FOXO3-Frataxin介导的肝脏线粒体酒精性损伤及槲皮素干预机制

Mitochondrial damage, as an early initiating event and an important pathological changes throughout the alcoholic liver disease (ALD), is the originating cause fate of hepatocytes. Hence, investigation the potential mechanism of mitochondiral damage is very important. In this study, frataxin responsible for mitochondrial key function, and FOXO3 which regulate themitochondrial homestasis in many aspects are our main focuses. We will carry out research on animals and cells to investigate the role of frataxin in ALD and mitochondial damage, as well as the mediated effect of FOXO3 and the upstream signaling pathways (AMPK, Akt, ERK and SIRT3), by a variety of molecular biology techniques including RNA trsanfection, inhibiting and activating, CHIP and EMSA. Quercetin is a ubiquitous plant-derived flavonoid with well-documented biological functions in both cell and animal systems, including alleviating ethanol-mediated oxidativedamage and inflammatory stress. Therefore, we also investigate the protective role of quercetin against alcoholic liver mitochondrial damage and the role of FOXO3-frataxin. Implementation of this project, is not only expected to further clarify the pathophysiological significance of FOXO3 and frataxin, will also provide a new theoretical basis for the research and application of quercetin in prevention of mitochondrial damage and mitochondrial-related diseases.

线粒体损伤不仅是酒精性肝病（ALD）的早期始发事件更是贯穿始终的重要病理改变，是肝细胞命运的重要原因，研究线粒体损伤机制是防治ALD的关键。本项目拟以维持线粒体功能的关键蛋白—Frataxin为切入点，以调节线粒体稳态的核心蛋白-FOXO3为调控点，借助基因转染、通路激动与阻断、ChIP、EMSA及荧光素酶报告等技术，在动物整体和细胞分子水平对frataxin在酒精性肝线粒体损伤的作用，FOXO3的介导效应以及AMPK、Akt、ERK和SIRT3等潜在信号通路层层剖析。在此基础上，选择前期研究显示对ALD具有保护效应且特异性富集线粒体的槲皮素进行干预，分析其对FOXO3-frataxin介导的酒精性线粒体损伤的拮抗效应及潜在作用靶点，从全新的角度深入揭示其拮抗ALD的分子机制。本项目的实施，将进一步丰富FOXO3和frataxin的病理生理学意义，还为槲皮素防治线粒体相关性疾病提供新思路。

线粒体损伤是酒精性肝损伤（alcoholic liver damage，ALD）早期且贯穿始终的重要病理改变，但具体机制和相关信号通路研究明显缺乏。深入研究线粒体结构与功能的改变在酒精性肝损伤中的作用对于更为深入地阐明ALD的发病机制具有特殊的病理生理学意义，还将为ALD的靶向性营养防治提供新的启示。Frataxin与线粒体相关性疾病密切相关，但是否介导肝线粒体的酒精性损伤，至今缺乏直接相关的证据。此外，Frataxin依赖FOXO3调节为深入研究酒精性肝线粒体损伤的防治开辟新的作用靶点，但相关研究尚是空白。同时，可富集于线粒体的槲皮素，其拮抗ALD效应是否与线粒体功能调节蛋白有关，如何调控，尚待深入研究。在此研究背景下，本项目以C57BL/6J小鼠为研究对象（含乙醇的Lieber De Carli liquid 饲料等能量配对喂养），结合细胞水平实验分析研究frataxin在酒精性肝线粒体损伤中的作用。本研究成功建立ALD动物和细胞模型，首先明确ERK、AMPK 、SIRT1和PI3K-FOXO3信号通路在酒精性肝线粒体损伤中的作用及机制，揭示了酒精性线粒体损伤的潜在机制。同时，鉴于线粒体铁过载的同时却铁利用不足，筛选线粒体铁代谢相关基因，以frataxin为核心，首次发现长期酒精喂养抑制frataxin的表达，构建frataxin缺失的稳转株HepG2细胞，发现酒精进一步抑制frataxin的表达，而frataxin缺失加剧酒精性肝线粒体毒性，frataxin缺失HepG2胞浆铁水平下降，而线粒体铁水平增加，酒精进一步降低胞浆铁水平，进一步增加线粒体铁水平。同时选用前期研究显示对酒精性肝损伤具有保护作用，且富集线粒体的槲皮素为干预对象，揭示了槲皮素对酒精性肝线粒体损伤的保护机制和作用靶点。动物和细胞结果均发现frataxin介导酒精对线粒体自噬的抑制作用，相关机制尚待进一步探讨。