重金属铅对药用动物蚂蟥抗凝血活性影响的机制研究

Leech is the main animal origin of Traditional Chinese medicine leech. According to reports,it has a phenomenon that the amount of present heavy metals exceed the standard of leech, the research group has also confirmed in the previous study of heavy metals in medicinal animal leech in the environment with water pollution,and surprisingly found that heavy metals may also affect its anticoagulant activity, in which heavy metal lead to its greatest impact.However, there is little research on the effects of heavy metals on the anticoagulant activity of leeches, and themechanism ofthe effects is not reported either. In this study, the effects of heavy metal lead on the anticoagulant activity of leech were studied by manually simulating the leech samples treated by heavy metal lead pollution including on the main metabolic studies, determine the anticoagulant activity of leech related metabolic pathways and key metabolites;and on the proteomics research, identify leech anticoagulant activity modules and associated key proteins; and researchby means of transcriptomics, localization of leech anticoagulant activity related to the expression pathway and key genes.In this study, weplan to study the effect of heavy metal lead on the anticoagulant activity of leech by in vitro experiments and reveal the mechanism of heavy metal lead on the anticoagulant activity of leeches from different levels.The research has great practical significance for the development of leech quality evaluation criteria, the establishment of leech breeding environment technical standards, and the protection of the quality of leech herbs.As well as quality ecology, germplasm resources, origin suitabilityand resource survey.

蚂蟥是中药材水蛭的主要动物来源。据报道，水蛭药材存在重金属超标现象，本课题组前期研究中也证实在水污染环境下药用动物蚂蟥的重金属超标，并且惊奇地发现重金属还可能影响其抗凝血活性，其中重金属铅的影响最大。但目前关于重金属对水蛭药材抗凝血活性影响的研究甚少，而影响机制的研究更是未见报道。本课题通过人工模拟重金属铅污染环境下获得处理的蚂蟥样品，研究重金属铅对蚂蟥抗凝血活性的影响。并且借助代谢组学研究，确定蚂蟥抗凝血活性相关的代谢通路及关键代谢物；借助蛋白组学研究，识别蚂蟥抗凝血活性相关的功能模块和关键蛋白；借助转录组学研究，定位蚂蟥抗凝血活性相关的表达通路及关键基因。本课题拟通过体外实验研究重金属铅对蚂蟥抗凝血活性的影响，从不同层面系统深入地揭示重金属铅影响蚂蟥抗凝血活性的机制。该研究对制定水蛭药材品质评价标准、建立水蛭养殖环境技术标准以及保障水蛭药材的品质具有重大的现实意义。

本课题组结合代谢组学、蛋白组学、转录组学方法，多层次探讨金属铅影响蚂蟥抗凝血效果的机制，研究结果包括：在转录组学层面，共得到1,110个差异表达基因，包括694个上调基因和416个下调基因。差异表达基因GO富集分析显著富集到85个GO条目。KEGG富集分析显著富集到5条通路，其中花生四烯酸代谢和溶酶体是重要的途径；蛋白组学方面，筛选到差异表达蛋白152个,显著富集在囊泡介导转运和转运正调节、内吞小泡、顶端质膜和碳水化合物结合等条目上；KEGG分析显示差异表达蛋白参与的通路76条,集中体现在代谢途径、次生代谢产物的生物合成和上皮细胞的细菌侵袭等途径。此外还得到了2个具有Antistasin结构域的抗凝蛋白；在代谢组学层面，发现铅污染的蚂蟥中脂类、核苷酸、二肽等24种代谢物含量有显著的变化，而这些化合物主要参与了甘油磷脂代谢、鞘磷脂代谢、核苷酸代谢等代谢途径。本课题从转录组学、蛋白组学、代谢组学方面地探讨了铅元素被蚂蟥摄入后所产生的一系列影响，揭示铅影响蚂蟥抗凝血的机制，为蚂蟥的养殖提供了参考依据。