Zinc Oxide Nanoparticles Induce Vascular Endothelial Injury Through Ferritinophagydependent Ferroptosis

Purpose:Zinc oxide nanoparticles(ZnONPs)are widely metal nanoparticles and have been proved to be cytotoxic.Previous studies suggest that inhalation of particulate matter can penetrate the alveolar epithelial cells and directly enter into the blood and cause or aggravate cardiovascular disease.Vascular endothelium is vulnerable once the entrance of ZnONPs into blood vessel.As such,it is of great scientific significance to reveal the molecular mechanism of vascular endothelium injury caused by ZnONPs.This study aims to elucidate the mechanism of vascular endothelial injury caused by ZnONPs through ferritinophagy-dependent ferroptosis in vitro and in vivo.Methods:1.We determined whether ZnONPs exposure induced cell death in vascular endothelial cell HUVEC and EA.hy926 by microscopy,MTS and FACS.We determined the iron content in cells by MDA assay and C11-BODIPY staining.We determined the change of key genes involved in ferroptosis by QPCR and WB.In addition,ferroptosis inhibitors such as Fer-1 and DFP were applied to reverse the cell death induced by ZnONPs.2.We determined whether ZnONPs treatment induced autophagy using TEM,immunofluorescent analysis and WB.Pharmacological intervention with 3-MA,CQ or Baf A1 and genetical intervention by ATG5 knockout were used to determine the complete of autophagy.In addition,we determined whether ZnONPs induced NCOA4-mediated,autophagy-dependent degradation of FTH1.sh RNA-mediated NCOA4 knockdown cell was used to verify this notion.3.We determined the key regulation factors(such as AMPK and MTOR and their downstream factors)that are involved in ZnONPs-activated autophagy by WB.si RNA-mediated indicated gene knockdown was used to verify this notion.Mitochondrial membrane potential,morphology and oxidative stress were detected to determine whether ZnONPs caused mitochondrial damage and the production of mitochondrial derived ROS(mtROS).Then,the ROS scavengers Mito TEMPOL and NAC were used to determine whether mtROS were the upstream mechanism of ferritinophagy and ferroptosis.4.To demonstrate the relationship between ferritinophagy-mediated ferroptosis and vascular injury when exposed to ZnONPs in vivo,a mouse model of endothelium injury induced by intratracheal exposure to ZnONPs was established,and the ultrapathological changes of abdominal aorta were detected by transmission electron microscopy,inflammatory related factors were detected by q PCR,MDA content was measured,and ferritinophagy and ferroptosis-related protein expression were detected by WB.Ferroptosis inhibitor Fer-1 was administrated to observe whether inhibition of ferroptosis contributing to alleviation of ZnONPs-induced vascular endothelium injury.Results:1.ZnONPs caused ferroptosis in vascular endothelial cells: ZnONPs exposure induced elevation of iron content,lipid peroxidation and cell death in HUVEC and EA.hy926 cells in a dose-and time-dependent manners.ROS scavenger Fer-1 and iron chelator DFP were efficient to repress cell death induced by ZnONPs.There results suggest that ZnONPs induce ferroptosis in vascular endothelial cells.2.ZnONPs induced ferroptosis through NCOA4-mediated,autophagy-dependent degradation of FTH1 in vascular endothelial cells:ZnONPs induced activation of autophagy,although blockade of autophagic flux might exist.Pharmacological intervention with 3-MA,CQ or Baf A1 and genetical intervention by ATG5 knockout were able to ameliorate cell death induced by ZnONPs,suggesting that autophagy plays pro-death role during ZnONPs treatment.Importantly,we demonstrated that ZnONPs-induced,autophagydependent degradation of FTH1,which led to elevation of iron content and ferroptosis.Knockdown of NCOA4 significantly repressed degradation of FTH1 and ferroptosis,indicating that NCOA4-mediated ferritinophagy is crucial for ZnONPs-induced ferroptosis in vascular endothelial cells.3.The mtROS-AMPK-ULK1 signaling axis is involved in ferritinophagy and ferroptosis induced by ZnONPs: Zinc ions released by ZnONPs led to mitochondrial damage and increase of mtROS,which in turn activated autophagy through AMPK-ULK1 signaling axis,caused elevation of iron content and ferroptosis.Elimination of mtROS by Mito TEMPOL and NAC,or knockdown of AMPK and ULK1 genes,significantly reduced intracellular lipid peroxidation and ferroptosis caused by ZNONPs,therefore suggest that mtROS-AMPK-ULK1 signaling axis is important for ZnONPs-induced ferroptosis.4.Intratracheal exposure of ZnONPs induced ferritinophagy-dependent vascular injury: Intratracheal exposure of ZnONPs caused ultrapathological deterioration and inflammation,accompanied by increase in LC3B-II,p-AMPK,p-ULK1 and decrease in FTH1,suggesting activation of ferritinophagy in blood vessel.Meanwhile,ZnONPs exposure caused decrease of negative regulator for ferroptosis such as GPX4 and SLC7A11 and increase of ferroptosis marker Ptgs2 and MDA,suggesting that ZnONPs caused ferroptosis in blood vessel.Supplementation of ferroptosis inhibitor Fer-1 profoundly mitigated vascular injury induced by ZnONPs,indicating ferroptosis might be causally linked with ZnONPs-induced vascular injury in vivo.Conclusions:1.We demonstrate that ZnONPs could promote ferroptosis and induce vascular injury through NCOA4-mediated ferritinophagy.2.mtROS activated AMPK-ULK1 signaling is crucial for ZnONPs-induced ferritinophagy and ferroptosis.3.This study highlights that ferritinophagy-induced ferroptosis is a novel mechanism of vascular endothelial injury caused by nanoparticles,which provides a novel perspective for the understanding of nano-bio interaction.