（P<0.01）,irregular arrangement,widened intercellular spaces and karyopyknosis of neurons in the ischemic side were found, and the expressions of MAP2 and VWF in the ischemic brain tissue were decreased（P<0.01）, the expression of GFAP was increased（P<0.01）,and a total of 82 differentially expressed（DE）miRNAs were observed（FC≥ 1.5 and P<0.05）. Compared with the model group,the neurobehavioral scores of rats in the BHD group were decreased（P<0.01）, the arrangement of neurons in the ischemic side was relatively regular, the intercellular spaces were decreased, the nucleoli were clear. The expressions of MAP2 and VWF in the ischemic side brain tissue were increased（P<0.01）, the expression of GFAP was decreased（P<0.01）, and nine DE miRNAs were identified（FC≥ 1.5 and P<0.05）. Bioinformatics analysis revealed that core miRNAs may exert therapeutic effects mainly through IL-17 signaling pathway, chemokine signaling pathway, and Hippo signaling pathway. Compared with the control group,qRT-PCR revealed that the expressions of rno-miR-532-5 p,rno-miR-338-3 p,and rnomiR-412-5 p were downregulated（P<0.01, P<0.05）, while rno-miR-211-3 p and rno-miR-494-3 p were upregulated（P<0.01,P<0.05）in the model group. Compared with the model group,the expressions of rno-miR-532-5 p,rno-miR-338-3 p and rno-miR-412-5 p were upregulated（P<0.01,P<0.05）,and the expressions of rno-miR-211-3 p and rno-miR-494-3 p were downregulated（P<0.01）in the BHD group,which were consistent with the trend of the microarray results.ConclusionBHD may play a role against CI injury by affecting the miRNA expression profile and protecting the NVU in rats with CI.更多还原