基于蛋白组学和代谢组学的皂苷类中药注射剂类过敏成分及监测方法研究

Clinically, anaphylactoid reaction accounts for 75% of the acute allergic reactions of Chinese medicine injections (CMI) and exhibits serious harm to patients. In the course of fulfilled NSFC (National Natural Science Foundation of China) project, the mechanism ambiguity of anaphylactoid reactions of CMI was recognised, especially the anaphyalctoid reaction of saponins possessed the semi-antigen property. Research indicated that the anaphylactoid reaction of CMI composed of saponins always exhibited higher rate and its surveillance method of anaphylactoid reaction is of necessary to be further studied. As the allergy reaction is mainly involved with immunoglobulins and reactive compounds with smaller molecular weight, the techniques of proteomics and metabonomics are just suitable for studying biomarkers of the CMI anaphylactoid reactions. Taken Xue-Sai-Tong and Sheng-Mai injections as examples and by way of techniques of proteomics and metabonomics, the anaphylactoid components and specific biomarkers of CMI composed of either one herbal drug or numerous herbal drugs have been studied in this project, and thereof, their anaphylactoid mechanism will be further elucidated by comparison of anaphylactoid -omics of typical substances. Furthermore, the methods for anaphylactoid surveillance of CMI will be established on the basis of combination of determination of chemical constituents, in vitro and in vivo anaphylactoid tests of CMI. It is for the first time that the techniques of proteomics and metabonomics are used for the mechanism study of anaphylactoid reaction of CMI and method establishment of anaphylactoid surveillance of CMI. The research will lay the solid basis for promoting the quality of CMI, reducing or eliminating the anaphylactoid ADR (adverse drug reactions) of CMI and also provide an example for the method establishment of anaphylactoid surveillance of CMI.

类过敏占中药注射剂急性过敏反应的75%以上，危害严重。在已完成的基金课题中，发现中药注射剂的类过敏机制尚不清楚，尤其是皂苷类成分本身可作为半抗原，其注射剂在临床上类过敏的发生率较高，监测方法有待于进一步研究。过敏反应主要是通过免疫蛋白和小分子效应物质而发生的，因而蛋白组学和代谢组学技术较适合类过敏生物标志物的研究。课题以临床上类过敏发生较严重的血塞通和生脉注射液为例，基于组学技术对单方和复方中药注射剂类过敏成分及其类过敏生物特征标志物进行研究，结合典型物质类过敏组学研究，探讨其类过敏机制，建立基于类过敏成分的化学定量、体外及基于类过敏特征标志物体内评价模型的皂苷类注射剂类过敏监测方法。课题首次采用组学的方法，通过同时进行多指标检测阐明中药注射剂类过敏机制，进而建立其类过敏监测方法。课题为全面提高中药注射剂质量，有效减少或根除其类过敏反应奠定基础，为中药注射液类过敏监测方法研究提供范例。

急性过敏为中药注射剂主要的严重不良反应之一，主要包括I型过敏和类过敏反应，后者占75%以上。目前《中国药典》中只有关于I型过敏评价方法。本研究首先通过典型物质及中药注射剂对BN大鼠、豚鼠、SD大鼠及ICR小鼠的类过敏进行综合评价，发现BN大鼠较适合对中药注射剂的类过敏反应评价。通过蛋白组学和代谢组学对典型物质及中药注射剂的类过敏机制进行研究，表明中药注射剂的类过敏可分为发生过程及效应过程。发生过程主要包括补体途径（经典途径和旁路途径）、直接刺激途径、凝血途径、激肽-激肽释放酶途径和共同途径5条途径，Sc5b9（C4d和Bb），Gpx1，F13，Kng1和IgE等蛋白分别可以作为以上途径的相关标志物, 其中Gpx1，F13，Kng1和IgE为首次鉴定的作为类过敏潜在生物标志物。研究首次明确了共同途径及发现各类过敏途径的标志物，进一步丰富和完善了类过敏发生机制。研究首次采用代谢组学对中药注射剂类过敏机制进行系统研究，发现类过敏的效应过程主要包括组氨酸代谢、能量代谢、嘌呤代谢和花生四烯酸代谢等途径，同时发现各途径相应的生物标志物。通过体外实验对中药注射剂活性成分的致敏性进行筛选，首次发现血塞通等注射剂中残留蛋白和鞣质为类过敏主要致敏成分，进一步发现致敏强弱跟蛋白种类相关，以血塞通注射剂为例，建立中药注射剂微量蛋白测定方法。研究首次通过体内BN大鼠模型、体外RBL-2H3细胞模型和人血清补体模型以及TSK凝胶柱蛋白定量分析等方法建立中药注射剂类过敏监测方法。研究发表论文13篇，其中SCI收载论文6篇;特邀国内外会议报告3人次，组织会议1次，行业特邀培训做报告2人次；出版专著1部; 项目负责人被评为辽宁省特聘教授、沈阳市优秀研究生导师；获得辽宁省优秀博士论文提名奖1人次，辽宁中医药大学优秀博士论文一等奖1人次；研究团队被评为辽宁省创新团队。研究为中药注射剂类过敏监测方法建立奠定基础。