基于代谢组学探讨ApoE基因敲除小鼠动脉粥样硬化易损斑块形成的动态机制及解毒活血法的干预作用

AS vulnerable plaque rupture and thrombosis is the most important pathological basis of acute cardiovascular clinical events,and effective control of accurate identification of vulnerable plaque is very important.This project takes "Jiedu Huoxue Method – inhibition of the reaction of inflammation- anti-atherosclerosis - plaque stabilization-diminution of cardio cerebral vascular events" as hypothesis. The project is from the point of view of TCM holistic, uses ApoE gene knockout mice with long-term high fat feeding as the research object, establish the each group of the animal blood metabolite fingerprint at experimental began, plaque, vulnerable plaque formation by UPLC-MS/MS、MS/TOF technology, analyse and identify the closely related to biomarkers of AS plaques progress and Simiao YonganDecoction efficacy by using pattern recognition, search closely related signaling pathway with biomarkers with the help of metabolic pathway database KEGG, GenMAPP, BioRag, combine with different time points in each group animal blood lipids ,aorta and brachiocephalic artery pathological structure, plaque imaging characteristics and blood inflammatory factors as verification.By doing so, we aimed to provide detection means of overall, real-time diagnosis of AS plaque progression,and lay the foundation for the dynamic mechanism of the formation of AS plaque and the mechanism of "Simiao Yongan Decoction" to stabilize plaque.

AS易损斑块的破裂合并血栓形成是造成心血管急性临床事件的最重要病理基础，易损斑块的准确识别与有效防治至关重要。本课题以“解毒活血-抑制炎症反应-抗动脉粥样硬化-稳定斑块-减少心脑血管事件”假说的前期研究结果为基础，从中医学整体观出发，以高脂高胆固醇饮食长时间喂养ApoE(-/-)小鼠为研究对象，运用UPLC-MS/MS、MS/TOF技术，通过构建实验开始、斑块形成时、易损斑块形成时3个时间点各组动物血液的代谢物指纹图谱，采用模式识别分析确定与AS斑块进展和四妙勇安汤药效密切相关的“生物标志物”；借助代谢途径数据库KEGG、GenMAPP 、BioRag，找寻与生物标志物密切相关的信号通路；结合不同时间点各组动物血脂、病理结构、斑块的显微成像特点及血液炎症因子的验证，为AS斑块进展的诊断提供整体、实时的检测手段；为系统揭示AS易损斑块形成的动态机制及“四妙勇安汤”稳定斑作用机理奠定基础。

动脉粥样硬化是严重危害人类健康的常见病和多发病，其中AS易损斑块的破裂合并血栓形成是造成心血管急性临床事件的最重要病理基础。对其形成机制、无创准确诊断及探索稳定斑块的治疗方法对防治由其破裂导致的急性冠状动脉综合征（ACS）等心血管不良事件的发生意义重大。本课题以具有丰富研究基础的解毒活血法代表方“四妙勇安汤”为研究对象，采用高脂高胆固醇饮食喂养ApoE(-/-)小鼠13周和26周分别制备AS斑块形成不同阶段动物模型，于斑块形成时和易损斑块形成时给予不同剂量中药及阳性药干预12周。采用基于超高效液相色谱-四级杆-飞行时间质谱仪（UPLC-Q-TOF/MS）联用技术构建高脂饲养前、斑块形成时、易损斑块形成时模型组、对照组及不同阶段药物干预各组的血清代谢轮廓图，运用主成分分析（PCA）和正交偏最小二乘法-判别分析（OPLS-DA）的方法对不同阶段正常组和模型组获得的内源性代谢物分析，寻找并鉴定了AS斑块动态演变过程中潜在生物标记物，包括脂肪酸、甘油脂、固醇脂质、Prenol脂质、脂肪酰基类等成分，其中以脂肪酸与As的关系较为密切，其次为甘油脂。进一步对不同阶段给药各组获得的内源性性代谢物分析，寻找并鉴定了与“四妙勇安汤”稳定AS斑块相关的潜在生物标记物主要是脂肪酸和甘油脂类，促进脂质代谢，起到抑制As的作用。本研究方法体现了中药治疗AS“整体性”特点，研究结果有望对四妙勇安汤治疗AS整体作用机制的研究奠定实验基础，同时为进一步研究确认与AS形成相关生物标志提供有意义的参考。