基于线粒体识别的中药拮抗阿霉素心脏毒性“多组分-多靶标”关联分析的方法学研究

Mitochondria is an important target of Traditional Chinese medicine (TCM) against doxorubicin-induced cardiotoxicity, and it is a good recognizer for the screening of the active components from TCM. To focus on the problems that the effective components and mechanisms of Sini decoction (SND) against doxorubicin-induced cardiotoxicity are unclear, we take the mitochondrial membrane as recognizer to screen the bioactive components and targets of TCM, based on the preliminary result that some components in SND can directly target mitochondria of cardiomyocytes. In this study, we will establish a new method for the screening of the bioactive components in TCM based on the combination of the mitochondria recognition with comprehensive two-dimensional liquid chromatography, and then identify the binding components of SND on mitochondrial membrane and evaluate their bioactivities against doxorubicin-induced cardiotoxicity. Meanwhile, by using the drug affinity responsive target stability (DARTS) technique, we will identify their target protein on mitochondrial membrane. Then, we further perform molecular docking and molecular dynamics simulation to profile the corresponding relationship between the binding components and the binding targeted proteins on mitochondrial membrane. Finally, we choose three to five binding components and perform the validation of the targeted proteins on the cell model and the molecular level using siRNA and surface plasmon resonance (SPR) technologies. The relationship between the components and the targets against doxorubicin-induced cardiotoxicity will further be confirmed. By this study, we will establish a new approach to study the multicomponent-multitarget relationship based on mitochondria recognition. The results are expected to provide the active lead compounds and new targets for the new drug development against doxorubicin-induced cardiotoxicity. It is also an important significance on the discovery of new drug to treat mitochondrial diseases .

线粒体是中药拮抗阿霉素心脏毒性的重要靶点，是活性组分筛选的良好载体。针对四逆汤拮抗阿霉素心脏毒性药效物质不清，机制不明的问题，在证实四逆汤存在直接作用于心肌细胞线粒体的组分和前期工作基础上，采用线粒体膜为活性组分和靶标识别载体，建立基于线粒体识别-二维色谱技术的中药活性组分筛选新方法，鉴定四逆汤线粒体膜结合组分，评价其拮抗阿霉素心脏毒性的活性；采用药物亲和反应靶标稳定技术，鉴定并解析四逆汤在线粒体膜上的结合靶蛋白；通过分子对接模拟，表征线粒体膜结合组分与结合靶蛋白的对应关系。遴选制备3-5种结合组分，采用siRNA干扰等实验手段，在细胞分子水平进行靶点反向试验验证，明确四逆汤拮抗阿霉素心脏毒性组分与靶标的对应关系。通过本研究，构建基于线粒体识别的中药“多组分-多靶标”关联分析的新方法，有望为拮抗阿霉素心脏毒性的新药开发提供活性先导化合物和新靶点，这也对治疗线粒体疾病的新药研究具有重要意义。

线粒体是中药拮抗阿霉素心脏毒性的重要靶点，是活性组分筛选的良好载体。针对四逆汤拮抗阿霉素心脏毒性药效物质不清，机制不明的问题，本项目以心肌线粒体膜为活性组分和靶标识别载体，制备了“氨丙基三乙氧基硅烷共价修饰硅胶-戊二醛交联”的心肌线粒体膜固定相，建立了基于线粒体识别-二维液相色谱技术的中药靶向线粒体活性组分筛选新方法，鉴定四逆汤中24种线粒体膜结合组分，验证了12种结合成分的抗阿霉素心脏毒性活性。综合结合成分对阿霉素心肌细胞损伤的细胞活力分析和一维心肌线粒体膜色谱柱的保留行为，确定了10种四逆汤靶向心肌线粒体膜的活性成分，结果显示宋果灵具有较优的活性，并且细胞活力分析显示其不降低阿霉素抗肿瘤效果。建立了基于药物亲和反应靶标稳定技术（DARTS）的中药组分在心肌线粒体膜上结合蛋白的分离识别方法，鉴定了四逆汤在线粒体膜上的10种结合靶蛋白。采用分子模拟对接技术，建立了四逆汤靶向心肌线粒体膜活性成分与结合靶标蛋白的潜在对应关系，并遴选宋果灵及其靶蛋白HADHA进行Western Blot、SPR等验证，发现宋果灵与HADHA具有很好的亲和作用（Kd=8.168×10-6M）。建立了心肌线粒体非标记定量蛋白组学研究方法，在体内阐明了宋果灵抗阿霉素心脏毒性作用机制。通过本项目研究，构建了基于线粒体识别的中药“多组分-多靶标”关联分析的新方法，为靶向线粒体的中药活性成分发现和靶点识别提供借鉴。另外，本项目还建立了心肌线粒体代谢谱分析方法，构建了四逆汤调控阿霉素心脏毒性的线粒体代谢物调控网络；整合GC/LC-MS代谢组学和生物信息学方法，构建了“四逆汤-药材-代谢通路”关联网络，解析了四逆汤抗阿霉素心脏毒性的配伍规律。项目研究已经发表论文9篇(第一标注7篇，第二标注2篇)，另有3篇项目直接相关SCI论文待发表；申请专利4项，其中授权1项；培养研究生3名（在读），本科毕业设计3名。