基于网络药理学和神经血管单元的三七治疗缺血性脑卒中整合调节机制研究

Radix notoginseng(sanchi) has definite therapeutic effect in the treatment of stroke with a long history. Presently, some active ingredients extracted from Radix notoginseng has been widely used in clinic treatment of ischemic stroke. Existing researches and our previous research showed that some active ingredients of Radix notoginseng can act on many links during pathological process of cerebral ischemia with multi-target. However, current research and treatment are mainly about some active ingredients of Radix notoginseng, which splits the overall concept of traditional Chinese medicine, and neglect integrative adjustment mechanism of synergy of some active ingredients of Radix notoginseng act on ischemic stroke. To use neurovascular unit which was proposed to act as a whole new concept of the treatment of stroke and network pharmacology which found the complex network pharmacology role between drugs and diseases, may be helpful to reveal and explain the integrative adjustment mechanism of Radix notoginseng for ischemic stroke. Thus, based on network pharmacology and neurovascular unit, modern technology like whole genome sequencing technology combined with traditional Chinese medicine treatment concept as a whole was used to detect deferentially expressed genes in the brain after Radix notoginseng intervention in rats with cerebral ischemia, build drug - target - disease network, and analyze targets and pathways. In addition, from the point of view of neurovascular unit units and network pharmacology, the integrated regulation mechanism of Radix notoginseng in the treatment of ischemic stroke was revealed and clarified, so as to provide theoretical basis for the clinical use of Radix notoginseng.

三七治疗中风历史悠久且有疗效，目前提取的三七一些活性成分（三七总皂苷等）已广泛用于缺血性脑卒中的临床治疗。现有研究和我们前期研究发现三七的某些活性成分可作用于脑缺血损伤后病理过程中多个环节。但是，当前的研究和临床治疗主要围绕三七某些活性成分开展，分裂中医药的整体观，忽视三七多活性成分协同作用于缺血性脑卒中的整合调节机制。利用近年提出的作为卒中整体治疗新概念的神经血管单元和发现药物与疾病间存在复杂网络调控作用的网络药理学，将有助于揭示和阐释三七在缺血性脑卒中的整合调节机制。为此，本课题将基于神经血管单元和网络药理学，采用全转录组测序技术等现代技术结合中医药传统整体观，检测三七干预后大鼠缺血脑组织差异表达的基因，构建药物-靶点-疾病网络，分析三七的作用靶点及通路，从神经血管单元和网络药理学角度揭示和阐明三七在缺血性脑卒中治疗中的整合调节机制，为三七的临床应用提供理论依据。

为从网络药理学和神经血管单元角度揭示三七在缺血性脑卒中治疗中的整合调节机制，阐明三七通过多靶点、多作用通路修复缺血性脑卒中后的神经血管单元，改善神经功能。本研究通过在体实验（动物实验）和离体实验（细胞培养）通过电镜和流式细胞学技术观察三七对缺血缺氧后神经血管单元形态学变化影响。利用RNA-Seq技术检测三七干预后大鼠脑组织的差异基因表达谱，DESeq2软件分析并筛选出三七干预后差异表达基因。采用qRT-PCR方法对差异表达基因进行验证。构建“三七有效成分-靶基因-神经血管单元”的整体网络，通过Cytoscape软件和DAVID数据库对整体网络进行分析，筛选三七与神经血管单元共同作用的基因和通路。. 研究结果：（1）三七明显降低大鼠神经功能评分，减小脑梗死体积，减轻细胞损伤，同时抑制神经元、微血管内皮细胞凋亡、降低血脑屏障的通透性，对神经血管单元具有整体保护作用。（2）三七干预后差异表达基因有817个（390个上调基因和427个下调基因），参与抗缺血性脑卒中调节的靶点有420个，参与缺血后神经血管单元保护作用的关键靶点有48个，其中以IL6、VEGFA和CXCL8为核心靶点；富集信号通路有54条，其中以PI3K-Akt信号通道、钙离子信号通道、cGMP-PKG信号通道和AMPK信号通路为主要通路。. 科学意义：中药三七在治疗缺血性脑卒中方面具有多活性成分、多作用靶点和多作用通路，将有助于三七中药现代化，为三七的临床应用提供实验依据。