Rab7介导的线粒体自噬和外泌体在酒精性肝铁过载的协同机制及槲皮素干预研究

The synergistic toxicity of iron overload in alcoholic liver disease (ALD) needs further elucidation. Recent research shows that iron status is a sensitive signal to mitophagy. However, the role of mitophagy in ALD is controversial and the potential mechanism is unclear. Exosomes, as emerging biological mechanisms, overlap with (mitochondrial) autophagy during the stages of formation, transport and fusion. Synergy between the two is expected to enrich the mechanism of alcoholic liver iron overload. The aim of this study is to be as the research background in iron overload, centered on mitophagy and exosomes, and to target Rab7 of the critical protein on regulation. We aimed to investigate the role of Rab7 -mediated mitophagy and exosomes in alcoholic liver iron overload comprehensively from animal and cell experiments with gene transfection, immunofluorescence and immunization co-precipitation, proteomic and bioinformatics analysis, and so on. Quercetin was selected to explore the protective effect and regulatory mechanism of ALD owing to its characteristics of iron chelating and concentration on mitochondria. The implementation of this project will provide an important theoretical basis for the new path of the prevention and intervention of alcoholic liver iron overload, which will further enrich the pathophysiological significance of mitophagy and exosomes and provide new ideas for polyphenols such as quercetin intervention.

铁过载在酒精性肝病（ALD）中的协同毒性亟待深入阐明。最新研究表明：铁状态是线粒体自噬的敏感信号。但是，线粒体自噬对ALD的作用尚不一致，且机制未阐明。外泌体，作为新兴的生物学机制，在形成、转运和融合等阶段与（线粒体）自噬交叉重叠。两者的协同作用有望丰富酒精继发性肝铁过载的机制。本项目以铁过载为切入点，以线粒体自噬和外泌体为核心，以协同调节两者的关键分子Rab7为靶点，借助免疫荧光共定位、活细胞成像，结合蛋白组学，通过基因沉默和转染等，在动物整体和细胞分子水平全面剖析线粒体自噬和外泌体协同调节酒精性肝铁过载及潜在的机制。在此基础上，选择对ALD及铁过载具有良好保护效应的铁螯合天然多酚--槲皮素进行干预，分析其保护效应及对线粒体自噬和外泌体的影响，揭示槲皮素的关键靶点。本项目的实施，对ALD及铁过载相关机制的阐明具有重要意义，且从线粒体自噬和外泌体角度为天然多酚的营养干预提供新思路。

近年来酒精性肝病(Alcoholic liver disease, ALD)患病率不断增加，造成了重大的公共卫生负担。ALD继发的铁过载，是肝脏产生氧化应激的重要原因之一。自噬和外泌体，也被证实参与了ALD的发生发展，但二者之间是否在ALD的进展中具有协同作用尚未可知。尽管槲皮素减轻ALD小鼠肝损伤，但潜在机制尚未完全阐明。.基于以上研究背景，本项目的主要研究内容是，探讨ALD继发性铁过载的相关机制，以及自噬与外泌体在ALD中的协同机制，同时确定Rab7 在其中的作用及相关信号通路；在此基础上，揭示槲皮素的作用机制。.该项目主要取得以下重要进展：.1) 槲皮素减轻ALD小鼠肝损伤及线粒体功能紊乱。ALD小鼠转氨酶水平明显上升，肝组织结构紊乱，大量脂滴蓄积，出现明显线粒体损伤，且线粒体自噬受阻；槲皮素显著改善ALD小鼠上述变化。.2) 槲皮素减轻ALD小鼠中线粒体功能紊乱与线粒体铁伴侣分子frataxin有关。ALD小鼠肝脏frataxin水平降低，frataxin缺乏进一步加剧酒精对线粒体自噬的抑制作用。槲皮素改善酒精对frataxin的抑制及随后的线粒体自噬受阻和线粒体铁过载。.3) 槲皮素通过调节铁噬水平缓解线粒体功能紊乱。ALD小鼠肝脏NCOA4介导的铁噬水平降低，同时，可变铁池LIP水平上升，线粒体功能紊乱。槲皮素干预改善上述变化。FAC处理降低槲皮素对线粒体膜电位的保护效应。.4) 槲皮素缓解ALD小鼠Rab7介导的脂噬受阻。ALD小鼠肝脏Rab7活性降低从而导致Rab7介导的脂噬受阻，槲皮素促进Rab7的周转，恢复Rab7-溶酶体轴的正常功能，从而缓解酒精导致的脂噬受阻。.5) 槲皮素通过TFEB介导的溶酶体修复调节自噬及外泌体释放从而缓解ALD。ALD小鼠肝脏自噬受阻，外泌体释放增加，二者协同促进ALD发生，转录组学提示溶酶体是协调二者的关键。槲皮素通过恢复TFEB介导的溶酶体修复，从而调节自噬及外泌体的变化，缓解ALD。.本项目阐明了槲皮素对ALD的保护效应及潜在机制，重点阐明了线粒体铁代谢，线粒体自噬，脂噬，Rab7-溶酶体，以及外泌体在其中的重要作用。同时，揭示了自噬与外泌体在槲皮素拮抗ALD中的协同作用，丰富了槲皮素缓解ALD的机制研究。