支链氨基酸及相关代谢物的动脉粥样硬化危险预测作用及机制研究

Atherosclerosis (AS) is a main pathological basis of cardiovascular and cerebral diseases; thus it is particularly crucial to discover and study new risk factors of AS. Our previous studies demonstrated that the elevated serum concentration of branched-chain amino acids (BCAAs) was independently associated with the increased risk of subclinical AS and coronary artery AS, and we suggested that BCAAs might be the potential risk factors of AS. Accumulation of BCAAs and abnormal changes of levels of BCAA related metabolites in circulation may result from BCAA catabolic defect. The defect may be the most important influencing factor when BCAAs are involved in AS. Therefore, monitoring BCAA and their related metabolites in the development of AS can not only help us further identify the association of BCAAs with AS and discover new BCAA related metabolite risk factors of AS, but also reveal the relevant mechanism of AS. Based on our early work, the current project aims to 1) develop a liquid chromatography tandem mass spectrometry method of simultaneously determining BCAAs and their related metabolites; 2) analyze the association of their baseline levels with the outcome of AS and evaluate the capability of risk prediction in our prospective cohort study; 3) observe their dynamic changes in AS progression and explore the effects of BCAA on AS development and relevant mechanisms by using the AS mice model. This project will offer us some scientific evidence to establish BCAA as a new risk factor of AS, and it will also provide new ideas and new targets for the prevention and treatment of AS.

动脉粥样硬化（AS）是心脑血管病的重要病理基础，寻找和研究新的AS危险因素具有重要意义。我们前期发现，血清支链氨基酸（BCAA）水平升高与亚临床AS和冠状AS风险增高独立相关，是潜在的AS新危险因素。BCAA代谢障碍引起体内BCAA蓄积及相关代谢物水平异常变化是其参与AS的主要因素，其代谢物可能也具有AS危险预测作用。故在AS进程中监测BCAA及相关代谢物，有助于发现AS新的BCAA代谢物类危险因素，确认BCAA与AS的关系，并揭示其作用机制。本项目拟在前期工作基础上建立液相色谱串联质谱同时测定BCAA及相关代谢物的准确方法；在我们已建立的前瞻性队列中分析其基线水平与AS终点事件发生的关系，评估危险预测能力；在AS小鼠模型中观察BCAA及相关代谢物在AS进程中的动态变化，探讨其在AS进程中的作用及机制。本项目有望为确立BCAA作为AS新危险因素提供科学依据，为AS防治提供新思路和新靶点。

国内外研究及我们前期研究提示，血清支链氨基酸（BCAA）及其相关代谢物水平异常可能是动脉粥样硬化（AS）新的危险因素，但目前缺乏简便可靠且适用临床应用的测定方法，BCAA在AS进展中的作用及机制也不清晰。本项目遵照课题申请中的内容及年度执行计划从以下三个方面展开工作：. 1）建立了基于LC/MS/MS靶向代谢组学的同时测定16种BCAA及相关代谢物的准确方法。质谱采用电喷雾离子化的多离子反应监测模式，用HILIC亲水相互作用色谱柱分离代谢物，16种代谢物均能在5min内得到有效分离。方法学评价结果显示，所有代谢物的线性相关系数均大于0.999，相对回收率在93.5-139.6%之间，平均为101.8%；总CV为2.10-10.19%。. 2）完成2009例行冠状动脉造影患者招募工作，建立了规范化的血液样本库和基线数据库，并对行冠状动脉造影术后人群开展了随访。用本项目建立的LC/MS/MS方法对基线人群的血清BCAA及相关代谢物进行测定，统计分析发现BCAA、Ala、C3、3-HIB、2-HIV和HIC水平与冠心病呈独立相关。. 3）在ApoE-/-小鼠高脂饲料诱导动脉粥样硬化模型实验中发现，膳食补充BCAA不显著增加主动脉斑块面积；但对血糖血脂异常有一定加重；对PI3K/Akt/mTOR信号通路、胆固醇合成、转运及清道夫受体关键蛋白无明显影响；对肠道菌群丰度无明显影响。提示膳食补充BCAA可能不直接影响高脂饲料诱导的动脉粥样硬化易感小鼠斑块进展，在AS发生发展中可能不是始动因素，其水平变化可能仅是疾病伴随状态。. 总之，本项目所建方法操作简单，样品用量少，分离时间短，精密、可靠，适合临床应用，有望作为临床AS危险预测的重要手段。BCAA及相关代谢物对AS事件的预测作用尚待三年随访完成后进一步的前瞻性队列研究来验证。.