Astragaloside Ⅳ Alleviates Cardiac Ferroptosis Caused By Doxorubicin In Rats By Activating Nrf2/GPX4 Pathway

Objective:Anthracycline drugs such as doxorubicin（DOX）have greatly improved the survival rate of cancer patients,but the clinical application is greatly limited due to severe myocardial toxicity.How to reduce the myocardial injury caused by doxorubicin has become an urgent problem to be solved.This study investigated the mechanism of astragaloside Ⅳ（ASⅣ）on ferroptosis in adriamycin induced myocardial injury in rats,so as to provide a new therapeutic strategy for the treatment of clinical myocardial injury.Methods:Thirty SD rats were injected intraperitoneally with DOX to establish a rat model of myocardial injury,and were treated with ASⅣ by gavage.Left ventricular ejection fraction（LVEF）was detected by cardiac color Doppler ultrasound,and the levels of B-type natriuretic peptide（BNP）and cardiac troponin Ⅰ（cTnⅠ）in tissues were detected by enzyme-linked immunosorbent assay（ELISA）.Hematoxylin and eosin（HE）staining,Masson staining and wheat germ agglutinin（WGA）staining were used to observe the morphological changes of the heart.The contents of malondialdehyde（MDA）,Fe²⁺and reduced glutathione（GSH）/oxidized glutathione（GSSG）were measured by colorimetry.The organelle structure in cardiomyocytes was observed by transmission electron microscope,and the difference of gene expression was detected by whole genome sequencing and enrichment analysis.Nuclear factor E2related factor 2（Nrf2）in cardiomyocytes was detected by Western blot（WB）and real time quantitative polymerase chain reaction（RT-qPCR）Expression of iron death proteins such as glutathione peroxidase 4（GPX4）.Results:1.Compared with the control group and sham operation group,the rats in DOX group had symptoms such as hair loss,subcutaneous bleeding and bone deformity,and their body weight decreased significantly compared with the control group.2.Compared with the control group and sham operation group,the heart weight of DOX group was significantly reduced,LVEF decreased,BNP and cTnⅠ increased;After ASⅣ intervention,compared with the model group,LVEF increased and BNP and cTnⅠ decreased（P<0.05）.3.HE staining showed that the muscle fibers in the control group were arranged orderly and the cytoplasm was full and uniform,while the myocardial fibers in the DOX group were arranged disorderly and the cytoplasm was loose.Masson staining showed that the degree of myocardial fibrosis in DOX group was significantly higher than that in the control group.The results of WGA immunofluorescence showed that the myocardial cross-sectional area of DOX rats increased.After ASⅣ treatment,myocardial fibrosis and cell area of rats were significantly improved（P<0.05）.4.Compared with the control group:the myocardial mitochondria of DOX group were swollen,ruptured,vacuolized and the mitochondrial cristae disappeared under electron microscope.Compared with the control group,the levels of Fe²⁺and MDA in DOX group increased and GSH/GSSG decreased.After ASⅣ intervention,the levels of Fe²⁺and MDA decreased and GSH/GSSG increased（P<0.05）.5.Whole genome sequencing showed that ASⅣ up-regulated the expression of genes such as antioxidant stress and anti lipid peroxidation.Compared with the control group,the protein and mRNA expression of Nrf2 and GPX4 in DOX group decreased significantly,and the protein expression of Nrf2 and GPX4 increased after ASⅣ intervention（P<0.05）.After administration of Nrf2 inhibitor,the expression of Nrf2 and GPX4 decreased in ASⅣ treatment group（P<0.05）.Conclusion:1.ASⅣ reverses DOX induced myocardial injury in rats.2.ASⅣ alleviates DOX mediated myocardial ferroptosis by activating Nrf2/GPX4 pathway.