Exploring The Mechanisms And Therapeutic Compounds Of Ferroptosis In Septic Myocardial Injury Based On Comprehensive Transcriptomic Analysis

Objective:The aim of this study is to explore the molecular biology mechanisms of ferroptosis in septic myocardial injury（SMI）and its therapeutic compounds based on the human heart transcriptome and experimental models.which is expected to provide preliminary evidence based on human specimens to clarify the pathological role of ferroptosis in SMI,and bringing new insights into the precise diagnostic and therapeutic targets for SMI.Methods:The study was conducted in a sequential pattern according to the following methods:1.Firstly,indepth bioinformatics analysis based on the human septic heart transc riptome was conducted.GSEA was performed to explore the variation of ferroptosis-r elated genes（FRGs）in the human septic heart transcriptome.The potential biological functions and functional pathways of FRGs were further explored through Gene Ont ology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analyses,and t he potential crosstalk between them was analyzed using the spearman correlation met hod.After constructing the protein-protein interaction（PPI）network,the key modules and hub genes among the differentially expressed FRGs（DEFRGs）were identified b y K-means clustering algorithm and MCC algorithm respectively.2.Then,a lipopolysaccharide（LPS）-induced myocardial injury mouse model wa s constructed to simulate SMI in vivo.Echocardiography,blood biochemistry,myocar dial biochemistry,immunohistochemistry,Dihydroethidium（DHE）fluorescent probe,transmission electron microscopy,Quantitative Real-time PCR（q RT-PCR）and West ern blotting were used to verify the multiple ferroptosis characteristics and expression variations of hub genes in the pathological state of SMI,as well as the identification of key FRGs.3.Subsequently,the diagnostic ability of key FRGs for the septic heart was furth er evaluated in human heart and blood sample cohorts by Receiver Operating Charact eristic（ROC）curve analysis,as well as the correlation with sepsis condition and prog nosis.The expression and distribution of key FRGs in human heart tissue was determi ned by analysis of human heart single cell transcriptome data.The proportion of imm une cells in the septic heart was estimated by the CIBERSORT algorithm and the key FRGs in the septic heart were analysed for immune infiltration correlation.4.Finally,potential compounds that may regulate ferroptosis in SMI were predic ted based on compound-target correlations of key FRGs in the DSig DB database,and the binding of target compound to the corresponding proteins of key FRGs was simul ated and predicted by molecular docking technique.In the LPS-induced H9c2 cardio myoblasts injury model,the protective effects and ferroptosis regulation of the target compound on cardiomyoblasts with simulated SMI condition were investigated by cel l viability,cell biochemistry,western blot,q RT-PCR,DCFH-DA and C¹¹BODIPY^581/591fluorescent probe staining,transmission electron microscopy and labile iron pool de tection.The effects of the target compound on the protein levels of key FRGs with hi gh predictive binding capacity were further investigated by western blot.Results:1.The indepth bioinformatics analysis based on the human sepsis heart transcriptome revealed that the ferroptosis related gene set was significantly altered in the septic heart transcriptome（FDR<0.05,NES=1.489）.A total of 71 DEFRGs（FDR<0.05,|log2Fold Change|≥0.25）were identified,and analyzed to be mainly participated in biological processes such as regulation of cellular iron homeostasis and responses to nutrients,oxygen and chemical stress（P<0.05）;mainly localized to cellular components such as apical and basolateral membranes,autophagosomes and autophagic lysosomes（P<0.05）;and their molecular functions mainly involved transcription factor-DNA binding,ubiquitin-protein ligase activity,transcription coregulator and coactivator,heat shock protein binding,and acyltransferase and oxidoreductase activity（P<0.05）.They were mainly enriched in ferroptosis,reactive oxygen species,autophagy,and cancer-related signaling pathways（P<0.05）.The spearman correlation analysis