【作者】 李翔；

【导师】 范骁辉；

【作者基本信息】 浙江大学 ， 药物分析学， 2014， 博士

【摘要】 中药复方药效物质及其作用机制的研究是中药现代化的关键科学问题之一。秉承中医整体论的治病理念,中药复方与复杂性疾病之间的相互作用是“系统-系统”的非线性、复杂作用模式,因而采用传统方法难以满足揭示这种复杂网络状关联关系。近年兴起的网络药理学和系统生物学试图从宏观或整体调控的角度诠释药物对生物网络的作用,为中药复方作用机制研究提供了新的研究思路和技术手段。在临床上芪参益气方用于治疗缺血性心脏病,其有效性已得到循证医学的研究证实,但芪参益气方抗心肌缺血的整合作用还缺乏系统性研究,阻碍了芪参益气方被国际市场认可和接受的进程。鉴于此,本论文运用网络药理学方法和技术,开展芪参益气方抗心肌缺血作用研究,通过整合分子、细胞及动物水平的多层次研究,初步诠释其多成分组合、多靶点、多途径的整合协同作用模式。主要研究内容如下：1、构建了冠心病网络药理学研究平台CHD@ZJU V2.0,并开展相关网络分析。通过整合文献、疾病相关公开数据库及蛋白-蛋白相互作用关系等资源,构建冠心病疾病网络,并采用网络拓扑学、子簇聚类及生物功能GO等开展网络分析,结果显示与疾病发病机制相关的生物学途径主要有：免疫炎症反应、血管生成、缺氧和氧化应激反应、能量代谢等；2、研究了芪参益气方主要代表性成分对疾病网络的调控作用。采用文本知识挖掘技术得到芪参益气方主要成分的作用靶点信息,构建药物-疾病网络,研究通过网络分析发现人参皂苷Rg1(Rg1)和丹参素(DSS)的共同作用靶点是JUN、MAPK8和MAPK3,黄芪甲苷(Ast)的心血管药理作用与免疫炎症反应、细胞迁移、粘附及血液循环等相关；3、研究了主要活性成分Ast、DSS、Rgl和反式橙花叔醇(ENL)单给及其组合给药抗大鼠急性心肌梗死(AMI)的药效作用,发现Ast和DSS能显著减小心肌梗死范围,组合给药能更显著地减小梗死范围。进一步的转录组学分析结果表明,免疫炎症、血管生成、心室重构和心肌修复、能量代谢等可能是四个成分发挥多靶点、多途径整合协同作用对抗AMI的关键生物学途径；4、采用H9C2心肌细胞缺氧、氧化应激损伤模型,研究四个主要成分对心肌细胞的协同保护作用。Ast,DSS和Rg1能对抗H9C2缺氧损伤及氧化应激损伤,四个成分的低浓度组合给药能协同增强抗心肌细胞缺氧损伤作用；5、采用LPS诱导RAW264.7炎症反应模型,研究四个主要成分的协同抗炎作用。研究发现倍半萜烯类成分ENL、SDL和RDL对LPS诱导NO分泌具有不同程度的抑制作用。DSS和Rg1两成分组合能协同增强对抗LPS诱导的NO分泌,且这一作用能被HO-1特异性抑制剂ZnPPIX减弱甚至取消。四个成分组合给药可以显著性地增强对LPS诱导NO分泌的抑制作用。更多还原

【Abstract】 Material basis and modes of action (MOA) of traditional Chinese medicine formula (TCMF) belong to the key issues of the TCM modernization. Under the guidance of holism concept, TCMF are established to treat diseases. Due to non-linear "system to system" interactions between TCMF and disease, traditional methods are not suitable to uncover these multi-complexed interactions. Emerging new concepts, such as systems biology and network pharmacology, offer new opportunity to investigate drug effects on a biological network from a perspective of holistic regulation, which provide novel rationale and techniques for TCMF study. QiShenYiQi formula (QSYQ) is commonly used in treatment of ischemic heart disease (IHD) in clinic and its efficacy has been confirmed by evidence-based medicine study. Despite previous studies on QSYQ, a systematic research is required to understand the integrated effect of QSYQ against myocardial ischemia, which would accelerate its approval and acceptance in the international pharmaceutical industry. Based on network pharmacology, this thesis was designed to investigate anti-myocardial ischemia action of QSYQ, elucidate the integrative and synergistic mode of action in a multi-component-multi-target-multi-pathway manner through an integrative study at molecular, cellular and in vivo levels. The main contents of this thesis are summarized as follows:1. A platform for coronary heart disease (CHD) network, namely CHD@ZJU V2.0, was constructed and further network analysis was carried out according to information in this database. Through integrated resources from published articles, available databases and protein-protein interaction (PPI), CHD disease network was established. Network analysis (including network topology, clustering classes and biological function Gene Ontology analysis) results showed that the biological pathways associated with the pathogenesis of CHD mainly include:immune and inflammation, angiogenesis, hypoxia and oxidative stress, energy metabolism, etc.2. Regulatory effects on disease network were studied for main components from QSYQ. Through text mining, targets information of ten main compounds from QSYQ was obtained and drug-disease network was constructed. Network analysis showed that the common targets of ginsenoside Rg1 (Rg1) and danshensu (DSS) were JUN, MAPK8 and MAPK3. Pharmacological effects of astragaloside IV (Ast) on The cardiovascular system were associated with immune and inflammation, cell migration, adhesion and blood circulation, etc.3. As the main active components Ast, DSS, Rgi and trans-nerolidol (ENL) were chosen and their protective effects against acute myocardial infarction (AMI) individually or in combinations were investigated on rats. The infarct size was obviously decreased after Ast and DSS administration (i.g.), while more significant reduction was observed with four compounds combination group. Using gene chip technology, transcriptomics study was performed to reveal the integrated and synergetic effects of four compounds in a multi-target-multi-pathway manner. Results showed that the critical biological pathways related with their anti-AMI activity may relate to immune and inflammation, angiogenesis, ventricular remodeling and myocardium repair, energy metabolism, etc.4. Synergetic effects of four compounds were then evaluated against hypoxia injury and oxidative stress injury in vitro. Results indicated that Ast, DSS and Rgi showed cardioprotective effects against injury, four compounds combination Synergistic effects against hypoxia injury were exhibited for four-compound combination.5. The LPS induced RAW264.7 inflammation model was used to investigate the synergistic anti-inflammatory effects of the four components. Three sesquiterpene components, ENL, SDL and RDL, inhibited LPS stimulated NO secretion at various concentrations. Enhanced inhibitoryactivity against LPS induced NO secretion was observed on the combination of DSS and Rgi, which could be alleviated or even abrogated by HO-1 specific antagonist ZnPPIX. Four components combination can significantly enhance the inhibitory effect against LPS stimulated NO secretion.更多还原