Autophagy-dependent release of zinc ions is critical for acute lung injury triggered by zinc oxide nanoparticles

Pulmonary exposure to zinc oxide nanoparticles (ZnONPs) could cause acute lung injury (ALI), but the underlying molecular mechanism remains unclear. Herein, we established a ZnONPs-induced ALI mouse model, characterized by the histopathological changes (edema and infiltration of inflammatory cells in lung tissues), and the elevation of total protein and cytokine interleukin-6 in bronchoalveolar lavage fluid in time- and dose-dependent manners. This model also exhibited features like the disturbance of redox-state (reduced of glutathione to glutathione disulfide ratio, elevation of heme oxygenase-1 and superoxide dismutase 2), the decrease of adenosine triphosphate synthesis and the release of zinc ions in the lung tissues. Interestingly, we found that ZnONPs exposure caused the accumulation of autophagic vacuoles and the elevation of microtubule-associated proteins 1A/1B light chain (LC)3B-II and p62, indicating the impairment of autophagic flux. Our data indicated that the above process might be regulated by the activation of AMP-activated protein kinase but not the mammalian target of rapamycin pathway. The association between ZnONPs-induced ALI and autophagy was further verified by a classical autophagy inhibitor, 3-methyladenine (3-MA). 3-MA administration reduced the accumulation of autophagic vacuoles, the expression of LC3B-II and p62, followed by a significant attenuation of histopathological changes, inflammation, and oxidative stress. More importantly, 3-MA could directly decrease the release of zinc ions in lung tissues. Taken together, our study provides the evidence that ZnONPs-induced pulmonary toxicity is autophagy-dependent, suggests that limiting the release of zinc ions by inhibiting autophagy could be a feasible strategy for the prevention of ZnONPs-associated pulmonary toxicity.

氧化锌纳米颗粒（ZnONPs）经呼吸道暴露可诱发急性肺损伤（ALI），但其潜在分子机制尚未阐明。本研究构建了ZnONPs诱导的ALI小鼠模型，该模型以肺组织病理学改变（肺水肿及炎性细胞浸润）、支气管肺泡灌洗液中总蛋白与细胞因子白细胞介素6呈时间与剂量依赖性升高为特征。此外，该模型还表现出氧化还原稳态紊乱（谷胱甘肽与谷胱甘肽二硫化物比值降低、血红素氧合酶-1与超氧化物歧化酶2表达上调）、肺组织三磷酸腺苷合成减少以及锌离子释放等特征。有趣的是，本研究发现ZnONPs暴露可导致自噬泡蓄积，同时使微管相关蛋白1A/1B轻链3B-II与p62水平升高，提示自噬流受损。本研究数据表明，上述过程可能由腺苷酸活化蛋白激酶的激活所调控，而非哺乳动物雷帕霉素靶蛋白通路。通过经典自噬抑制剂3-甲基腺嘌呤（3-MA）进一步验证了ZnONPs诱导的ALI与自噬之间的关联：3-MA给药可减少自噬泡蓄积与LC3B-II、p62的表达，随后显著减轻肺组织病理学改变、炎症反应与氧化应激。更重要的是，3-MA可直接降低肺组织中锌离子的释放。综上，本研究证实ZnONPs诱导的肺毒性具有自噬依赖性，并提示通过抑制自噬以限制锌离子释放，或可成为预防ZnONPs相关肺毒性的可行策略。