N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, voltage-dependent channels of the ionotropic glutamate receptor family. The present study explored whether NMDAR activation induced ferroptosis in vascular endothelial cells and its complicated mechanisms in vivo and in vitro. Various detection approaches were used to determine the ferroptosis-related cellular iron content, lipid reactive oxygen species (LOS), siRNA molecules, RNA-sequence, MDA, GSH, and western blotting. The AMPK activator Acadesine (AICAR), HMGB1 inhibitor glycyrrhizin (GLY), PP2A inhibitor LB-100, and NMDAR inhibitor MK801 were used to investigate the involved in vivo and in vitro pathways. The activation of NMDAR with L-glutamic acid (GLU) or NMDA significantly promoted cellular ferroptosis, iron content, MDA, and the PTGS2 expression, while decreasing GPX4 expression and GSH concentration in human umbilical vein endothelial cells (HUVECs), which was reversed by ferroptosis inhibitors Ferrostatin-1(Fer-1), Liproxstatin-1 (Lip-1), or Deferoxamine (DFO). RNA-seq revealed that ferroptosis and SLC7A11 participate in NMDA or GLU-mediated NMDAR activation. The PP2A-AMPK-HMGB1 pathway was majorly associated with NMDAR activation-induced ferroptosis, validated using the PP2A inhibitor LB-100, AMPK activator AICAR, or HMGB1 siRNA. The role of NMDAR in ferroptosis was validated in HUVECs induced with the ferroptosis activator errasin or RSL3 and counteracted by the NMDAR inhibitor MK-801. The in vivo results showed that NMDA- or GLU-induced ferroptosis and LOS production was reversed by MK-801, LB-100, AICAR, MK-801, and GLY, confirming that the PP2A-AMPK-HMGB1 pathway is involved in NMDAR activation-induced vascular endothelium ferroptosis. In conclusion, the present study demonstrated a novel role of NMDAR in endothelial cell injury by regulating ferroptosis via the PP2A-AMPK-HMGB1 pathway.

N-甲基-D-天冬氨酸受体 (NMDAR) 是离子型谷氨酸受体家族的配体门控、电压依赖性通道。本研究探讨NMDAR激活是否诱导血管内皮细胞铁死亡及其体内外复杂机制。使用各种检测方法来测定铁死亡相关的细胞铁含量、脂质活性氧 (LOS)、siRNA 分子、RNA 序列、MDA、GSH 和蛋白质印迹。 AMPK激活剂Acadesine (AICAR)、HMGB1抑制剂甘草甜素(GLY)、PP2A抑制剂LB-100和NMDAR抑制剂MK801用于研究所涉及的体内和体外途径。 L-谷氨酸 (GLU) 或 NMDA 激活 NMDAR 显着促进细胞铁死亡、铁含量、MDA 和 PTGS2 表达，同时降低人脐静脉内皮细胞 (HUVEC) 中的 GPX4 表达和 GSH 浓度，而这一现象可通过铁死亡抑制剂 Ferrostatin-1(Fer-1)、Liproxstatin-1 (Lip-1) 或去铁胺 (DFO)。 RNA-seq 揭示铁死亡和 SLC7A11 参与 NMDA 或 GLU 介导的 NMDAR 激活。 PP2A-AMPK-HMGB1 通路主要与 NMDAR 激活诱导的铁死亡相关，已使用 PP2A 抑制剂 LB-100、AMPK 激活剂 AICAR 或 HMGB1 siRNA 进行验证。 NMDAR 在铁死亡中的作用在用铁死亡激活剂 errasin 或 RSL3 诱导并被 NMDAR 抑制剂 MK-801 抵消的 HUVEC 中得到了验证。体内结果表明，MK-801、LB-100、AICAR、MK-801 和 GLY 可逆转 NMDA 或 GLU 诱导的铁死亡和 LOS 产生，证实 PP2A-AMPK-HMGB1 途径参与 NMDAR 激活诱导的血管内皮细胞铁死亡。 总之，本研究证明了 NMDAR 通过 PP2A-AMPK-HMGB1 途径调节铁死亡，在内皮细胞损伤中发挥新作用。