基于“2R网络药理学”的四妙勇安汤治疗心肌肥厚有效组分及作用机制研究

Traditional Chinese medicine clinical practice and previous experimental results showed that Simiao Yongan Decoction could improve the symptoms of cardiac hypertrophy, which is closely related to hypoxic inducible factor 1 (HIF-1) signaling pathway. However, the effective components and mechanisms of action remain unknown. Based on the the previous works, the hypothesis is put forward and proposes that Simiao Yongan Decoction is capable of treating cardiac hypertrophy owing to the regulatory effect of effective components, formed by the compatibility of various effective constituents, on hypertrophy-related genes expressions via regulating PI3K/Akt/mTOR/HIF-1 signaling pathway. The research model of “2R network pharmacology” is constructed based on “dominant constituents prediction (Reducing constituents)-potential targets screening (Reducing targets)”. Dominant constituents are screened by ADMET prediction with serum pharmacochemistry and tissue distribution, and potential targets are screened based on HIF-1 signaling pathway. Network relationships are established by taking dominant constituents and potential targets as the core so as to study the effective components and targets of Simiao Yongan Decoction in the treatment of cardiac hypertrophy, and then further confirmatory and analytical experiments are conducted with respect to the function, mechanisms and compatibility. This project aims at providing a new method for the discovery and application of effective components from traditional Chinese medical formulae and laying a foundation for the research and development of therapeutic drugs in cardiac hypertrophy based on the effective components of Simiao Yongan Decoction.

中医临床和前期实验结果表明四妙勇安汤能够改善心肌肥厚症状，且与缺氧诱导因子1（HIF-1）信号通路密切相关，但其有效组分和作用机制尚不清楚。基于前期工作基础，提出假说“四妙勇安汤治疗心肌肥厚是其多种有效成分配伍组合形成的有效组分通过调节PI3K/Akt/mTOR/HIF-1信号通路，调控肥厚相关基因表达实现的”，构建基于“优势成分预测（减成分，Reducing constituents）-潜在靶点筛选（减靶点，Reducing targets）”的“2R网络药理学”研究模式。通过ADMET性质预测结合血清化学和组织分布研究筛选优势成分，基于HIF-1信号通路筛选潜在靶点。以优势成分和潜在靶点为核心建立网络关系，研究四妙勇安汤治疗心肌肥厚的有效组分及其作用靶点，并予以功能、机理及配伍等验证与分析。为中药复方有效组分发现与应用提供新方法，为基于四妙勇安汤有效组分研发心肌肥厚治疗药物奠定基础。

构建了基于“优势成分预测-潜在靶点筛选”的“2R网络药理学”研究模式，研究阐明了四妙勇安汤改善心肌肥大的有效组分及其作用机制。.采用UPLC-MS/MS技术，对四妙勇安汤及其组方药味化学成分进行研究，从四妙勇安汤水提物中鉴定出138个化学成分，从玄参中分离鉴定出116个化合物，从金银花水提物中鉴定出72个化学成分。在此基础上，通过对四妙勇安汤及其组方药味主要化学成分的体内过程（胃肠道代谢、血清化学、药代动力学、组织分布等）研究，为建立四妙勇安汤“2R网络药理学”研究模式中的“优势成分预测”奠定基础。 .采用admetSAR对四妙勇安汤中135个结构明确的化学成分的ADMET性质预测，确定67个具有较好肠吸收且无AMES毒性和致癌性的成分，结合体内过程研究结果，选定32个原型成分与67个ADMET性质良好的化学成分进行对比分析，确定31个成分为潜在有效成分。.利用Discovery studio 4.0软件，结合KEGG、UniProt、PDB等数据库，对前期研究发现的四妙勇安汤主要作用通路——HIF-1通路进行分子网络分析，筛选潜在靶点。共筛选出17个靶蛋白，采用分子对接技术，命中27个潜在有效成分。选择其中含量可测、在各药味中具有代表性的18个不同类型的化合物，检测其对ISO诱导的H9c2心肌细胞表面积的影响。结果显示绿原酸、新绿原酸、隐绿原酸、异绿原酸C、安格洛苷C、异绿原酸A、当药苷和哈巴苷等能够显著抑制ISO诱导的心肌细胞表面积的增加，确定该8个成分为该复方中改善心肌肥大的有效成分。.通过测定8个成分在不同浓度下对ISO诱导的H9c2心肌细胞ANP mRNA表达的影响，选定6个主要有效成分，分为A组（新绿原酸、绿原酸、隐绿原酸和异绿原酸C）、B组（当药苷）和C组（哈巴苷）进行配伍组合研究。结果显示配伍组分ABC能够明显抑制心肌细胞肥大和ANP mRNA的过表达，确定为有效组分。进一步研究了不同剂量有效组分ABC对ISO诱导的H9c2心肌细胞中ANP、β-MHC、PI3Kα、p-ERK、p-Akt、p-mTOR和HIF-1α表达的影响，结果显示有效组分ABC可通过抑制Akt/mTOR/HIF-1α通路的活化发挥抑制ISO诱导的心肌细胞肥大的作用。