基于UPLC-MS/MS联合MRM的靶向与非靶向代谢组学方法研究蛇伤中毒标志物

Poisonous snakebite is emergency and critical ill. The number of snakebites that occur each year in china may be more than 300,000. The accurate diagnosis of snakebite is currently very necessary and urgent work. There have been no research works on metabolic markers of snakebite were reported. This project intends to screening, verify and confirm the metabolic markers of snakebite by metabolomics.. After inject the Naja naja atra, Trimeresurus stejnegeri and Bungarus multicinctus venom into the Guangxi Bama pigs, respectively, the pig urine and plasma collected at a predetermined time point. Both targeted and non-targeted metabolomic methods, that is, UPLC-QTOF-MS/MS and MRM, are used to achieve complementary information of biological samples for the reliability of biomarkers screening, identify and verify. Quantitative variation of endogenous metabolites will be integrated by metabolomics approaches to acquire the biochemical information in dynamic information through the whole snakebite process. Then, confirms the biomarkers with MRM technology in clinical samples. Finally, performs MS, WB and QPCR to quantitative analysis of the core metabolites, regulatory proteins and genes associated with the markers in the metabolic pathways, and further investigate the reliability of the textual metabolic markers and the relationship between the snakebite and markers of metabolic pathways.. This work will solve the problem of the lack of reliable markers in the clinical diagnosis of snakebite, and provide reference for the accurate diagnosis, risk evaluation and toxicity mechanism of snakebite.

毒蛇咬伤（蛇伤）属于急重症，我国每年蛇伤患者达30多万人，而临床诊断缺乏准确可靠的标志物。当前国内外尚未见有蛇伤的代谢标志物的研究报道。本项目拟通过构建蛇伤的动物模型，采用代谢组学研究技术筛选、鉴定、验证和确认蛇伤的代谢标志物：首先，构建中华眼镜蛇、竹叶青蛇及银环蛇咬伤的广西巴马香猪模型；然后，采用UPLC-QTOF-MS/MS的非靶向代谢组学方法，分析蛇伤不同中毒时间点猪的尿液和血清中各类代谢物的动态变化，筛选出潜在代谢标志物；而后，通过以MRM技术为核心的靶向代谢组学方法靶向验证潜在标志物，并结合临床样本绝对定量确认标志物；最后，通过对标志物关联的代谢通路上的核心代谢物、调控蛋白和基因进行MS、WB和QPCR等定量分析，深入考证代谢标志物的可靠性，探讨蛇伤与标志物代谢通路的关系。以期解决蛇伤临床诊断缺乏可靠标志物的突出问题，为蛇伤的精准治疗、风险评估和毒性机制研究提供参考。

项目的背景：蛇伤属于急重症，我国每年蛇伤患者达30多万人，而临床诊断缺乏准确可靠的标志物。当前国内外尚未见有蛇伤的代谢标志物的研究报道。. 主要研究内容：本项目已构建中华眼镜蛇、竹叶青蛇及银环蛇咬伤的广西巴马香猪模型；并采用UPLC-QTOF-MS/MS的非靶向代谢组学方法，分析蛇伤猪的尿液和血清中各类代谢物的动态变化，筛选潜在代谢标志物；再以靶向代谢组学方法验证潜在标志物，并结合临床样本绝对定量确认标志物；最后对标志物关联的代谢通路上的核心代谢物、调控蛋白和基因进行分析，探讨蛇伤与标志物代谢通路的关系。.重要结果和关键数据：通过在巴马小型猪右腿皮下注射中华眼镜蛇、竹叶青蛇及银环蛇的蛇毒成功构建了上述3种蛇伤猪模型。上述3种蛇伤猪的最小致死量为0.08mg/kg、0.05mg/kg和0.04mg/kg。以上述猪模型对蛇伤猪的血清及尿液进行非靶向和靶向代谢组筛选、鉴定及验证代谢标志物，并结合临床初步确认志物。结果表明：肌苷、L-苯丙氨酸、D-脯氨酸3种代谢物有望成为中华眼镜蛇咬伤的代谢标志物。马尿酸、亚油酸、肌肽、脯氨酸、亮氨酸、谷氨酰胺6种代谢物有望成为银环蛇咬伤的代谢标志物。氢脂酰胺和5-羟基吲哚乙酸有望成为竹叶青蛇咬伤代谢标志物。.对银环蛇伤中毒小鼠补充前述获得的代谢标志物Gln，可以降低小鼠死亡率，减轻心肺组织损伤，Gln代谢异常得到缓解。Gln通过促进心肺组织中HSP70的表达，抑制肺组织中NF-κB信号通路的活化，抑制心肌组织中P53/PUMA信号通路的活化，抵御银环蛇咬伤中毒导致的心肺损伤。.科学意义：本项目的完成弥补了蛇伤研究领域没有中华眼镜蛇、竹叶青蛇及银环蛇咬伤大型实验动物模型的不足。并有望为临床诊断区分各种蛇伤提供代谢标志物，为蛇伤的精准治疗奠定基础。从Gln代谢出发，外援性补充Gln与银环蛇伤中毒救治联系在一起，研究结果将为临床银环蛇伤救治提供新的思路。