Fig. 1: Ferroptosis participates in NMDAR activation-mediated endothelial cell damage in vitro.

RNA sequencing was performed after in HUVECs treated with GLU (20 mM) or NMDA (1 mM) for 24 h, and the results of volcano plot (A, B) and KEGG analysis (C) were shown. D Ultrastructure of the mitochondria in HUVECs treated with GLU or NMDA, visualized by TEM. Black arrowheads: shrunken mitochondria (Scale bars, 1 μm). E MTT assay and (F) ferrous ion levels visualized by FerroOrange staining (Scale bars, 200 μm) and (G) corresponding quantification showing cell viability and ferrous ion levels in HUVECs pretreated with ferroptosis inhibitors (Fer-1: 10 μM, Lip-1: 2 μM, or DFO: 10 μM) for 1 h followed by GLU and NMDA treatment for 24 h. The relative levels of iron content (H), flow cytometry plots (I), and quantification of lipid ROS (LOS) production (J), MDA (K), and GSH (L) in HUVECs pretreated with Fer-1 (10 μM), Lip-1 (2 μM), or DFO (10 μM) for 1 h followed by GLU or NMDA treatment for 24 h. The relative levels of ferroptosis biomarkers PTGS2 and GPX4 shown by immunoprotein blots and quantification in the presence of GLU (M) or NMDA (N) and treatment with Fer-1 (10 μM), Lip-1 (2 μM), or DFO (10 μM). ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs control; ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs GLU; ^&P < 0.05, ^&&P < 0.01 and ^&&&P < 0.001 vs NMDA.