基于谱效关系和代谢组学研究斑蝥肝毒性的毒效物质组成及致毒机理

Mylabris, a strong toxic traditional Chinese medicine (TCM), is widely been used in the therapy of hepatocarcinoma and other tumor. At the same, mylabris has an obvious curative effect. However, the hepatotoxicity is easily induced by mylabris because of its narrow therapeutic window and hepatic affinity. The toxicological subtance basis and mechanism have not been well elucidated till now. The GC-MS and.HPLC finger printings were established by our recent studies, which indicated that there were great differences on the ingredient and content of Mylabris. Under the previous state and experiment results of Mylabris, we propose that we could study on the hepatotoxic subtance and mechanism of Mylabris based on multi-dimensional spectrum-effect relationship and metabonomics. Therefore the present study is designed to investigate the differences of chemical groups by the establishment of GC-MS, HPLC and IC finger printings of different polar parts of Mylabris; And then, the differences of multi-dimensional toxicity indexes are determined using normal rat model; These finger printings and toxicity indexes are analyzed by mathematic technique to explore multi-dimensional spectrum-effect relationship and further identification of toxicological subtance basis; From this, we use LC-MS and samples of LO2 cells exposed to the toxicological subtance of Mylabri to study the changes of endogenous metabolites based on PCA and PLS-DA analysis, then the hepatotoxicity mechanism of Mylabri is further detected after finding the potential metabolic markers. The present project is aimed to explore the hepatotoxic component and mechanism and to give a reference for the safe and rational use of Mylabris.

大毒中药斑蝥临床广泛用于治疗肝癌等，药效显著。然而其治疗窗窄且有肝脏亲和性，极易引起肝毒性。迄今斑蝥的肝毒效物质及致毒机理尚未明了。本项目组前期通过GC-MS,HPLC指纹图谱，发现不同批次斑蝥成分和含量差异较大;可能是引起毒性的主要原因之一。据此本研究拟通过谱效关系和代谢组学探索斑蝥肝毒效物质组成及致毒机理。首先建立斑蝥不同极性组分的HPLC及离子色谱指纹图谱，研究不同批次的多源化学成分差异；同时采用正常大鼠模型观察不同组分的毒性差异；继而通过数学方法整合指纹图谱和毒性指标，探索肝毒性的多维谱效关系，辨识毒效组分物质组成和比例；最后，采用基于LC-MS技术的代谢组学方法，观察毒效物质对大鼠血浆及正常肝LO2细胞内代谢物组的影响，寻找潜在毒性生物标志物，深入研究斑蝥肝毒性的致毒机理。本项目旨在探索斑蝥的肝毒效物质及机理，为其临床安全使用提供依据。

大毒中药斑蝥临床广泛用于治疗肝癌等，药效显著。然而其治疗窗窄且有肝脏亲和性，极易引起肝毒性。迄今斑蝥的肝毒效物质及致毒机理尚未明了。本项目采用ICP-MS检测了不同产地斑蝥中的20种微量元素，进一步聚类分析发现不同地区不同种类的斑蝥中微量元素存在差异；GC-MS检测分析也发现南方大斑蝥与黄黑小斑蝥之间的挥发性成分存在一定差异。同时建立了HPLC指纹图谱，匹配了22个峰，并测定了了尿嘧啶、尿嘧啶核苷和次黄嘌呤的含量，总含量在1.83-2.44%之间。将斑蝥的水层组分（SC）、氯仿层组分、斑蝥素（CTD）干预大鼠，结果发现SC和斑蝥素组分CTD的肝脏系数增加，ALT、AST、ALP、T-Bil、LDH上升，氯仿层组分未见明显肝毒性。代谢组学发现基于发现SC致大鼠肝毒性早期生物标志物有LysoPC (22:6(4Z,7Z,10Z,13Z,16Z,19Z))等5个，CTD致大鼠肝毒性早期生物标志物有Arachidonic acid及PE(14:1(9Z)/16:1(9Z))共2个。此外还发现SC致大鼠肝毒性晚期生物标志物有Sulfatide等6个，涉及甘油磷脂代谢等9条代谢通路；CTD致大鼠肝毒性晚期生物标志物有Sulfatide及Cortexolone等4个，涉及甘油磷脂代谢等6条代谢通路。对肝脏组织代谢物检测，共鉴定识别出Phytosphingosine及Sphinganine等17个生物标志物，主要涉及鞘脂代谢、类固醇激素生物合成等10条代谢通路。CTD干预后鉴定出LysoPC(22:1(13Z))等15个差异代谢物，主要涉及甘油磷脂代谢、类固醇激素生物合成等10条代谢通路。进一步基于LO2细胞的脂质组学分析发现，CTD高剂量组发现差异代谢物33个，且上述代谢水平被显著上调；低剂量组发现差异代谢物6个，且代谢水平显著上调。此外，本项目延伸探索研究发现CTD和SC可升高GRP78、CHOP和ATF4表达；CTD和SC各剂量组大鼠肝脏Bax/Bcl-2、Caspase-3显著上升；SC各剂量组大鼠肝脏Bax/Bcl-2、Caspase-3显著上升；CTDM和SCM组大鼠肝脏Caspase-8显著上升；CTD可升高大鼠肝脏LC3Ⅱ/LC3Ⅰ、Beclin-1。本项目旨在探索斑蝥的肝毒效物质及机理，为其临床安全使用提供依据。