水牛角对发热生物标记物的干预及其效应物质基础研究

Water buffalo horn (Bubali Cornu, WBH) tastes bitter and has cold properties, can remove toxic-heat, reduce heat in the blood, and arrest convulsions. WBH was documented firstly in China pharmacopeia (1977 edition), which was used as substitute for rhinoceros horn (Rhinoceri Asiatici Cornu and Rhinoceri Africani Cornu) in clinical applications and produced satisfactory therapeutic efficacy. Despite its long history of use, the mechanism of action and active components of WBH are still unclear. For this reason and based on Chinese medical principle and our previous study, methods and ideas of multi-indicator bioactive evaluation study were introduced to investigate the mechanism of action and active components of WBH: (1) Reproducible fever animal model was used to evaluate the antipyretic activity of WBH on regulating key fever-related cytokine and transmitter; (2) Based on the metabolomic strategy, fever-related biomarkers, metabolism related network and metabolism pathway were identified, and used to evaluate the regulation activity of WBH on fever metabolism pathway; (3) Ultrafiltration, MS/MS, De novo sequencing were used to analyze and identify the components of WBH, further association analysis was used to confirm the antipyretic active components of WBH. Based on these investigations, antipyretic efficacy evaluation method and bioactive components research pattern were established, the comprehensive action on fever model of WBH was confirmed. The antipyretic activity mechanism and bioactive components of WBH were revealed, which might provide proper approach and evidence on further horn-derived TCM investigations.

水牛角性苦，寒，具清热凉血，解毒定惊之功，77版《中国药典》始收载水牛角浓缩粉作为犀、广角类效品用于临床治疗发热，且疗效确切。然而多年来水牛角解热作用机制及效应物质基础仍不明确，为此本课题组在前期研究基础上，基于多指标生物效应评价的研究思路，围绕水牛角中医功效特点，深入开展水牛角解热效应物质基础研究：（1）建立客观的发热病理学模型，评价水牛角对发热指标性成分的影响；（2）基于代谢组学研究思路与方法，寻找发热生物标记物组，明确发热代谢途径，综合评价水牛角对代谢途径的干预作用；（3）以分子截留、串联质谱、氨基酸序列分析等现代分离分析技术研究水牛角物质基础，以物质基础与效应关联分析探讨解热效应物质基础。通过本课题研究，建立水牛角解热效应评价方法及效应物质基础研究模式，明确水牛角对发热机体的综合干预情况，系统揭示客观水牛角解热效应的科学内涵，并为角类动物药的研究提供思路与依据。

水牛角性苦，寒，具清热凉血，解毒定惊之功，临床用于治疗发热，疗效确切。然而多年来水牛角解热作用机制及效应物质基础仍不明确，为此本课题组在前期研究基础上，基于多指标生物效应评价的研究思路，围绕水牛角中医功效特点，深入开展水牛角解热效应物质基础研究：.1. 建立多种生物效应指标综合评价水牛角解热效应的方法.水牛角具有良好的解热功效，能降低发热大鼠体温，其解热效应可能的作用方式有：降低外周炎症因子TNF-α、IL-1β的含量；同时降低外周与中枢的PGE2含量；对5-HT、GABA、NA及cAMP等中枢神经递质具有一定的干预作用。.2. 水牛角对发热动物体内生物标记物干预研究.建立发热大鼠代谢组学研究方法，共发现41个与发热相关的生物标记物（p < 0.05，p < 0.01），评价水牛角对发热大鼠体内生物标记物的干预，水牛角能够干预其中18个生物标记物（p < 0.05，p < 0.01），水牛角的解热作用主要通过干预arachidonic acid代谢通路与氧化应激相关，其中关键的生物标记物有PGs类物质、LTs类物质及半胱氨酸等。.3. 水牛角物质基础研究.建立基于shotgun技术的混合蛋白鉴定方法，建立基于UHPLC–Q-TOF MS快速鉴定混合多肽的方法，通过这些方法从水牛角中鉴定了264个蛋白质，从水牛角水煎液中鉴定了877个多肽，完成水牛角中14个核苷类成分的定量研究。.4. 水牛角解热效应物质基础研究.结合代谢组学与多肽组学的思路，研究水牛角的活性部位物质基础，确定水牛角解热活性部位的多肽组成及其形成过程，活性多肽主要源于蛋白质的末端肽段。水牛角两个活性部位的解热效应与可能与调节Glutathione代谢、Fatty acid代谢、Glycerophospholipid代谢、Arachidonic acid代谢相关，但F1偏向于调节prostaglandin A2的水平，而F2偏向于调节Allocholic acid的水平。.5. 珍稀角类动物药替代资源的寻找与评价研究.基于角类药材的蛋白质组轮廓比较研究，寻找与评价犀角、羚羊角等珍稀药材的替代资源，从蛋白质分子功能、细胞成分等方面比较7种角类动物药的相关性，结果表明，水牛角、牦牛角与犀角蛋白质组轮廓接近，但犀角的可溶性蛋白质含量较高；山羊角与羚羊角蛋白质组轮廓接近。