靶向蛋白质组学在中药药代-药效物质基础及其作用机理研究中的应用

The research on the active substances and pharmacological mechanism for traditional Chinese medicine (TCM) is the key point and bottleneck in the process of its modernization and internationalization. The study on active substances for TCM based on the regulation of protein expression and modifications will play an important role on the revealing the multi-component, multi-faceted and multi-target pharmacodynamic mechanism of TCM. .In this study, we will establish a robust methodology for target-proteomic study based on 2D-NanoLC-Q-TOF MS、LC-MS/MS、MALDI-TOF-TOF MS combined with Skyline software, SILAC technique, etc. Schisandra chinensis will be chosen as a model TCM, and the target-proteomic methodology will be applied into the pharmacological mechanism study of protective effect on liver injury produced by Schisandra chinensis. In this process, the target proteins related to liver injury will be identified by comparing the proteins expressing levels of normal liver cells and CCl4 / TTA induced necrotic damage liver cells (in vitro). Besides, the proteins expressing levels in liver tissues of normal and model (CCl4 / TTA induced damage liver) rats were compared to investigate the target-proteomics (in vivo). Then, the Schisandra component groups (I, II, III….) will be prepared by high preparative performance liquid chromatography, and will be used to screen the active substances of Schisandra chinensis by studying the regulating effect on the target proteins in vitro and in vivo. Based on the research above, the active substances and pharmacodynamic mechanism of Schisandra chinensis will be revealed thoroughly. Last but not least, pharmacokinetics of Schisandra chinensis will be studied to further validate the results of active-ingredient screening..In sum, we can provide a universal and feasible strategy and methodology for the PK & PD study for TCMs based on the research above.

中药药代-药效物质基础及其作用机理研究是中药现代化的瓶颈与关键。以靶蛋白的调控为指标进行中药多环节、多靶点的药理活性研究，对揭示中药药代-药效物质基础、诠释其作用机制具有重要意义。.本项目拟采用2D-NanoLC-Q-TOF MS、MALDI-TOF MS结合SILAC等技术建立蛋白组学研究方法；以五味子为模型药、以五味子肝损伤保护作用为切入点，通过比较正常肝细胞与CCl4/ TTA诱导坏死性损伤肝细胞、正常大鼠与肝损伤模型大鼠靶组织中蛋白组的变化，寻找肝损伤相关的靶蛋白；再通过制备五味子成分组群，以五味子各成分组对靶蛋白组的调控为指标，从in vitro和in vivo系统地考察五味子治疗肝损伤的作用靶点并筛选其药效物质组；最后通过药代动力学研究在揭示五味子体内药代物质组的同时进一步验证其药效物质组。.该项目为中药药代-药效物质基础及其作用机理研究研究提供普适、可行的研究思路与技术体系。

目前，中药有效物质基础不清、药理机制不明、评价中药药效药理模型缺乏。五味子为临床常见的保肝中药，对CCl4、 TTA等造成肝损害引起的SGPT升高均有降低作用，但其发挥肝保护的物质基础、作用靶标与机理仍不清晰。.本研究首先进行了五味子物质基础研究，分别采用10%、50%和90%乙醇制备的SLE考察五味子的最佳提取工艺，结果发现，50%乙醇为五味子最佳提取溶剂；再采用制备液相分离制备五味子低、中、高极性成分组，结果发现，中等极性的五味子木脂素的药理活性强于高极性和低极性的木脂素。 此外，五味子醇乙（Sol B）和酯甲（Stn A）被发现具有明显的肝损伤保护作用，且Stn A的肝保护活性高于Sol B。为了寻找五味子的作用靶标，本研究分别建立了基于NanoLC-MS-Obitrap和MicroLC-QTOF-MS的非靶向Label-Free蛋白质组学研究技术，在小鼠肝脏中鉴定到的1405个蛋白。正常组小鼠和APAP诱导的肝损伤组小鼠肝脏中蛋白组存在显著的差异，APAP诱导的肝损伤小鼠经五味子给药后，肝脏中蛋白组趋近正常组小鼠。正常组、APAP造模组和50%SLE给药组小鼠肝脏中差异蛋白富集到了57个KEGG数据库中的信号通路，其中13条通路有明显的影响。与APAP诱导的肝损伤大鼠肝脏中的蛋白表达量相比，五味子给药后，肝损伤大鼠肝脏中脂肪酸代谢、脂质代谢、蛋白水解、血小板聚集、谷胱甘肽代谢、含硫化合物代谢相关蛋白的下调得到了显著逆转。通过蛋白组学和代谢组学研究发现，五味子对多种脂质均具有显著的调节作用。APAP造模情况下，小鼠肝脏中甘油二酯与甘油三酯的变化最为显著。Lipin1，DGAT2在APAP肝损伤下表达显著上调，可能导致了DAG和TAG在细胞中的蓄积，五味子对Lipin1，DGAT2的具有显著的逆转作用。.因此，本研究通过利用多组学技术（蛋白组，脂质组，代谢组）,从不同角度阐明五味子发挥肝损伤保护作用的 PK-PD物质基础与作用靶标研究的同时，建立一整套普适的中药作用靶标与机制研究技术与思路。.