Screening And Verification Of Key Ferroptosis Genes In Myocardial Ischemia-Reperfusion Injury Based On Bioinformatics Analysis

Objective:Cardiovascular disease has been the leading cause of death in China for many years,with Acute Myocardial Infarction(AMI)being one of the most lethal and disabling cases.Percutaneous coronary intervention(PCI)is the mainstay of reperfusion therapy,which can greatly reduce mortality and improve the prognosis of AMI patients.The presence of myocardial ischemia/reperfusion injury(MI/RI)can significantly reduce the effectiveness of reperfusion therapy,and existing interventions for MI/RI,including pharmacological treatment,ischemic preadaptation and postadaptation,have had little success.The exact mechanism of MI/RI is not fully understood,but studies have shown that oxidative stress,apoptosis,calcium overload and ferroptosis are all involved in the pathophysiological process of MI/RI.Ferroptosis has become a hot topic in the study of MI/RI mechanisms,therefore,starting from the regulation of ferroptosis,exploring the deeper underlying mechanisms of MI/RI and finding effective therapeutic targets can provide a new theoretical basis for the prevention and treatment of MI/RI.This study focuses on screening and validating the role of ferroptosis related genes in MI/RI and their mechanisms through bioinformatic methods,and exploring potential targets for ferroptosis therapy in MI/RI.Methods:1.Two ischemia-reperfusion(I/R)related datasets,GSE61592 and GSE83472,were screened for differentially expressed genes(DEGs)by bioinformatics methods,and ferroptosis-related differentially expressed genes(FRDEGs)were obtained by intersecting the DEGs with the ferroptosis gene set.The resulting genes were used for Gene Ontology(GO)enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and protein-protein interaction(PPI)network analysis to screen for ferroptosis-related hub genes and core protein networks in I/R.Validation of the binding ability of the herbal components currently used in the H/R model study with the core FRDEGs obtained from the above screening using molecular docking techniques.2.A model of hypoxia-reoxygenation injury in H9c2 cells was established to simulate myocardial ischemia-reperfusion injury.A hypoxia time gradient and a reoxygenation gradient were set to observe the expression levels of HO-1 protein under different time conditions and to determine the optimal time point for modelling.After transfection with Hmox1-si RNA for 24 h,the expression levels of HO-1,Gpx4,Fth1 and Slc7a11 were detected by Western Blot,and intracellular MDA,ROS and iron ion concentration levels were measured after treatment with dosing and hypoxia-reoxygenation models.Results:1.By bioinformatics analysis,1786 and 58 DEGs were screened in the I/R dataset GSE61592 and GSE83472,respectively,yielding 55 FRDEGs.GO and KEGG enrichment analysis showed that the 55 FRDEGs were mainly enriched in Fox O,m TOR,HIF-1 and other signaling pathways.Ten core genes contained in the key networks in I/R were screened by PPI network analysis,including Egfr,Hmox1,Acsl1,Nras,Timp1,Tgfbr1,Idh2,Ptpn6,Cs and Gpx4.Based on the results of MCC analysis,we selected Hmox1 as the main studied gene.Molecular docking results shown that Hmox1 can bind freely to each drug component.2.The results of the hypoxia-reoxygenation time gradient experiment selected hypoxia 6h reoxygenation 4h as the modeling time point for H/R in H9c2 cells.Compared with the control group,the expression of HO-1 was upregulated and the expression level of Gpx4 was downregulated in H/R group,the expression level of HO-1 was significantly downregulated and Gpx4 expression was significantly increased in the H/R+Hmox1-si RNA group,the cellular concentrations of MDA,ROS and iron ion were reduced in the H/R+Hmox1-si RNA group compared to the control group and H/R group.Conclusion:In this study,the key genes of iron death in I/R were screened in the core network by bioinformatics analysis,and the H/R model was established by H9c2 cells,and iron death was found to be involved in the acute MI/RI process.HO-1 was highly expressed in H/R,and the iron death index improved after inhibition of its expression,suggesting that the high expression of HO-1 in MI/RI may have promoted the development of iron death.Hmox1 is a key ferroptosis gene in MIRI.