Astragalosides Ⅳ Inhibits Ferroptosis Of Cardiomyocytes Induced By Doxorubicin Through Nrf2/GPX4 Pathway

Objective:Advances in drug research and development technology have improved survival in cancer patients,however,a number of chemotherapeutic agents cause adverse drug reactions,as a threat to life,the cardiovascular toxicity is especially common in adverse reactions.Doxorubicin（DOX）is the most widely used anti-tumor drug in clinical practice,however,because of its fatal cardiotoxic effects and cardiac suppression,the use of DOX in clinical practice has been limited.Therefore,to reduce the cardiotoxicity of DOX becomes the focus of clinical research.Research studies have found that ferroptosis plays a significance role in doxorubicin-associated myocaridial injury and other cardiovascular diseases.Astragaloside IV（ASIV）has a number of potencies like antioxidant,anti-inflammatory,immune regulation,ischemic protection and so on.In this study,DOX myocardial injury model was established at cytological level,the effect of ASIV on ferroptosis in doxorbucin-induced myocardial injury was determined,and its molecular mechanism was preliminarily expounded.Methods:（1）The H9c2 cardiomyocytes were interposed with different concentrations of doxorubicin,Astragaloside IV,ferrostatin-1（Fer-1）,and myocardial viability was gauged by cell counting kit 8（CCK8）,to determine the optimum condition of models.（2）The experiment was split into Control group（blank）,ASIV group（100μmol/L）,DOX group（2μmol/L）,and D+A100 group（DOX 2μmol/L+ASIV100μmol/L）.Cell morphology was observed by the optical microscope and the secretory volume of type B natriuretic peptide（BNP）and lactate dehydrogenase（LDH）were calculated.Lipid peroxidation sensor（C11-BODIPY lipid probes）to evaluate reactive oxygen species（ROS）level;Mitochondrial membrane potential detection with JC-1 probes（JC-1）to judge function of mitochondrial,ultrastructure of mitochondria observed by transmission electron microscopy.The contents of total iron,ferrous ion(ferrous iron,Fe²⁺)and malondialdehyde（MDA）were detected by ELISA.The transcription factor expression of the nuclear factor-erythroid 2 related factor2（Nrf2）,glutathione peroxidase 4（GPX4）,was mensurated by real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）.The level of the translation of ferroptosis-related proteins Nrf2,GPX4,ferritin heavy chain 1（FTH1）,Ferroportin1（FPN1）and 4-hydroxylnannelaldehyde（4HNE）were measured by western blot（WB）.（3）Groups were divided into Control,Fer-1（Fer-1 10μmol/L）,DOX（2μmol/L）,D+Fer-1（DOX2μmol/L+Fer-1 10μmol/L）and D+F+A（dox2μmol/L+Fer-1 10μmol/L+ASIV100μmol/L）.JC-1 were used to detect mitochondrial function changes,the contents of total iron,ferrous ion(Fe²⁺)were detected by colorimetry.The levels of Nrf2,GPX4,FTH1,FPN1,and 4HNE were measured by WB.（4）Different concentratinons of adenovirus containing Nrf2 sh RNA were transinfected in H9c2 cells.Then the Nrf2-negative control sh RNA（NC）and Nrf2sh RNA（sh RNA）were received Control,ASIV,DOX and D+A100 intervention.And the levels of genes and protein of Nrf2 and GPX4 were assessed by RT-q PCR and WB.Results:（1）DOX decreases the relative activity of H9c2cardiomyocytes（P<0.05）,The optimal dose was 2μmol/L and the intervention time was 24h.There were no major differences in different concentrations of ASIV and Fer-1 had on the relative cellular activity of H9c2 cells（P>0.05）.The reduced cardiomyocyte activity caused by DOX could be inhibited by ASIV and Fer-1（P<0.05）.The 100μmol/L ASIV and10μmol/L Fer-1 were enrolled as the optimal intervention concentration.（2）Cell damage occurred in H9c2 cells in the DOX group,and the LDH and BNP levels were markedly elevated when compared to the Control group（P<0.05）.High levels of LDH,BNP resulting from DOX can be inhibited by ASIV（P<0.05）.（3）DOX significantly increased the level of lipid peroxidation of ROS,MDA,and 4HNE（P<0.05）,compared with the DOX group,ASIV significantly decreased the lipid peroxidation indexes of ROS,MDA,and 4HNE（P<0.05）.（4）DOX caused robust up-regulation of the total iron and Fe²⁺levels,and reduced the levels of FTH1 and FPN1which reflecting iron storage and export capacity（P<0.05）.ASIV lowered the total iron and Fe²⁺levels and increased the levels of FTH1 and FPN1 caused by DOX（P<0.05）.（5）DOX lowered the mitochondrial membrane potential markedly（P<0.05）,and the ASIV reduced the mitochondrial membrane potential damage caused by the DOX（P<0.05）.DOX caused mitochondrial damage,while ASIV attenuates mitochondrial structural damage caused by DOX.（6）After lentiviral infection of H9c2cells with NC and Nrf2 sh RNA（sh RNA-1,sh RNA-2 and sh RNA-3）,the level of Nrf2 genes and proteins in Nrf2 sh RNA group was lowered when contrasted with NC group（P<0.05）.（7）Contrasted with NC group,the levels of Nrf2,GPX4 proteins and genes in the low-expression model of Nrf2 have decreased markedly（P<0.05）.Compared with the sh RNA+Control group,Nrf2,GPX4 protein and m RNA levels were no major differences from the sh RNA+DOX and sh RNA+D+A groups（P>0.05）.Conclusion:（1）ASIV inhibits DOX-induced cardiomyocyte injury.（2）DOX promotes ferroptosis in cardiomyocytes via the Nrf2/GPX4signaling pathway.（3）ASIV inhibited DOX-associated ferroptosis via the Nrf2/GPX4 signaling pathway,and attenuates cardiomyocyte injury caused by DOX.