RFK调控TNF-α-Nox2-ROS信号轴在冠脉微栓塞后心肌损伤中的作用与机制研究

Myocardial injury after coronary microembolization (CME) leads to poor prognosis. Studies have shown that TNF-α is a key pathogenic factor of CME; NADPH oxidase 2 (Nox2)-mediated oxidative stress is involved in the pathological process of myocardial injury. Our previous studies found that CME mice and TNF-α-stimulated primary cardiomyocytes had Nox2 activation and reactive oxygen species (ROS) overexpression, suggesting that TNF-α-Nox2-ROS signal axis had important pathogenic effect in CME, but its regulatory mechanism is not yet clear. It is reported that riboflavin kinase (RFK) has binding sites for TNF-α receptor-associated death domain protein (TRADD), and can combine to p22phox, a transmembrane subunit of Nox2, converting riboflavin to flavin mononucleotide and flavin adenine dinucleotide, and then participates in biological oxidation. Therefore, we deduce that after CME, RFK activates Nox2 by directly coupling TRADD and p22phox, inducing oxidative stress injury. This present program intends to clarify the role of TNF-α-Nox2-ROS signal axis in myocardial injury after CME and reveal the specific mechanism of how RFK regulates the signal axis, by constructing CME mouse model and culturing primary cardiomyocytes, via molecular biological techniques, such as recombinant adeno-associated virus/lentiviral transfection, co-immunoprecipitation and high performance liquid chromatography, in order to provide new ideas and targets for the prevention and treatment of CME.

冠脉微栓塞（CME）后心肌损伤可导致不良预后。TNF-α是CME关键致病因子；NADPH氧化酶2（Nox2）介导的氧化应激反应参与心肌损伤病理过程。前期研究发现CME小鼠及TNF-α刺激心肌细胞有Nox2激活及活性氧（ROS）过表达，提示TNF-α-Nox2-ROS信号轴是CME的重要致病环节，但其调控机制尚不明确。研究表明核黄素激酶（RFK）与TNF-α受体相关死亡域结构蛋白（TRADD）存在结合位点，并可与Nox2亚单位p22phox结合，使核黄素活化，参与生物氧化。我们推论CME后，RFK通过偶联TRADD和p22phox，激活Nox2，诱发氧化应激损伤。本项目以CME小鼠、原代心肌细胞为研究对象，通过基因修饰、免疫共沉淀及高效液相色谱法等手段，阐明TNF-α-Nox2-ROS信号轴在CME后心肌损伤中的作用，揭示RFK调控该信号轴的具体机制，以期对CME防治提供新的思路及靶点。

冠脉微栓塞（CME）后心肌损伤可导致不良预后。本研究拟阐明TNF-α-Nox2-ROS信号轴在CME后心肌损伤中的重要作用及调控机制。研究以CME 小鼠、原代心肌细胞为研究对象，通过基因修饰，组织病理分析，荧光染色，Western Blot，ELISA，实时定量PCR及免疫共沉淀等手段，以揭示TNF-α-Nox2-ROS信号轴在CME后心肌损伤中的作用，并明确RFK调控该信号轴的具体机制。.结果：通过病毒转染CME小鼠使Nox2 过表达及靶向沉默，发现Nox2低表达组ROS表达水平降低，心肌损伤及凋亡的表现减轻；通过抗体干预使CME小鼠心肌分别高低表达TNF-α，发现在TNF-α低表达组可见ROS水平下降，Nox2表达下降及心肌损伤与凋亡的减轻。通过病毒转染原代心肌细胞使Nox2高低表达，再应用TNF-α干预心肌细胞，在Nox2低表达组，其对ROS的表达及心肌细胞的损伤及凋亡均有减低，在Nox2高表达组则相反。通过病毒转染使小鼠RFK沉默，发现CME后RFK低表达组组织内炎症反应及纤维化程度更低，ROS表达水平更低，细胞损伤及凋亡程度在也更低，提示RFK可能对TNF-α-Nox2-ROS 信号轴起到影响作用；对CME小鼠进行心肌组织免疫共沉淀检测，证实TNF-α受体TNFR1死亡结构域TRADD、RFK以及p22phox存在相互作用。.结论：本研究证实了TNF-α-Nox2-ROS信号轴在CME后心肌损伤中的作用，明确了RFK与TNFR1死亡结构域TRADD以及p22phox之间存在相互作用，并揭示了RFK可调控TNF-α-Nox2-ROS 信号轴的作用。