revealed widespread crosstalks between gene expression,biological function,pathway,and protein interactions in DEFRGs（P<0.05）.Finally,two key modules and 10 hub genes（STAT3,NOX4,TP53,HIF1A,NFE2L2,MAPK3,RELA,PTEN,EGFR,PPARA）were screened from the PPI network constructed by DEFRGs2.The LPS-induced myocardial injury mouse model was constructed to simulate the pathological state of SMI.Echocardiography showed a significant decrease in left ventricular ejection fraction and fractional shortening,and circulating inflammatory c ytokines（IL-6,IL-1β,TNF-α）and myocardial injury markers（LDH,CK-MB,c Tn T）were significantly increased（P<0.05）.Meanwhile,the myocardium of mice in exper imental SMI model was also found to exhibit multiple biomarker,morphological and biochemical features of ferroptosis,including including significantly up-regulated PT GS2 expression levels,increased iron content,elevated ROS levels,significantly incr eased 4-HNE and MDA levels,extensive mitochondrial damage characterized by atro phy and matrix loss,and significant down-regulation（P<0.05）of the main physiolog ical ferroptosis defense systems-SOD,GSH-Px,GSH/GSSG.Fer-1 pretreatment sig nificantly reversed the ferroptosis characteristics induced by LPS treatment（P<0.05）,but did not significantly reduce the iron accumulation in the myocardium（P>0.05）.Echocardiography showed that Fer-1 pretreatment significantly improved mouse card iac function,and correspondingly,circulating inflammatory cytokines and myocardial injury markers were significantly reduced（P<0.05）.HIF1A,MAPK3,NOX4,PPA RA,PTEN,RELA,STAT3 and TP53 were identified as key FRGs in SMI by q RT-PC R（P<0.05）.3.The ROC analysis determined that key FRGs have excellent diagnostic capabi lities for the septic heart,the expression levels of several key FRGs correlated signific antly with the prognosis of septic patients（P<0.05）.All key FRGs were expressed to varying degrees in human fetal and adult hearts,and they are also expressed to varyin g degrees in various immune cells such as neutrophils and macrophages.Additionally,the spearman correlation analysis determined several significant correlations between key FRGs expression and immune cell infiltration proportion in septic hearts（P<0.05）.4.Resveratrol was predicted to be a potential ferroptosis regulatory compound in SMI based on the key FRGs identified（P<0.05,combined score=2975802）.Furthe r in vitro results showed that resveratrol alleviated LPS-induced iron overload in H9c2 cardiomyoblasts by down-regulating TFR1 expression and up-regulating FPN1 expr ession,while reducing LPS-induced increase in intracellular labile iron pool levels by decreasing FTL expression and increasing FTH expression（P<0.05）.In addition,re sveratrol inhibited LPS-induced ferroptosis in H9c2 cardiomyoblasts by attenuating d amage to the physiological ferroptosis defence system and reducing ROS and lipid pe roxidation levels（P<0.05）.Molecular docking results showed that the protein struct ures of RELA（-CDOCKER interaction energy=33.5562）,PPARA（-CDOCKER inte raction energy=32.3970）and NOX4（-CDOCKER interaction energy=30.7867）in key FRGs were more strongly bound to resveratrol.（P<0.05）,while additional resve ratrol pretreatment significantly inhibited LPS-induced variations in NOX4 and PPAR A expression（P<0.05）,but no significant variations in RELA protein levels were fo und after LPS and resveratrol treatment（P>0.05）Conclusion:Significant dysregulation of FRGs was observed in the human septic heart transcriptome,with extensive gene expression correlations,biological functions,and signaling pathway interactions among DEFRGs.The SMI model in mice exhibited features of ferroptosis in the myocardium,and inhibition of ferroptosis improved cardiac function in the SMI model mice.HIF1A,MAPK3,NOX4,PPARA,PTEN,RELA,STAT3,and TP53 were identified as key FRGs in SMI,and these key FRGs demonstrated potential clinical relevance in SMI.Res inhibited ferroptosis and exerted protective effects on cardiomyocytes in an in vitro experimental SMI model,while NOX4 downregulation and PPARA upregulation mediated the protective effects of Res.Taken altogether,this study provides experimental and theoretical basis for further exploring the role of ferroptosis in SMI.