基于NMR代谢组学技术研究电针内关对大鼠心肌缺血再灌注损伤的预保护作用机制

Myocardial ischemia is one of clinically significant disease, myocardialischemia-reperfusion injury (MIRI) is an important pathological process of myocardialischemia, so reperfusion injury prevention is the key to the treatment of myocardial ischemia.A lot of literature and our previous studies confirmed that electroacupuncture(EA) on Neiguanpoint has the good control effect of MIRI, can regulate a variety of proteins and geneexpression the expression, etc, but the overall adjustment mechanisms of EA preventionof MIRI have not yet been revealed. Metabonomics is a systems biology researchmethods with the overall concept, and the characteristic is consistent with theoverallregulation of acupuncture. This thesis put forward the hypothesis: " electroacupuncturepreconditioning at Neiguan has protective effect on myocardium of MIRI rats, andchanged the metabolism of MIRI mode, and there are specific metabolic regulatory networks". To establish the MIRI rat model by coronary artery embolization,, and to observe theprotective effect of EA preconditioning at Neiguan on myocardium of MIRI rats by electronmicroscopy and molecular biology methods; at the same time, this project uses NMR-basedmetabonomic analysis to observe the effect of EA preconditioning at Neiguan onmetabolome of myocardialtissue、blood and urine in rat, screening of specific metabolites toexplore the metabolism regulation pathways and molecular mechanisms ofEA preconditioning at Neiguan relieves MIRI. This thesis provides theoretical andexperimental basis for the prevention of EA Neiguan on MIRI, which is important for thefurther development of acupuncture prevention of myocardial ischemia.

心肌缺血是临床重大疾病之一，心肌缺血再灌注损伤（MIRI）是心肌缺血重要病理环节，防治再灌注损伤是治疗心肌缺血的关键。本课题组前期研究证实电针内关对MIRI有很好的防治作用，可调节多种蛋白和基因表达等，然而电针防治MIRI的整体调节机制尚未揭示。代谢组学是具有整体观念的系统生物学研究方法，与针灸整体调节特点相契合。本课题提出假说：“电针内关穴预处理对MIRI大鼠心肌具有保护效应，并改变了MIRI代谢模式，存在特定的代谢调控网络”。拟采用冠状动脉栓塞法建立MIRI大鼠模型，应用电镜、分子生物学观察电针内关穴预处理对MIRI大鼠心肌的保护效应；同时，本项目采用基于核磁共振的代谢组学技术，观察电针内关预处理对大鼠心肌组织、血液、尿液代谢组的影响，探讨电针内关预处理减轻MIRI的代谢调控途径和分子机制，为电针内关防治MIRI提供理论基础和实验依据，这对于进一步拓展心肌缺血的针灸防治具有重要意义。

本研究是在前期研究基础上，建立大鼠心肌缺血再灌注损伤模型，采用电针预处理（电针内关/合谷穴），以缺血预适应心脏保护作用为阳性对照，监测心电图变化，运用HE染色、透射电镜、高效液相色谱法、核磁共振氢谱技术（1H NMR）、代谢组学等多种技术手段，检测心肌组织的细胞形态学的变化，腺苷酸（ATP、ADP、AMP）的含量变化；以及利用1H NMR进行心肌组织或血清中代谢物检测并利用小二乘分析（PLS-DA）、正交偏最小二乘分析（OPLS-DA）进行模式识别，寻找MIRI大鼠的特征性代谢物。研究发现：1.电针预处理（电针内关/合谷穴）7d能减轻MIRI大鼠心肌组织的病理损伤，其预防保护作用可能与升高其心肌组织中腺苷酸的含量有关。电针合谷穴比电针内关穴的预防保护效果更佳。2.电针内关预处理可以显著地改变MIRI大鼠的血清和心肌代谢模式，其可能通过调节糖代谢、丙酮酸代谢、氨基酸代谢、酮体代谢等多个途径完成对MIRI大鼠的保护；电针合谷未能改变MIRI大鼠的血清和心肌代谢模式，进一步证实内关穴在防治心脏疾病的穴位特异性。为针灸“治未病”理论机制在临床应用提供了实验依据。本项目已在国内外学术期刊发表论文11篇，培养硕士研究生7名。