基于体液药理学和成分敲出-敲入策略的柴胡皂苷类成分治疗老年痴呆药效物质发现研究

Chronic neuroinflammation is recognized as the major cause of Alzheimer's disease(AD). Radix Bupleuri exhibits great development potential in AD treatment due to its remarkable anti-inflammatory effect and anti-AD clinical use of Radix Bupleuri prescription. In preliminary study, we found total saikosaponins in Radix Bupleuri could significantly ameliorate learning and memory impairment through inhibiting inflammatory factors release and microglial activation in AD mice brain. Thus we demonstrated that total saikosaponins had remarkable anti-AD effect. In order to investigate the anti-AD effective substances in total saikosaponins, this project is designed based on body fluid pharmacology methods and substance knock-out/knock-in strategy. Firstly, liquid chromatography-mass spectrometry will be used to establish fingerprint of dynamic content in the blood and cerebrospinal fluid after total saikosaponins administration. Secondly, anti-AD activity of drug containing plasma and cerebrospinal fluid will be tested in N9 microglial cells. Then substance knock-out/knock-in strategy will be performanced according to the correlation of substances and activities using preparative HPLC to identify anti-AD effective substances. Finally, the mechanism of anti-AD effect of effective substances will be discussed using microglial cell model. Based on the definite anti-AD efficacy of total saikosaponins, this project innovatively presents a method combining body fluid pharmacology and substance knock-out/knock-in strategy, which provides a new way for the effective substances study for traditional Chinese medicine, especially for those used in brain disease treatment. Meanwhile this project could provide scientific basis for the development of saikosaponin in clinical AD treatment.

慢性神经炎症被公认为老年痴呆（AD）的主要致病原因，柴胡因其卓越的抗炎功效加之柴胡方剂临床治疗AD的良好疗效而极具开发潜力。前期研究中我们发现柴胡总皂苷可显著抑制脑内小胶质细胞的活化及炎症因子的释放，有效缓解神经炎症，而对AD小鼠学习记忆损伤起到良好治疗作用。在此基础上，本课题使用液相-质谱联用手段对柴胡总皂苷给药后入血和入脑脊液成分进行分析；采用体液药理学方法，利用小胶质细胞活化模型对柴胡总皂苷含药血浆和含药脑脊液抗AD活性进行测试；同时结合使用制备液相分离的“成分敲出-敲入”策略来寻找柴胡总皂苷入血和入脑脊液成分中的抗AD药效物质；最后在细胞模型中探讨柴胡总皂苷抗AD药效物质的作用机制。本课题创新性的将体液药理学方法与“成分敲出-敲入”策略相结合，可为中药尤其是治疗脑部疾病药物的药效物质研究提供新思路，为将柴胡皂苷开发成为治疗老年痴呆的新型药物提供科学依据。

慢性神经炎症被公认为老年痴呆（AD）的主要致病原因，柴胡因其卓越的抗炎功效加之柴胡方剂临床治疗AD的良好疗效而极具开发潜力。前期研究中我们发现柴胡总皂苷可显著抑制脑内小胶质细胞的活化及炎症因子的释放，有效缓解神经炎症，从而对AD小鼠学习记忆损伤起到良好治疗作用。但柴胡皂苷体内代谢过程和抗AD作用之间的关系尚不明确，其作用机制也有待进一步探索。. 本项目利用液相-质谱联用技术建立了柴胡总皂苷成分图谱、兔空白血浆和空白脑脊液成分图谱及给药后血浆和脑脊液成分图谱。通过图谱比对确定了柴胡总皂苷入血有7个原型成分和15个代谢产物，入脑脊液有4个原型成分和5个代谢产物。利用质谱碎片信息分析、标准品对照、数据库和文献检索等方法对上述入血和入脑脊液成分进行了结构鉴定。选取含药血浆中含量较高的9个成分作为目标，运用“成分敲出-敲入”的方法，结合细胞药效学实验，综合分析确定了柴胡苷元D、E、F、G为柴胡总皂苷入血的主要药效成分。利用LPS激活的小胶质细胞模型进一步探讨了柴胡总皂苷药效物质治疗AD的相关作用机制，研究结果表明柴胡总皂苷药效物质能够明显抑制LPS诱导的炎症因子释放和NADPH氧化酶gp91phox、p47phox等亚基蛋白表达。AD转基因动物实验结果显示，柴胡总皂苷药效物质能明显缓解AD小鼠认知功能损伤，其作用机制与抑制胶质细胞活化、降低炎症因子水平从而保护脑内神经突触有关。. 本项目在明确柴胡总皂苷抗AD药效的基础上，创新性的将体液药理学与“成分敲出-敲入”策略相结合，初步探明了柴胡总皂苷入血成分中抗AD药效物质，可为深入了解柴胡皂苷体内过程和抗AD作用之间的关系奠定基础，为将柴胡皂苷开发成为治疗AD的新型药物提供科学依据，对中药研究现代化和柴胡的进一步开发利用具有重要意义。