基于DARTS技术的柴胡总皂苷治疗老年痴呆的CX3CR1相关作用机制研究

On the basis of the significant correlation between traditional Chinese medicine theory"injury of brain collaterals by toxins" and western medical theory "chronic neuroinflammation" in the pathogenesis of Alzheimer's disease(AD), our previous studies revealed that clearing heat and detoxification Chinese medicine Radix Bupleur had significant protective effect on learning and memory impairment in AD mice. Total saikosaponins, which are the main anti-inflammation ingredients in Radix Bupleur, exhibited extremely remarkable anti-AD effects stronger than first line AD treatment medicine donepezil. Further study suggested that total saikosaponins exerted cognitive protection effect through neuroinflammation inhibition by up-regulating protein expression of CX3CR1, one of the key neuroinflammation regulating receptors. Therefore we presume that anti-AD effect of total saikosaponins is possibly mediated by CX3CR1 and related neuroinflammation. In this study, traditional AD mice model and microglial activation model will be employed; a novel and high efficient drug targets screening technology, drug affinity responsive target stability (DARTS), combined with bioinformatics analysis will be performanced to confirm the role of CX3CR1 in the anti-AD effect of total saikosaponins and analyze CX3CR1 signaling pathway related proteins in the direct targets of total saikosaponins. Then high content analysis, Western blot and PCR will be used for clarifying molecular mechanisms of the pharmacological effects. This study will provide scientific basis for establishing CX3CR1 as a new target for AD treatment and developing anti-AD clinical applications for saikosaponins.

本课题以老年痴呆（AD）发病的“毒损脑络”中医学说和“慢性神经炎症”西医理论的相关性为指导，前期研究发现清热解毒抗炎中药柴胡对AD小鼠治疗效果良好，其抗炎活性成分柴胡总皂苷抗AD作用尤为突出，较临床一线AD治疗药物多奈哌齐效果更优。进一步研究表明，柴胡总皂苷通过上调AD小鼠脑内炎症关键调节受体CX3CR1的表达，抑制神经炎症，而起到保护学习记忆损伤的作用。由此提出假说：炎症关键调节受体CX3CR1对柴胡总皂苷保护AD小鼠学习记忆的功能起到介导作用。本研究拟采用经典的AD小鼠模型和小胶质细胞活化模型，运用新型高效药物寻靶技术—DARTS结合生物信息学分析，确证CX3CR1对柴胡总皂苷抗AD作用的介导，研究柴胡总皂苷作用靶点中与CX3CR1信号通路相关的目标蛋白，进一步明确相关分子作用机制。这将为确立CX3CR1作为AD治疗的新靶点提供科学依据，为将柴胡总皂苷开发为新型抗AD药物奠定理论基础。

本课题以老年痴呆（AD）发病的“毒损脑络”中医学说和“慢性神经炎症”西医理论的相关性为指导，前期研究发现清热解毒抗炎中药柴胡对AD小鼠治疗效果良好，其抗炎活性成分柴胡总皂苷抗AD作用尤为突出。进一步研究表明，柴胡总皂苷可上调AD小鼠脑内炎症关键调节受体CX3CR1的表达，抑制神经炎症。由此提出假说：炎症关键调节受体CX3CR1对柴胡总皂苷保护AD小鼠学习记忆的功能起到介导作用。. 本课题采用经典的AD神经炎症小鼠模型，采用Morris水迷宫、NOR实验等行为学方法，确证了柴胡总皂苷对老年痴呆小鼠的学习记忆损伤有明显治疗作用；通过免疫组化、ELISA等实验，证实了柴胡总皂苷可显著抑制AD小鼠脑内海马区小胶质细胞活化，明显降低AD小鼠脑内多种炎症因子的释放。离体细胞实验结果进一步确证了柴胡总皂苷能够显著抑制LPS诱导的小胶质细胞活化，以及TNF-α、IL-1β等炎症因子的释放，从而发挥抑制神经炎症的作用。在机制研究中我们发现：柴胡总皂苷能够明显抑制LPS和FKN（CX3CR1的特异性配体）诱导的小胶质细胞炎症因子释放，且柴胡总皂苷能够显著抑制LPS诱导的小胶质细胞CX3CR1蛋白表达下调，同时，柴胡总皂苷对老年痴呆模型小鼠脑内海马区CX3CR1的表达显著上调。蛋白芯片和DARTS实验结果表明，柴胡皂苷并不直接结合CX3CR1，而与转录因子SOD2有高度亲和力，并且在LPS诱导的AD模型小鼠脑内SOD2蛋白的表达显著下调，柴胡总皂苷可显著抑制SOD2蛋白表达的下调。通过检索文献结合生物信息学分析，我们发现SOD2和CX3CR1之间存在密切联系，这提示我们柴胡总皂苷保护AD小鼠认知功能障碍的作用可能与激活SOD2的转录活性进而提高CX3CR1的表达有关。. 本课题从多方面诠释了中医的“毒损脑络”学说和“慢性神经炎症”现代医学理论之间的相关性,证实了柴胡总皂苷治疗老年痴呆的作用通过抑制“毒损脑络”相关的神经炎症来实现，并确证了小胶质细胞炎症关键调节受体CX3CR1对柴胡总皂苷作用的介导。这将为开发柴胡总皂苷治疗老年痴呆的临床新用途奠定理论基础、为确立CX3CR1作为老年痴呆治疗的新靶点提供科学证据。