一种利用线粒体集“识别-鉴定-评价-寻靶”于一体的中药抗肿瘤活性成分群快速筛选体系的构建研究

Mitochondria is the vital organelle of energy genesis in cells, which plays a key role in several important physiological and biochemical processes. Mitochondria dysfunction might lead to pathogenesis of many diseases, as a result of which, prevention and alleviation of mitochondria dysfunction has become an effective treatment. Nowadays, several drug targets have been found on mitochondria, such as mPTP, VDAC, PDK, LDH, IDH, and so on. Many traditional Chinese medicines (TCMs), such as ginseng, Sophorae Flavescentis Radix, Smilacis Glabrae Rhizoma, Scutellariae Barbatae Herba, could exhibit anti-tumor effect through regulation the function of mitochondria. However, whether TCMs could exhibit their therapeutical effects through those targets, which component in TCMs could directly act on those targets, or what is the mechanism, are still unknown. Besides, rapid and effective screening of active components in TCMs is still a bottleneck problem in the development of TCMs. Therefore, the present project will focus on the establishment of a rapid screening system, which combines recognition, identification, evaluation and target-searching, for discovery of novel anti-tumor components or components group in TCMs. And the new screening system will be used in the active components group searching in several TCMs or formulas, such as Smilacis Glabrae Rhizoma and Kushen injection, as well as their activity evaluations. Furthermore, this project will also apply molecular docking, molecular probe, and binding competition, for target searching, and and explain the mechanism of those active components. This project will provide a universal idea and method for high-performance active components searching, as well as the theoretical basis and experimental proof for pharmacology of TCMs.

线粒体是细胞能量生成的关键细胞器，其功能障碍在肿瘤发生、发展过程中起重要作用。线粒体存在众多药物靶点，如渗透转换孔复合体、电子传递链复合物及多种能量代谢酶等。很多中药可通过调节线粒体功能发挥抗肿瘤作用。但中药能否通过直接作用于线粒体发挥药效，哪些活性成分可作用于线粒体及作用机理尚不明确，此外线粒体为靶标的快速筛选方法的缺乏，严重限制中药线粒体靶向活性成分的发现及相关机制研究。本项目拟采用线粒体靶向筛选及LC/MS技术，建立筛选周期短、样品用量少、分析成本低，集“识别-鉴定-评价-寻靶”于一体的中药抗肿瘤活性成分群快速筛选体系，筛选土茯苓、桂枝茯苓丸等中药或复方中抗肿瘤活性成分群，并结合分子探针、结合竞争等技术、体内外研究探索其作用靶点及药效机制。本项目成果可提供一种活性物质筛选的通用性思路及方法，为中药药效物质基础及作用机制研究提供理论、技术支持，为先导化合物发现及抗肿瘤新药研发提供依据。

中药可通过引起线粒体损伤发挥抗肿瘤功效，但未见作用于线粒体的关键活性药效物质群及其靶点的方法报道，为克服传统筛选方法的费时费力，本项目利用不同来源（如人肝癌细胞、人结肠癌细胞等）的线粒体，结合亲和超滤与液质联用技术，成功构建一不经分离纯化的、以线粒体为靶点的集“识别-分离-鉴定-寻靶”于一体的抗肿瘤活性成分快速筛选新体系，应用于十余种中药来筛选其作用于线粒体的活性成分，结果从中筛到69种首次发现的、可与线粒体结合的活性物质，其中44种化合物具有抗肿瘤活性，皆为中药发挥以线粒体为靶点的抗癌物质基础。. 基于线粒体肿胀、膜电位变化、癌细胞活力与细胞凋亡检测等实验并结合线粒体专属抑制剂（包括亲环素D抑制剂、钙离子转运抑制剂、电压依赖阴离子通道抑制剂），发现8种化合物（大黄酸、苦参醇I、苦参酮、白杨素葡萄糖醛酸苷、槲皮苷、黄杞苷、芦荟大黄素-1-O-葡萄糖苷、大黄酚-1-O-葡萄糖苷）可诱导线粒体通透性转换孔开放，引起线粒体膜电位降低、癌细胞增殖抑制、凋亡等作用而发挥抗肿瘤药效；其中4种为首次发现可抑制癌细胞的化合物；6种化合物可与亲环素D（通透性转换孔主要蛋白之一）相结合。. 进一步纯化重组蛋白亲环素D，并建立其磁珠固定亲和色谱法和表面等离子共振高通量筛选法应用于中药中的活性成分筛选，发现可与亲环素D结合的中药成分28种。其中表没食子酸儿茶素没食子酸酯可别构结合于阳性药环孢菌素A结合位点以外的位点、抑制亲环素D的酶活力、降低线粒体的钙滞留能力。以上结果不仅为亲环素D结合药物的开发提供了新的化学结构类型，也为与亲环素D结合小分子的在抗癌等疾病作用机制提供了实验依据。. 本项目所建方法实现了抗癌中药这一复杂体系中以线粒体为靶点活性物质的快速、有效筛选，从线粒体整体到线粒体蛋白，逐步揭示了中药作用于线粒体的主要物质基础及其作用靶点亲环素D，为深入探讨中药药理机制及中药质量控制、中药新药发现提供了科学依据。