白芍-甘草药对“酸甘化阴”治疗Alzheimer病神经炎症协同增效的物质基础和作用机理研究

The neuroinflammation is considered to play a critical role in the pathogenesis of Alzheimer’s disease (AD).Previous study showed that herb pairs ofRadix Paeoniae Alba and Glycyrrhizae Radix et Rhizoma(Baishao-Gancao) and its single herb can inhibit neuroinflammation and oxidation in AD. However, its underlying compatibility mechanism and material basis have not been clearly investigated. Based on theory of ‘ Pungent and Swee Flavors Transforming into Yang, Sour and Sweet Flavors Transforming into Yin’ and earlier research results: material Basis and Mechanism of Cassia twig and Liquorice in myocardium ischemia-reperfusion injury (MIRI), this project will study multi-target synergy mechanism of herb pairs of Radix Paeoniae Alba and Glycyrrhizae Radix et Rhizoma(Baishao-Gancao) on expression of p38 MAPK/NF-κB signaling pathway in AD neuroinflammation, from whole animals, tissues, cellular and molecular levels, to represent the theory of ‘Sour and Sweet Flavors Transforming into Yin’ . Secondly, the project will study the active ingredients of Radix Paeoniae Alba and Glycyrrhizae Radix et Rhizoma(Baishao-Gancao) to compare the differences in the effect and chemical composition from different compatibility proportions and extracting methods. And it’s evaluated though Herb Pair compatibily-Single herb-Ingredient(s) compatibility-Monomer compositions as the main line. Thirdly, based on metabolomics and network pharmacology technology, the project will study the relationship among effective ingredients-drug effluent-action mechanism.Finally, from a new perspective,we systematically study the material basis and action mechanism of herb pairs of PRadix Paeoniae Alba and Glycyrrhizae Radix et Rhizoma(Baishao-Gancao) in the improvent effect on neuroinflammation of AD with system biology. We hope to propose some innovative thoughts for the development of TCM treatment of difficulty treated disease such as Alzheimer’s disease and the theory compound compatibility of TCM.

神经炎症是阿尔茨海默病(AD)重要的病理基础。既往研究表明，“酸甘化阴”经典药对“白芍-甘草”能有效抑制AD神经炎症反应，但两者配伍“相使为用”治疗AD神经炎症的物质基础和作用机制尚不清楚。基于前期对“辛甘化阳”药对配伍的研究基础，本课题拟在整体动物-细胞-分子不同水平，从p38 MAPK/NF-κB信号通路/炎性因子表达评价药对白芍-甘草抗AD神经炎症的协同作用机制；围绕药对-单味药-成分（群）配伍-单体成分，比较白芍-甘草配伍在药效、化学成分上的差异，探讨药对白芍-甘草“酸甘化阴” 配伍的物质基础；结合 LC-MS 串联色谱分析技术和网络药理学方法，以系统生物学新视角，分析药对白芍-甘草治疗AD神经炎症的药效物质-药物效应-效应作用机制之间的关系；阐释白芍-甘草”酸甘化阴“治疗AD神经炎症的协同增效作用机理和物质基础 ，以期为中医药防治AD提供可靠的实验依据，指导临床的合理治疗。

神经炎症是AD重要的病理基础，药对“芍药-甘草”组成药味均能有效抑制AD神经炎症反应。但两者配伍，防治AD神经炎症的作用机制和物质基础尚不清楚。基于此，我们以酸甘化阴理论为指导，从动物、细胞、分子不同层次开展芍甘配伍防治AD神经炎症的药效物质-药物效应-作用机制研究。动物实验结果表明，不同配伍比例芍药甘草可改善AD模型大鼠认知功能；降低Aβ诱导的炎症因子IL-1β、IL-6、TNF-α表达；改善AD模型大鼠脑区病理变化。进一步的研究表明，以1:1配伍比例组方，即芍药甘草汤能显著降低 AD 模型大鼠 Aβ1-40和Aβ1-42的含量，并通过调控p38 MAPK/NF-κB信号通路，抑制AD神经炎症反应。细胞实验结果表明，不同配伍比例芍药甘草含药血清可抑制AD细胞模型炎症因子TNF-ɑ、IL-6、IL-1β、iNOS的表达；经过含药血清活化的小胶质细胞条件培养液可降低BACE1 mRNA的表达，抑制PC12细胞Aβ1-40/Aβ1-42的分泌。体内外实验均揭示了药对芍药-甘草配伍对于AD神经炎症的防治具有一定作用，其机制与干预p38MAPK/NF-kB信号通路表达、减少神经元损伤、干预小胶质细胞活化、抑制 Aβ 分泌及炎症因子表达等相关。基于此，本研究采用UPLC-QTOF/MS色谱-质谱联用技术，鉴定了“白芍-甘草”药对的31个主要化学成分及12个血中移行成分。结果表明两者配伍后，能提高31个主要化学成分的相对溶出率，并且白芍-甘草1:1 配伍后有27种成分的相对溶出率要高于2:1 组和1:2组，与药理研究结果相一致。网络药理学和分子生物学验证结果均证实MAPK14(P38α)是芍药甘草发挥防治AD的关键靶点。本研究从整体-细胞-分子水平诠释了白芍-甘草配伍“酸甘化阴”治疗AD神经炎症协同增效分子机制和物质基础，拓展了芍药甘草汤的临床使用范围，为临床应用“酸甘化阴”防治AD提供科学依据。