酒精性肝铁过载中NCOA4-铁蛋白自噬与LIP联动失衡机制及槲皮素的干预效应

NCOA4 serves as cargo receptor to recognize ferritin and targets its lososomal autophagy, which is defined as ferritinophagy (FP), a novel regulative mechanism on iron homeostasis independent of but closed to hepcidin. Aiming at iron overload secondary to alcoholic liver disease, the current is designed to explore discreetly the joint effect of NCOA4-FP (including FP flux and type) and LIP followed chronic ethanol exposure in vivo and in vitro by using a variety of molecular biological methods. Focusing on the lysosomal LIP, the pathophysiological mechanisms, as well as signaling pathways, associated with joint disequilibrium between NCOA4-FP and LIP would be elucidated, according to the program. The crosstalk between NCOA4-FP/LIP and hepcidin, would also be discussed from a new angle. Subsequently, a naturally occurring quercetin is chosen for the intervention of alcoholic iron over load based on its iron-chelating effect. The protective effect, especially the potential mechanism and target of quercetin would be illustrated by studying its intervention for hepatic alcoholic iron over load. Such findings and results would shed a new light on the further elucidation of pathophysiological mechanism of iron over load secondary to alcoholic liver disease, extend hepcidin-mediaing iron regulation theory, and highlight the development and application of iron-chelating nature polyphenols.

NCOA4作为底物受体特异性识别铁蛋白并介导其靶向溶酶体自噬――铁蛋白自噬（FP），是独立于hepcidin但又与之紧密联系的铁稳态调控新机制。本项目针对酒精性肝铁过载，以NCOA4-FP为切入点，以溶酶体LIP为中心，借助多种分子生物学手段，从动物和细胞、分子水平层层剖析长期乙醇暴露对肝脏NCOA4-FP通量与途径及LIP水平的联动影响，分析两者联动失衡的分子机制、潜在信号通路与病理生理学意义，以及hepcidin在此过程中的作用；在此基础上，选择具有铁螯合效应的天然槲皮素作为干预物，从NCOA4－FP及LIP联动调控角度进一步揭示槲皮素预防酒精性肝铁过载的分子机制与作用靶点。本项目的实实施，不仅有助于从FP与LIP角度进一步阐释酒精性肝铁过载的分子机制，丰富hepcidin铁调控理论，还将为具有铁螯合性能的天然功能多酚的开发应用提供新的启示。

酒精性肝病（ALD）表现为剂量依赖性的铁过载（iron overload），而酒精性铁过载与NCOA4介导的铁噬的关系有待探究。本项目以NCOA4-ferritinophagy (FP)为切入点，探究肝脏铁过载与铁噬与LIP平衡的扰动的关系。此外我们也探究了NCOA4的上游转录调控及溶酶体功能及与之相关的LIP引起的铁死亡在酒精肝模型下变化及槲皮素的保护效应。选用C57BL/6J小鼠，通过慢加急（chronic-plus-single-binge ethanol feeding）ALD建模方法建立慢性ALD继发铁过载动物模型。在此基础上，我们利用高铁膳食喂养建立铁过载模型。借助基因过表达、信号通路激活与阻断等方法，结合细胞水平分子实验验证，阐明NCOA4-FP在ALD铁过载发生发展中的病理生理学意义与信号调控通路以及槲皮素在这一过程中对铁的调控作用。.经过2年的努力，取得的主要研究成果有： .1. 慢性酒精肝继发铁过载模型表现为NCOA4介导的铁噬水平的降低。.2. 慢性酒精通过增加FOXO1磷酸化，从抑制其转录活性，进而下调NCOA4介导的铁噬，导致铁蛋白蓄积。.3. FOXO1转录活性的降低导致了TFEB蛋白表达水平降低，使溶酶体功能受损，更新再生受阻。.4. 由于溶酶体酸化能力不足导致溶酶体铁输出障碍，ROS和LIP水平升高，诱发铁死亡。.5. 槲皮素通过调控FOXO1恢复NCOA4介导的铁噬，从而缓解酒精肝继发铁过载。.6. 槲皮素通过FOXO1升高TFEB蛋白表达，恢复溶酶体功能，缓解由于溶酶体ROS和LIP升高诱发的铁死亡。. 本研究的开展，不仅深入探讨酒精肝继发铁过载分子机制及NCOA4介导的铁噬的作用机制，同时发现并阐明了FOXO1通过调控NCOA4和TFEB而调控铁噬和铁死亡的相关分子机制。进一步发现并证实槲皮素有效抑制酒精肝铁过载缓解铁死亡的保护作用，为ALD的预防和治疗提供了充分的理论依据并开辟了新的路径。