Endothelial Knockdown of NARFL Promotes Ferroptosis and Ferrostatin‐1 Alleviates Oxidative Stress Injury Caused by NARFL Gene Deletion. A) Dynamic observation under a microscope and daily photographing of HPMEC wild‐type cells and NARFL mutant cells recorded significant differences in cell death patterns between the two groups. NARFL mutant cells exhibited clear morphological changes, gradually transitioning to an enlarged and fattened morphology with increased granularity, and their death morphology was similar to that of cells undergoing ferroptosis. B) FerroOrange fluorescence probe was taken by confocal fluorescence microscopy for the detection of HPMEC, NARFL−/− HPMEC, and NARFL−/− HPMEC cells treated with Ferrostatin‐1 (6 µM). The darker orange color indicated the higher content of ferrous ions in the cells. C) Quantitative results were measured by the FerroOrange fluorescent probe; ferrous ion content in HPMEC increased significantly after NARFL deletion, but decreased significantly after Ferrostatin‐1 was added. D‐F) The quantitative results of cytoplasmic cisaconitase (D), glutamine (E) and glutathione (F) in HPMEC, NARFL−/−HPMEC and 6 µM Ferrostatin‐1 treated NARFL−/− HPMEC cells, which showed that NARFL deletion can also cause the decrease of cytoplasmic cis‐aconitase activity and intracellular glutathione and glutamine content in endothelial cells, which can be partially saved by ferroptosis inhibitor Ferrostatin‐1. G) Transmission electron microscopy showed that the mitochondrial morphology of NARFL−/−HPMEC was obviously different from that of wild type cells. The black arrows indicate mitochondria exhibiting typical ferroptotic morphology, including becoming smaller, increased membrane density, and diminished cristae. H) Survival analysis showed that 8 µM Ferrostatin‐1 can extend the survival time of narfl−/− zebrafish from 13 days to 21 days. I) Detection of 5 dpf zebrafish treated with 8 µM Ferrostatin‐1 by DPPP fluorescent probe; the results showed that 8 µM Ferrostatin‐1 inhibits lipid peroxidation levels caused by narfl deletion. Statistical significance was assessed using one‐way ANOVA with Tukey's multiple comparison correction. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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