Resveratrol Alleviated 5-FU-Induced Cardiotoxicity By Attenuating GPX4-Dependent Ferroptosis

5-Fluorouracil（5-FU）,a commonly clinical chemotherapeutic drug,was used for head and neck cancer,breast cancer and other cancers.However,long-term administration of 5-FU will lead to cardiotoxicity.Cardiotoxicity caused by 5-FU includes arrhythmia,heart failure and even cardiac arrest.The pathogenesis of5-FU-induced cardiotoxicity mainly include oxidative stress,inflammation,autophagy and apoptosis.Ferroptosis is a type of cell death that depends on iron and reactive oxygen species（ROS）.It is characterized by mitochondrial membrane shrinkage and cristae rupture.Ferroptosis is involved in the occurrence and development of cancer,acute organ failure and central diseases.However,the role of ferroptosis in chemotherapeutic-induced cardiotoxicity remains to be studied.The cardiotoxicity caused by chemotherapy drugs could be alleviated by reducing the drug dose clinically.However,the efficiency of drug will be reduced.It is necessary to find safe and effective cardioprotective agents.Resveratrol（Res）,a natural substance,has a polyphenol structure and exists in grapes,knotweed and peanuts.Res has pharmacological effects including anti-inflammatory,anti-tumor and antibacterial effects.In addition,Res has protective effects on cardiac and endothelial function.However,the role of Res in 5-FU-induced cardiotoxicity remains to be studied.The aim of the study was to investigate the protective effects of Res on 5-FU-induced cardiotoxicity,and to further explore whether the protective effect was related to GPX4-dependent ferroptosis.Animal experiments:8-week-old male C57BL6/J mice were injected with 30mg/kg/d 5-FU by intraperitoneal injection for 7 days to establish a cardiotoxicity model.The model mice were randomly divided into 5 groups:（1）Model（5-FU）group:intragastric administration normal saline solution,（2）Res low-dose group:intragastric administration of 1 mg/kg/d Res solution,（3）Res medium-dose group:intragastric administration of 2 mg/kg/d Res solution,（4）Res high-dose group:intragastric administration of 4 mg/kg/d Res solution,（5）Ferroptosis inhibitor（Fer-1）group:intraperitoneal injection of 2.5 mg/kg/d Fer-1 solution.The rest of the mice were used as Control group:the normal saline solution was administered by intragastric administration.The administration period was 3 weeks.Ejection fraction（EF）,fractional shortening（FS）,cardiac output（CO）,left ventricular end-diastolic diameter（LVID）and left ventricular end-systolic diameter（LVAW）were used to monitor the left ventricle of the heart.After the left ventricular function indexes were recovered,the mice were anesthetized and sacrificed.The protective effects of Res on 5-FU-induced cardiotoxicity were evaluated by body weight,cardiac weight index,serum myocardial enzyme,myocardial morphology and myocardial fiber injury.Cardiac lipid deposition,oxidative stress level and mitochondrial structure were used to explore whether ferroptosis was involved in the cardioprotective effect of Res.Immunohistochemistry and Western-Blot assay were used to detect the expression of protein with GPX4-dependent ferroptosis.Cell experiments:H9c2 cells were used in cell experiments and divided into six groups.Control group:treated with medium for 48 h,Model（5-FU）group:treated with5μM 5-FU solution for 48 h,the low,medium,high dose of Res group:treated with12.5μM,25μM,50μM Res solution for 12 h before using 5μM 5-FU solution for 48 h,Ferroptosis inhibitor（Fer-1）group was treated with 20μM Fer-1 solution for 12 h,and then treated with 5μM 5-FU solution 48 h.MTT assay was used to investigate the effect of Res and 5-FU on the proliferation activity of H9c2 cells.DCFH-DA,Calcein-AM and TMRE probes were used to observe the levels of reactive oxygen species（ROS）,iron and mitochondrial membrane potential in H9c2 cells.Protein levels associated with GPX4 in H9c2 cells were assessed by Western-Blot.The results of in vivo experiments showed that 1)Res could alleviated5-FU-induced cardiotoxicity.Specifically,increased cardiac weight index,restored the function of the left ventricle,normalized the myocardial enzyme levels and cardiomyocyte morphology,decreased cardiomyocyte vacuolization.In addition,the expression of fibrotic proteins Col-Ⅰand Col-Ⅲwere down-regulated,thereby myocardial fibrosis was reduced.2)Res was attenuated 5-FU-induced cardiotoxicity by inhibiting GPX4-dependent ferroptosis.Specifically,reduced oxidative stress levels,reduced mitochondrial membrane rupture.The results of Western-Blot assay showed that Res regulates the GPX4-dependent ferroptosis,including up-regulated the expression of antioxidant proteins GPX4,NQO1,Nrf2 and iron storage protein FTH1,down-regulated p53 related to lipid peroxidation and Tf R related to iron transport.The results of Fer-1 group were basically consistent with the Res group.The results of in vitro experiments showed that Res enhanced the proliferative activity of H9c2 cells,decreased the levels of intracellular ROS and Fe²⁺and restored mitochondrial function.Res regulated the GPX4 dependent ferroptosis.Specifically,the5-FU-induced decrease of GPX4,FTH1,Nrf2 and NQO1 protein expression and increase of Tf R and p53 protein expression were reversed.In summary,Res alleviated 5-FU-induced cardiotoxicity by increasing antioxidant levels,increasing iron storage and reducing the expression of iron transport related proteins,thereby inhibiting the GPX4-dependent ferroptosis.Therefore,the study provided theoretical support for the protective effects of Res against 5-FU-induced cardiotoxicity clinically.