This study aimed to elucidate the molecular mechanisms of hypoxia/reoxygenation (H/R) injury in human cardiac microvascular endothelial cells (HCMECs) by regulating ferroptosis. H/R model was established with HCMECs and before the reperfusion, ferroptosis inhibitor ferrostatin-1 or ferroptosis inducer erastin was all administered. Wound-healing assay was performed to detect the migration ability of cells in each group, and the angiogenesis ability was determined by tube formation assay. The level of reactive oxygen species (ROS) was detected by flow cytometry. Transmission electron microscopy (TEM) was used to observe the state of mitochondria. The expressions of related proteins in HCMECs were assessed by Western blot. From the results, H/R injury could inhibit the migration and angiogenesis, induce the ROS production, and cause the mitochondrial damage of HCMECs. Ferroptosis activator erastin could aggravate H/R injury in HCMECs, while the ferroptosis inhibitor ferrostatin-1 could reverse the effects of H/R on HCMECs. Western blot results showed that H/R or/and erastin treatment could significantly induce ACSL4, HGF, VEGF, p-ERK, and uPA protein expression and inhibit GPX4 expression. The addition of ferrostatin-1 resulted in the opposite trend of the proteins expression above to erastin treatment. What is more, overexpression of ENPP2 markedly suppressed the damaging effect of H/R on HCMECs and reversed the effects of H/R or erastin treatment on the expression of related proteins. These results demonstrated a great therapeutic efficacy of ENPP2 overexpression in preventing the development of H/R injury through inhibiting oxidative stress and ferroptosis.

本研究旨在通过调节铁死亡阐明人心脏微血管内皮细胞 (HCMECs) 缺氧/复氧 (H/R) 损伤的分子机制。用HCMECs建立H/R模型，再灌注前均给予铁死亡抑制剂ferrostatin-1或铁死亡诱导剂erastin。划痕实验检测各组细胞的迁移能力，成管实验检测血管生成能力。通过流式细胞术检测活性氧（ROS）水平。透射电子显微镜（TEM）用于观察线粒体的状态。通过蛋白质印迹评估HCMEC中相关蛋白的表达。从结果来看，H/R 损伤可以抑制迁移和血管生成，诱导 ROS 的产生，并引起 HCMEC 的线粒体损伤。铁死亡激活剂 erastin 可加重 HCMEC 的 H/R 损伤，而铁死亡抑制剂 ferrostatin-1 可逆转 H/R 对 HCMEC 的影响。Western blot 结果表明，H/R 或/和 erastin 处理可显着诱导 ACSL4、HGF、VEGF、p-ERK 和 uPA 蛋白表达并抑制 GPX4 表达。添加 ferrostatin-1 导致上述蛋白质表达的趋势与 erastin 处理相反。此外，ENPP2 的过表达显着抑制了 H/R 对 HCMEC 的破坏作用，并逆转了 H/R 或 erastin 处理对相关蛋白表达的影响。这些结果证明了 ENPP2 过表达通过抑制氧化应激和铁死亡来预防 H/R 损伤发展的巨大治疗效果。