表皮儿茶素没食子酸酯（EGCG）具有抗肿瘤多药耐药、抗氧化和抗炎等多种生物学效应。铁死亡是缺血性心脏损伤的主要驱动因素，因此抑制铁死亡可能是心血管疾病的有效治疗策略。然而，EGCG对缺血性心肌组织中铁死亡的作用及其潜在机制仍不清楚。本研究旨在研究EGCG对体外和体内心肌缺血诱导的铁死亡的影响和潜在机制。体外和体内分别建立了心肌细胞缺氧模型和小鼠急性心肌梗死（AMI）模型。转染miR-450b-5p和ACSL4沉默或过表达质粒，预处理或不预处理EGCG。细胞存活率通过CCK-8实验确定。用血红素和嗜铬酸染色法（HE）和透射电子显微镜（TEM）评估形态学变化。用TTC染色观察梗死面积，用超声心动图评估心功能。采用流式细胞术评估反应性氧化物（ROS）的存在。使用试剂盒检测心肌肌钙蛋白I（cTn I）、谷胱甘肽（GSH）、丙二醛（MDA）、二价铁离子（Fe2+）和超氧化物歧化酶（SOD）的含量。通过荧光素酶活性实验评估miR-450b-5p与ACSL4的结合能力。相关基因和蛋白的表达分别通过RT-qPCR和免疫印迹法测量。EGCG减轻了AMI引起的铁死亡，并改善了心肌缺血损伤，与减少铁沉积和cTn I、抑制脂质过氧化、降低TFR1和ACSL4、上调SLC7A11、FTH1和GPX4有关。同时，EGCG预处理增加了缺血性心肌组织中miR-450b-5p的表达。进一步研究发现，miR-450b-5p的沉默部分削弱了EGCG在缺氧HL-1细胞中产生的保护效应，而与miR-450b-5p模拟物结合能增强EGCG在缺血性心肌组织中的效力。双荧光素酶试验表明miR-450b-5p与ACSL4结合。此外，ACSL4的沉默与EGCG的心脏保护作用具有协同增强效果。更重要的是，EGCG处理通过miR-450b-5p/ACSL4轴调节铁死亡相关蛋白的表达。总之，本研究明确表明EGCG通过作用于铁死亡减轻心肌缺血损伤。我们的研究首次揭示了miR-450b-5p/ACSL4轴在AMI中的作用。此外，本研究还阐明了EGCG抑制铁死亡的分子机制在很大程度上依赖于miR-450b-5p/ACSL4轴，提示EGCG可能作为一种新型抗铁死亡药物在AMI中发挥治疗作用。

Epigallocatechin gallate (EGCG) has multiple biological effects such as anti-tumor multiple drug resistance, antioxidation and anti-inflammatory properties. Ferroptosis is the main driving factor of ischemic heart injury, thus inhibiting ferroptosis may prove to be an effective treatment strategy for cardiovascular diseases. However, the role of EGCG on ferroptosis in ischemic myocardium and underlying mechanisms remain uncertain.This study was aimed to investigate the effects and potential mechanisms of EGCG on myocardial ischemic-induced ferroptosis both in vitro and in vivo.Cardiomyocyte hypoxia model and mouse acute myocardial infarction (AMI) model were established in vitro and in vivo. MiR-450b-5p and ACSL4 silencing or overexpression plasmids were transfected, with or without EGCG pretreatment. Cell viability was determined by the CCK-8 assay. Hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM) were used to evaluate the morphologic alterations. TTC staining was used to observe the infarction area, and echocardiography was adopted to appraise the heart function. Using flow cytometry, the presence of reactive oxygen species (ROS) was assessed. The content of cardiac troponin I (cTn I), glutathione (GSH), malondialdehyde (MDA), divalent iron ions (Fe2+) and superoxide dismutase (SOD) were detected using reagent kits. A luciferase activity assay was performed to assess the binding ability of miR-450b-5p to ACSL4. Expressions of related genes and proteins were measured by RT-qPCR and western blotting respectively.EGCG attenuated AMI-induced ferroptosis and improved myocardial ischemia injury, which was associated with reducing iron deposition and cTn I, inhibition of lipid peroxidation, decreasing TFR1 and ACSL4, and upregulating SLC7A11, FTH1 and GPX4. Meanwhile, EGCG pretreatment increased miR-450b-5p expression in ischemic myocardium. Further researches discovered that knockdown of miR-450b-5p partially compromised EGCG-generated protective effect in hypoxia HL-1 cells, while combination with miR-450b-5p mimic could strengthen the potency of EGCG on ischemic myocardium. The dual-luciferase test demonstrated that miR-450b-5p has binding to ACSL4. Furthermore, silencing of ACSL4 synergistically increased the cardioprotective effect of EGCG. More significantly, EGCG treatment regulated the ferroptosis-related proteins expression via miR-450b-5p/ACSL4 axis.In summary, the present study evidently demonstrated that EGCG attenuates myocardial ischemia injury by targeting ferroptosis. Our work revealed the role of miR-450b-5p/ACSL4 axis in AMI for the first time. Further, it also elucidated the molecular mechanisms of EGCG on inhibiting ferroptosis greatly depend on the miR-450b-5p/ACSL4 axis, suggesting that EGCG may act as a novel anti-ferroptosis agent and exert a therapeutic role in AMI.Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.