基于“差异蛋白质组-生物效应关联”探讨动物药水蛭高温炮制的科学合理性

Leech is a classic breaking stagnant and eliminating blood stasis animal medicine, the processing methods of leech in the current authoritative law are all require to be treated in high temperature system, the quality control of those procedure just base on some experiential feeling such as the flexible state or departure from the pot depending on heating degrees, it is difficult to link those experiential methods with modern technology of qualification, so as to make the whole processing lack of effective evaluation tool, the scientific rationality of high temperature processing still needs to be proved. As biology assay, molecular simulation and proteomics techniques are superior in the quality control of complex protein system, and according to former research, this project tries to construct a new method to evaluate the feasibility of the high temperature processing system by the linkage of differential proteomics with biological effect. Based on "the systematically processing ", our group wants to focus on the following research points: (1) To establish differential proteomics of the main procedure of high temperature processing system; (2) To study on virtual screening of the individual molecules in protein group by molecular simulation; (3) In vivo biological potency detection; (4) Research on the spectrum-effect relationship between the dynamic change of the active ingredients during processing and biological effect of leech. This project provides a new pattern which try to solve the hard problem in the processing of TCM. It is a best reference for medicinal ingredients from animals and for medicine that contains active peptides and that needs to by heating during processing procedure.

水蛭是经典的破血逐瘀类动物药，目前权威法典收载该药材的炮制方法均需高温处理，炮制过程仅靠“灵活状态”、“看火候”出锅等人为的经验进行控制，缺乏有效的工艺过程评价手段，高温炮制的科学合理性有待论证。基于动物药物质内涵上的特殊性，结合蛋白质组学、生物检定、分子模拟等技术在复杂蛋白质、多肽群混合体系质量控制中的独特优势，课题以“差异蛋白质组-生物效应”联动方法构建动物药水蛭高温炮制工艺可行与否的评估手段。在“系统炮制”的基础上。通过（1）水蛭高温炮制过程各关键工艺环节差异蛋白组的构建；（2）蛋白质群/组中个体分子活性的虚拟筛选；（3）在体生物效价检测；（4） 炮制工艺过程活性成分动态变化与生物效应谱之间的“谱-效”关系研究，诠释该工艺的合理性，构建评价动物药高温炮制工艺可行性的新方法，为中药炮制的薄弱环节和工艺难题提供新的思路，同时也为以蛋白质、多肽为主要成分的动物药的合理炮制提供理论依据。

动物药高温炮制的合理性，一直是中医药学者反复探讨的热点。本项目以经典的破血逐瘀类动物药水蛭为研究对象，以“差异蛋白质组-生物效应关联”探讨水蛭高温炮制的科学合理性。研究过程中，针对水蛭炮制过程投药、炒制、“看火候”出锅等诸多环节，构建基于凝血级联通路和活血化瘀功效的水蛭生物活性评价方法；以SDS-PAGE、2-DE、差异蛋白质组学Label Free技术整体表征水蛭高温炮制前后的大分子体系的差异性；构建蛋白电泳指纹图谱，以 “谱效关系”表征炮制过程中活性成分动态变化与有效性之间的相关性；采用网络药理学方法推断了水蛭小分子成分活血化瘀的作用机制；基于大鼠急性血瘀模型，以高通量转录组测序（RNA-seq）技术，分析水蛭口服给药后差异表达基因，并采用GO和KEGG通路富集对差异表达的基因进行生物信息学分析，同时采用代谢组学，基于LC-Q/TOF-MS分析方法，筛选水蛭组与血瘀模型组间的差异代谢物，确定水蛭组干预大鼠血瘀的靶点代谢物，联合分析明确血小板为其重要靶细胞。以抗血小板聚集活性为评价指标，建立关联功效和机制的生物评价新方法；基于凝血酶亲和色谱-液质联用技术筛选水蛭中大分子的抗凝血活性成分，经体外活性测定验证发现，多肽 SYELPDGQVIIGNER具有良好的抗凝血酶活性，为首次从水蛭中发现新活性肽；采用分子对接技术预测该多肽与凝血酶的结合方式和亲和力，并对其在炮制过程中的动态变化进行研究。基于动物药物质内涵上的特殊性，项目研究结果表明：动物药高温炮制工艺存在大/小分子两个体系的不同变化，其中大分子体系存在肽键断裂释放活性肽段等物理化学变化，产生新的物质群。其科学意义在于：利用生物评价和多组学联用技术阐释了传统加工工艺的科学内涵，为以蛋白质和活性肽为主要成分的动物药的合理炮制提供理论依据与研究范例。