Zinc oxide nanoparticles (ZnONPs) induce in endothelial cells, leading to cell death.
ZnONPs exposure increases intracellular iron levels and lipid peroxidation in a dose- and time-dependent manner.
Cell death caused by ZnONPs can be attenuated by both a lipid reactive oxygen species scavenger and an iron chelator.
Ferroptosis induced by ZnONPs depends on macroautophagy, as inhibiting autophagy reduces cell death.
NCOA4-mediated is necessary for ZnONPs-induced ferroptosis, as its knockdown decreases iron levels and lipid peroxidation.
Mitochondrial reactive oxygen species may activate the AMPK-ULK1 pathway to promote ferritinophagy.
Pulmonary exposure to ZnONPs results in vascular inflammation and ferritinophagy in mice, with ferrostatin-1 mitigating vascular injury.
Simplified
Zinc oxide nanoparticles (ZnONPs) hold great promise for biomedical applications. Previous studies have revealed that ZnONPs exposure can induce toxicity in endothelial cells, but the underlying mechanisms have not been fully elucidated. In this study, we report that ZnONPs can induce of both HUVECs and EA.hy926 cells, as evidenced by the elevation of intracellular iron levels, lipid peroxidation and cell death in a dose- and time-dependent manner. In addition, both the lipid reactive oxygen species (ROS) scavenger ferrostatin-1 and the iron chelator deferiprone attenuated ZnONPs-induced cell death. Intriguingly, we found that ZnONPs-induced ferroptosis is macroautophagy/autophagy-dependent, because the inhibition of autophagy with a pharmacological inhibitor or bygene knockout profoundly mitigated ZnONPs-induced ferroptosis. We further demonstrated that NCOA4 (nuclear receptor coactivator 4)-mediated (autophagic degradation of the major intracellular iron storage protein ferritin) was required for the ferroptosis induced by ZnONPs, by showing thatknockdown can reduce the intracellular iron level and lipid peroxidation, and subsequently alleviate ZnONPs-induced cell death. Furthermore, we showed that ROS originating from mitochondria (mtROS) probably activated the AMPK-ULK1 axis to trigger ferritinophagy. Most importantly, pulmonary ZnONPs exposure caused vascular inflammation and ferritinophagy in mice, and ferrostatin-1 supplementation significantly reversed the vascular injury induced by pulmonary ZnONPs exposure. Overall, our study indicates that ferroptosis is a novel mechanism for ZnONPs-induced endothelial cytotoxicity, and that NCOA4-mediated ferritinophagy is required for ZnONPs-induced ferroptotic cell death.3-MA: 3-methyladenine; ACTB: Actin beta; AMPK: AMP-activated protein kinase; ATG: Autophagy-related; BafA1: Bafilomycin A1; CQ: Choloroquine; DFP: Deferiprone; FACS: Fluorescence-activated cell sorting; Fer-1: Ferrostatin-1; FTH1: Ferritin heavy chain 1; GPX4: Glutathione peroxidase 4; GSH: Glutathione; IREB2/IRP2: Iron responsive element binding protein 2; LIP: Labile iron pool; MAP1LC3B/LC3B: Microtubule associated protein 1 light chain 3 beta; MTOR: Mechanistic target of rapamycin kinase; NCOA4: Nuclear receptor coactivator 4; NFE2L2/NRF2: Nuclear factor, erythroid 2 like 2; PGSK: Phen Green™ SK; ROS: Reactive oxygen species; siRNA: Small interfering RNA; SQSTM1/p62: Sequestosome 1; TEM: Transmission electron microscopy; ULK1: Unc-51 like autophagy activating kinase 1; ZnONPs: Zinc oxide nanoparticles. ATG5NCOA4Abbreviations:
Key numbers
MDA content
Increase in lipid peroxidation
Measured in HUVECs and EA.hy926 cells after ZnONPs treatment.
12 μg/mice
12 μg
Administered to mice during pulmonary exposure experiments.
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