基于P-gp调控相关的多靶点脂筏色谱的构建、中药活性组分群的筛选及其作用机制研究

P-glycoprotein (P-gp) is enriched in lipid rafts. It is of great significance to selectively regulate P-gp in clinical application. Many Traditional Chinese Medicines (TCMs) had P-gp regulation activity. However, investigations on their active components have been stuck in the research mode of “separation first and activity analysis second”. So far, there have been considerable achievements using monotarget affinity chromatography to separate active ingredients from TCMs. Our previous research which employed lipid rafts as the stationary phase has found that there were multiple target proteins on the lipid raft-based stationary phase. Moreover, the extractions screened by this lipid raft-based stationary phase showed action mechanisms that were achieved by pathways mediated through the same proteins in the lipid rafts. Therefore, lipid raft chromatography is essentially a multitargets affinity system. On this basis, this project will construct a P-gp enriched novel multitarget lipid raft system. With the study on microenvironment of lipids and proteins on the lipid raft-based stationary phase as an entry point, this project aims to explore the influence of this microenvironment on chromatographic retention, and the affinity property of the multitargets lipid raft chromatography, employ this system to sift out active component groups associated with P-gp regulation from TCMs, and analyze the effects and interactions of the active component groups that have inhibitive effects on P-gp. This project will provide new strategies and insights for the development of multitargets affinity chromatography and its application in screening active component groups from TCMs as well as the mechanism research.

P-糖蛋白（P-gp）富含在脂筏区域，选择性调控P-gp的临床意义重大。许多中药具有P-gp 调控活性，但其有效组分的筛选研究尚停留在“先分离再行活性分析”的研究思路。亲和色谱法针对单一靶点筛选中药中的有效成分已取得一定成果。然而，我们在前期以脂筏为固定相的亲和色谱研究中，发现脂筏固定相上存在多个靶蛋白，且筛选出的中药提取物的作用机制正是通过这些靶蛋白介导的信号通路实现的。因此，我们推测脂筏色谱本质上是多靶点的亲和体系。在此基础上，本项目拟构建富含P-gp的新型多靶点脂筏色谱系统，以脂筏固定相上的脂质、蛋白微环境研究为切入点，探讨脂筏微环境对色谱保留行为的影响以及脂筏色谱多靶点的亲和特性。将构建的脂筏色谱用于以P-gp调控为目的的中药活性组分群筛选，讨论具有P-gp抑制作用的活性组分群间的功效关联和相互作用，为发展多靶点亲和色谱技术及其在中药活性组分群筛选和作用机制研究提供新的策略和思路。

项目以Caco-2细胞为主要模型，首次针对脂筏的总蛋白、脂筏特征蛋白、细胞特征蛋白和胆固醇等因素进行较为深入的分析研究。实验测定了不同方法提取的各层脂筏中的蛋白含量以进一步优化脂筏的抽提方法，优化后，不仅脂筏层数较多提取较充分，且总蛋白含量最高。同时发现，脂筏各层总蛋白含量与Flotillin-1和P-gp表达规律呈现一致性。这不仅为脂筏提取条件的优化提供了量化的补充说明，同样也为探讨脂筏中蛋白含量与疾病的关系提供了新的思路与理论支撑。项目还测定了脂筏中的胆固醇含量，为脂筏成分含量的研究及提供了数据。在此基础上，项目成功构建了富含P-糖蛋白、专属性、稳定性良好的多靶点脂筏色谱柱，比较不同脂筏固定相的蛋白等信号分子的构成与色谱上保留组分间的关系，讨论脂筏色谱微环境对筛选结果的影响。初步表明了脂筏吸附性能与脂筏成分中蛋白的含量有关，为脂筏色谱进行P-糖蛋白底物和抑制剂的筛选奠定了基础。实验以构建的脂筏色谱对人参、杨梅叶、大黄等中药的萃取部位进行筛选，着重围绕大黄活性成分进行研究，筛选出了潜在的含有P-gp的底物或抑制剂的大黄活性部位。P-gp功能的体外评价结果表明，脂筏柱筛选出的大黄浮选部位Ⅲ可明显增加细胞内Rho-123的蓄积量，具有抑制P-gp的功能，进一步验证了实验前期构建的P-gp脂筏色谱高通量筛选模型具有切实可行性。实验结果提示脂筏色谱固定相的微环境以及多个靶蛋白、活性酶等与中药活性成分色谱保留行为的具有关联性，建立的以P-gp调控为目的的脂筏色谱实现了多靶点的亲和筛选。