基于中药成分敲除和糖蛋白组学的姜黄抗前列腺癌活性物质组研究

By integration of the new theory and technology of multidisciplinary, and coupled with the imitation of the concept of gene knockout, this project intends to develop a platform based on chemical marker knockout and quantitative glycoproteomics for the study of the biological activity components of Curcuma longa against PC3 prostate cancer cell line. The potential biological activity components were firstly screened by PC3 cell fishing followed by chemical marker knockout to obtain the sample sets missing one or more components. The sample sets were then evaluated of the bioactivity against the cells to obtain the contribution of each component to total bioactivity as well as their interaction. Meanwhile, the glycoproteomes of cell lines treated by the sample sets were analyzed to figure out the differentially expressed glycoproteins before and after treating by the sample sets. The sample sets that missing different components may influence different glycoproteins which mean different components in Curcuma longa may involve in various protein pathways. By using this platform, the biological activity components of Curcuma longa and their mechanism of action against PC3 cell could be illustrated on glycoprotein level. Therefore, this project will provide the new idea and technology for the study of multi-components and multi-targets character of traditional Chinese medicines.

本项目集成多学科的新理论和新技术，借鉴基因敲除概念，建立中药成分敲除融合定量糖蛋白组学的研究平台，以人前列腺癌PC3细胞及姜黄为研究对象，通过靶细胞垂钓技术筛选出姜黄提取物中与PC3细胞结合的潜在活性成分群，之后通过成分敲除技术获得PC3细胞结合成分群以及缺失某个成分的样品集，经细胞活性评价后获得各个成分的活性贡献度及之间的相互作用（协同/拮抗）；同时开展细胞糖蛋白组学研究，观察成分敲除前后样品对糖蛋白组组成和含量的变化，阐释中药活性成分影响的生物标志物，不同成分可能影响不同的生物标志物，即不同的蛋白通路，从而阐明姜黄抗PC3 肿瘤细胞的活性物质组和作用机理，从而为中药多成分、多靶点整合作用研究提供新的研究思路和技术支撑。

在国家自然科学基金的资助下，本项目以姜黄为研究对象，借鉴基因敲除概念，建立了中药成分敲除融合定量糖蛋白组学的研究平台，研究姜黄抗人前列腺癌PC3细胞的活性物质组，取得了如下重要结果：①采用PC3靶细胞垂钓方法从姜黄提取物中筛选得到3个PC3细胞结合成分，经鉴定为姜黄素（CUR）、去甲氧基姜黄素（DMC）和双去甲氧基姜黄素（BDMC）；这3个成分均具有很强的PC3细胞抑制活性，IC50值分别为13.59、12.45、14.32 μg/mL，3个活性成分是姜黄提取物抗PC3细胞的主要活性物质组，其活性贡献度为64.25%；②协同指数法分析发现，按照姜黄提取物中的天然比例，CUR与DMC或BDMC具有叠加作用，而DMC与BDMC具有一定的协同作用；但3个成分比例为1:1:1时呈现出很强的协同作用，且BDMC和DMC是协同作用的必要成分；③糖蛋白组学研究发现3个姜黄素类成分能使31条糖肽表达下调，经功能注释和富集分析发现这些差异糖肽主要涉及内质网蛋白加工、ECM-受体相互作用、粘附斑、PI3K-Akt等信号通路。蛋白组学研究显示这3个活性成分主要作用于PC3细胞的核糖体、糖酵解/糖异生、氨基酸合成等信号通路，且3个成分同时给药时下调（糖）蛋白的能力最强。本项目的研究揭示了姜黄抗前列腺癌的主要活性物质组及其协同作用机制，为中药多成分、多靶点整合作用研究提供新的研究思路和技术支撑。研究结果以论文形式发表，发表SCI论文4篇，培养了1名博士研究生和2名硕士研究生。