同位素示踪代谢流分析技术研究银杏叶提取物心肌缺血保护作用的代谢机制和物质基础

Myocardial energy metabolism therapy is a new direction for the development of ischemic heart disease drugs. Targeted metabolomic profiling of cardioprotective effect of Ginkgo biloba L. extract (GBE) on myocardial ischemia (MI) in rats was well studied in our previous research. It was found that citric acid, the intermediate of tricarboxylic acid cycle (TCA), with asparate, arginine, ornithine and citrulline, which can be metabolized into TCA, were associated with MI. GBE can significantly regulate their levels and play a cardioprotective effect. Due to the carbon source of TCA metabolites may have three pathways: glycolysis, fatty acid catabolism and amino acid catabolism. So which pathways were these key metabolites involved in the damage procession of MI, and how GBE played a cardioprotective effect? In order to solve this problem, isotopic tracing metabolic flux analysis technique will be used to quantitatively study the dynamic changes of energy metabolic network in cardiomyocytes. A quantitative analytical method of isotopic metabolites and a rapid data processing method of metabolic flux covering the cell energy metabolism network will be developed. Then cardiomyocytes under physiological and pathological conditions will be treated with GBE and its main monomers, and then cultured in the medium containing isotope tracer. After collecting the cells, all intermediate metabolites will be detected by HPLC-HRMS. After analyzing the changes of metabolites isotope incorporation ratio and concentration with time, the source, metabolic rate and direction of key metabolites associated with cardiomyocyte injury and GBE protection can be obtained. On the basis of this, key enzymes that affected the dynamic metabolism changes and possible synergies of GBE monomers which can regulate them will be found. In addition, cell energy metabolism real-time monitoring technology will be used to verify the pathway and subcellular position of metabolic changes. This research can not only explore metabolic mechanism, material basis and potential targets of protective effect of GBE on MI, but also provide novel idea and strategy for the follow-up study of herbal medicine metabolomics and development of anti-myocardial ischemic natural medicines.

心肌能量代谢治疗是心肌缺血（MI）药物研发的新方向。我们前期开展了银杏叶提取物（GBE）对MI大鼠心脏保护作用的代谢组学研究，发现直接和间接参与TCA循环的柠檬酸和四种氨基酸与MI相关，而GBE能显著调节这些代谢物。由于TCA循环有多条碳源，究竟它们是通过哪条途径如何参与MI损伤，GBE如何发挥保护作用？针对该瓶颈问题，本课题拟采用同位素示踪代谢流分析技术，建立覆盖能量代谢网络中同位素代谢物定量分析和代谢流数据快速解析方法，研究MI心肌细胞给予GBE及单体保护并加入示踪物培养后代谢物同位素掺入比例和含量的动态变化，获得相关代谢物来源、代谢速率和方向改变，发现相关调控酶和调节酶的GBE单体及可能的协同作用。同时采用细胞能量代谢实时监测技术验证代谢变化途径和亚细胞场所，深入阐述GBE发挥MI保护作用的代谢机制、物质基础和作用靶点，为中草药代谢组学的拓展和心肌缺血天然药物的研究提供新策略。

心肌缺血(MI)作为一种高发病率和高死亡率的疾病迫切需要阐明其发病机制并开发新药。本研究从能量代谢流调控角度，研究银杏叶提取物(GBE)抗心肌缺血的机制及物质基础。采用代谢流分析技术(MFA)研究异丙肾上腺素(ISO)诱导的缺血样心肌细胞的能量代谢流紊乱和GBE成分的调节节点。结果发现在缺血样损伤心肌细胞中来自[U-13C]葡萄糖为碳源的代谢物同位素异数体m+2在三羧酸(TCA)循环上游显著蓄积，而下游代谢物则相反，但它们总的细胞浓度均增加。这表明缺血损伤后心肌细胞来自糖酵解的碳源在TCA循环受阻，其他碳源补偿性增加。采用活细胞能量代谢实时监测技术（Seahorse）筛选对线粒体有氧呼吸功能障碍有调节作用的GBE成分。结果发现白果内酯对损伤心肌细胞的线粒体有氧呼吸具有显著保护作用。MFA结果还发现白果内酯能够显著调节损伤心肌细胞的TCA循环代谢流，减少异常代谢物积累，平衡细胞对不同碳源的需求。Western blotting和PCR分析结果显示白果内酯显著降低了损伤细胞中关键能量代谢酶的增强表达。上述结果进一步在心肌缺血模型大鼠体内得到了验证。研究结果显示银杏叶提取物的活性成分白果内酯能够通过调节受损心肌细胞的TCA循环代谢流发挥心脏保护作用。这为心肌缺血的治疗提供了一种新的机制和潜在的药物治疗方法，也表明MFA/Seahorse技术结合在中草药作用机制研究中具有很大潜力。