HO-1调控ferritin自噬依赖性降解在氧化锌纳米颗粒诱发血管内皮细胞铁死亡和血管内皮损伤中的机制研究

Zinc oxide nanoparticles (ZnONPs) may penetrate blood–air barrier into circulation system, deposit at vascular endothelium and cause cytotoxicity, and consequently induce or aggravate cardiovascular-related diseases. In the previous studies (Cell Death and Disease 2017; Nanotoxicology 2018), we found that ZnONPs could induced non-apoptotic cell death and oxidative stress, these characterizations were similar as newly defined cell death-ferroptosis. Ferroptosis is iron-dependent cell death, the degradation of ferritin will lead to the elevation of intracellular iron concentration, which triggers ferroptosis. In the current project, we plan to demonstrate that ZnONPs could induce ferroptosis in vascular endothelial cell HUVEC and cause vascular endothelium dysfunction. Application of ferroptosis inhibitors, such as ferrostatin-1 or deferoxamine (DFO) would result in ameliorated vascular endothelium dysfunction caused by ZnONPs exposure. Moreover, multiply strategies such as autophagy activator/inhibitor, autophagy key gene knock-out cell line/mouse(ATG5 KO)，knockdown/knock out/stable ferritin/heme oxygenase-1 (HO-1) will be applied to elucidate the underlying mechanism of ZnONPs-induced vascular endothelium dysfunction. The project firstly demonstrate that ZnONPs could induce ferroptosis and vascular endothelium dysfunction in vitro and in vivo, and reveal that HO-1 mediated autophagy-dependent degradation of ferritin is responsible for ZnONPs-induced ferroptosis. Our project will contribute to wider and safer application of ZnONPs in biomedical fields.

氧化锌纳米颗粒（ZnONPs）可能穿透气血屏障入血，沉积于血管内皮造成细胞毒性，继而引发/加重心血管相关疾病。我们前期发现ZnONPs可导致非凋亡性细胞死亡、并伴随氧化应激状态激活，其特征与新型细胞死亡方式-铁死亡相契合。铁死亡是铁离子依赖的细胞死亡，铁蛋白ferritin自噬依赖性降解可促进铁离子浓度升高并诱发铁死亡，血红素氧合酶-1（HO-1）可能调控了此过程。据此我们提出假说：HO-1调控ferritin自噬依赖性降解促进ZnONPs诱发的铁死亡和血管内皮损伤。本项目拟在细胞和动物水平证明ZnONPs可诱发血管内皮细胞铁死亡和小鼠血管内皮损伤；并通过干预自噬、上/下调ferritin和HO-1等方法，证明HO-1调控ferritin自噬依赖性降解促进了ZnONPs诱发的血管内皮损伤。本项目旨在揭示新的ZnONPs导致血管内皮损伤的分子机制，为ZnONPs安全和广泛的应用提供科学依据。

纳米氧化锌（Zinc oxide nanoparticles, ZnONPs）是一种使用广泛的金属纳米材料。职业暴露或/和接触暴露后，ZnONPs可能穿透肺泡上皮细胞直接入血继而引发/加重心血管疾病。因此深入解析ZnONPs造成血管内皮损伤的分子机制，具有重要的科学意义。本研究证明了纳米氧化锌可引发血管内皮细胞依赖于自噬激活的、剂量和时间依赖性的铁死亡，自噬在此过程中起着促进血管内皮细胞死亡的作用。特别的是，我们证明了NCOA4介导的铁自噬（一种自噬依赖性降解储铁蛋白的选择性自噬）会导致血管内皮细胞内的铁离子增高，从而促进脂质过氧化和铁死亡的发生。机制研究表明，由于锌离子释放造成的线粒体损伤和mtROS升高会通过AMPK-ULK1通路激活铁自噬及铁死亡。小鼠模型的试验也证实了纳米氧化锌可能通过激活铁自噬和铁死亡引发血管内皮损伤。脂质过氧化清除剂Fer-1可以在细胞和动物水平显著缓解纳米氧化锌造成的血管内皮细胞铁死亡和血管内皮损伤。Nrf2-HO-1轴的激活对于保护ZnONPs造成的血管内皮损伤亦具有重要作用。总之，纳米颗粒诱导的坏死、凋亡和自噬被认为是纳米材料与控制细胞命运和活性的细胞机制相互作用的机制基础。我们扩展了这一概念，并提出铁自噬诱导的铁死亡是纳米颗粒在体内和体外诱导的一种新的细胞死亡形式，为深入理解纳米材料的生物学效应提供了新观点。..本项目的研究成果形成了4篇SCI论文，发表在颇有影响力的国际SCI期刊杂志Autophagy、Int J Nanomedicine、Nanotoxicology、Am J Respir Cell Mol Biol，4篇SCI论著皆标注本基金资助，并获得了同行的肯定，已被引用94次。申请人亦获得重庆市中青年医学高端人才和重庆市优秀临床药师荣誉称号。