基于网络药理学和实验验证的血栓通改善缺血性脑微循环障碍作用机制研究  

Xueshuantong improves cerebral microcirculation disorder:action mechanism based on network pharmacology and experimental validation

作　　者：王高瑞 陈姿羽 吴辉[1] 刘莹萍 陈明 赖树生 吴晓俊[1] 王峥涛[1] WANG Gao-rui;CHEN Zi-yu;WU Hui;LIU Ying-ping;CHEN Ming;LAI Shu-sheng;WU Xiao-jun;WANG Zheng-tao(Institute of Chinese Materia Medica of Shanghai University of Traditional Chinese Medicine and Shanghai Key Laboratory of Compound Chinese Medicines,Shanghai 201203,China;Guangxi Key Laboratory of Comprehensive Utilization Technology of Pseudo-ginseng,Wuzhou 543002,China)

机构地区：[1]上海中医药大学中药研究所暨上海市复方中药重点实验室,上海201203 [2]广西三七综合利用技术重点实验室,广西梧州543002

出　　处：《药学学报》2022年第7期2077-2086,共10页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81920108033);国家级大学生创新训练项目(202110268033);广西科技基地和人才专项项目(桂科AD20297068)。

摘　　要：本文通过网络药理学和实验验证结合,探讨血栓通主要皂苷成分改善脑微循环障碍的作用机制。从网络药理数据库获取血栓通主要三七皂苷成分结构式,预测药物潜在作用靶点和脑微循环障碍疾病靶点,将药物与疾病的交集靶点导入STRING在线分析平台和Cytoscape软件构建蛋白质互作分析图及拓扑图,获得作用核心靶点,并进行GO和KEGG富集分析;通过免疫组织染色检测大脑中动脉阻塞再灌注(MCAO/R)模型大鼠大脑缺血侧皮层血小板内皮细胞黏附分子-1(PECAM-1/CD31)的表达,RT-qPCR、Western blot检测MCAO/R模型大鼠缺血侧大脑皮层微血管药物作用潜在靶点基因的mRNA及蛋白表达水平。基于网络药理学,获取242个血栓通潜在作用靶点,425个脑微循环障碍疾病靶点,得到35个交集靶点,并筛选出蛋白激酶B(AKT1)、血管内皮生长因子A(VEGFA)、半胱氨酸天冬氨酸蛋白酶-3(CASP3)、基质金属蛋白酶9(MMP-9)、磷脂酰肌醇-3-激酶催化亚基α(PIK3CA)、信号传导及转录激活蛋白-3(STAT3)等关键靶点。在MCAO/R大鼠中实验验证发现,血栓通在48 mg·kg^(-1)剂量下,可显著改善模型大鼠的神经行为损伤和脑微血管密度和形态;并在脑微血管中下调AKT1、MMP-9的mRNA水平,上调血管内皮细胞标志物CD31及磷酸化AKT、磷酯酰肌醇-3-激酶(PI3K)蛋白表达水平和B淋巴细胞瘤-2/Bcl-2相关X蛋白(Bcl-2/Bax)比值,下调磷酸化STAT3、MMP-9、cleaved caspase-3的蛋白表达水平。结果表明血栓通可能通过调节脑微血管PI3K、AKT、MMP-9、STAT3、caspase-3相关信号通路改善缺血再灌注大鼠脑微循环障碍,减轻神经功能损伤。本研究严格遵守实验动物在医学研究过程中应该遵守的所有伦理原则。This study is to explore the mechanism of Xueshuantong improving cerebral microcirculation disorder through the combination of network pharmacology and experimental validation in vivo.Structural formulas of main Panax notoginseng saponins,including notoginsenoside R1,and ginsenoside Rg1,Re,Rb1 and Rd were obtained from Pubchem website and their potential targets were predicted by Swiss Target Prediction database.Potential molecular targets of brain microcirculation disorder were acquired from OMIM and GeneCards database.The overlapped molecular targets between the drug and disease were analyzed.Protein interaction analysis and topology maps were constructed through the STRING online analysis platform and Cytoscape software.Core action targets were selected.GO function and KEGG pathway were analyzed by DAVID database.Immunohistochemical method was used to examine the expression of platelet endothelial cell adhesion molecule-1(CD31)in the ischemic cortex of middle cerebral artery occlusion and reperfusion(MCAO/R)rats.The levels of mRNA and protein expressions of core action targets in MCAO/R model rats′brain microvessels were verified by RT-qPCR and Western blot.Based on network pharmacology,242 targets of Xueshuantong,425 targets of brain microcirculation disorder,and 35 overlapped targets were obtained.The potential key targets of Xueshuantong,protein kinase B(AKT1),vascular endothelial growth factor A(VEGFA),caspase 3(CASP3),matrix metallopeptidase 9(MMP-9),phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA),signal transducer and activator of transcription 3(STAT3)involved in the alleviation of cerebral microcirculation disorder were obtained by setting degree and betweenness centrality as screening parameters.Xueshuantong at the dose of 48 mg·kg^(-1)was shown to significantly improve the injury of neurological behaviors,as well as the density and morphology of microvessels of MCAO/R model rats.Xueshuantong could down-regulate the mRNA levels of AKT1,MMP-9,and STAT3,increase the pro

关 键 词：网络药理学 血栓通 三七皂苷 脑缺血 微循环障碍 

分 类 号：R966[医药卫生—药理学]