构建CYP2E1基因修饰大鼠模型，研究CYP2E1介导的糖代谢在心衰发生发展中的作用与机制

Metabolic reprogramming is an important pathomechanism, and glycometabolic disorder is core pivot in prevention and treatment of heart failure (HF) and multiple cardiovascular disease (CVD). The effort of study on the genes involved in energy metabolism would help us to understand the gene network and the prevention and cure of HF. Classical studies indicated that CYP2E1 catalyze the metabolism of a vast array of endogenous and exogenous substrates and tissue injury. We found that it CYP2E1 upregulated in multiple CVD, and overexpression of CYP2E1 induce reactive oxygen species (ROS) and apoptosis, while knockdown of CYP2E1 inhibited the development of dilated cardiomyopathy. Furthermore, we found that the expression of CYP2E1 is related to the morphology of mitochondria and several enzyme involved in energy metabolism. All those fact indicate the regulation of CYP2E1 on energy metabolism. To elucidate the mechanism of CYP2E1 on the pathological progress of glucose and energy metabolism, we plan to establish the cardiac-specific CYP2E1 overexpression and knockout rat, and induced HF by pressure and drug stimulation. Then the modulation of CYP2E1 on glycolysis, pyruvate metabolism and ATP synthesis were performed by contrastive analysis, and the probable molecules, PDH，PDK and ATP5F1 would be studied by biochemical analysis, protein-protein interaction analysis, and signal transduction analysis.The biological function of CYP2E1 on balance of ROS and energy supply would be studied from global, histoorgan and molecular level, and these would provide theoretical foundation of CYP2E1 regulation in glycometabolism and the prevention and cure for HF.

代谢重编程是心血管病重要发病机制，糖代谢异常是心衰及多种心血管病终末期防控重要节点，研究参与能量代谢基因及调控网络有利于心衰防治。经典研究认为CYP2E1参与内外源物质代谢和组织损伤。我们前期发现CYP2E1于多种心血管病时上调，其高表达可引起氧化应激和凋亡，表达敲低可抑制扩心病病理进程，其表达水平与线粒体形态及多种代谢关键酶直接相关，这些均揭示它对心肌能量代谢重要性。为阐明该基因参与心肌糖代谢及能量调控机制，拟建立心肌组织特异性CYP2E1过表达及敲除大鼠，通过压力负荷及药物刺激诱导心衰，利用生化分析，蛋白质互作及信号转导等技术，分析其与心肌糖酵解、丙酮酸代谢及线粒体ATP合成相关酶及蛋白，如PDH，PDK及ATP5F1等的互作及调控关系，从整体、组织及分子水平分析CYP2E1在氧化应激和能量供应平衡中的角色，最终明确其介导的糖代谢在心衰发生发展中的生物学作用并为心衰防治提供理论依据。

代谢重编程是心血管病重要发病机制，糖代谢异常是心衰及多种心血管病终末期防控重要节点，研究参与能量代谢基因及调控网络有利于心衰防治。经典研究认为CYP2E1参与内外源物质代谢和组织损伤。我们前期发现CYP2E1于多种心血管病时上调，其高表达可引起氧化应激和凋亡，表达敲低可抑制扩心病病理进程，其表达水平与线粒体形态及多种代谢关键酶直接相关，这些均揭示它对心肌能量代谢重要性。.为阐明该基因参与心肌糖代谢及能量调控机制，本项目首先完成了心肌组织特异性CYP2E1过表达和敲除大鼠的建立鉴定，并繁育工具大鼠年龄梯队。首先分析了在生理状态下CYP2E1的表达对心肌组织氧化应激和能量供应的影响，明确了CYP2E1对生理状态下心肌组织ROS生成及线粒体ATP生成的时程关系。随后对NTG/CYP2E1-ov/CYP2E1-ko 三组大鼠给予病理刺激处理，我们发现CYP2E1高表达可以加速，而CYP2E1表达缺失后可以抑制，心脏的病理发展进程。最后通过组学数据分析及蛋白质免疫沉淀及免疫印迹验证，我们发现CYP2E1通过影响线粒体分裂融合及自噬进程，来参与心肌损伤，并且确定了CYP2E1的互作蛋白分子TALDO1，即CYP2E1可能通过该分子，参与心肌病理情况下的糖代谢调控。.该项目明确了CYP2E1参与心肌损伤的分子机制及其介导的糖代谢在心衰发生发展中的生物学作用，为心衰防治提供了理论依据和实验室数据。