ALOX5 Mediating Macrophage Oxidative Stress Promote Atherosclerosis

Objective:The study aimed to identify biomarkers related to oxidative stress and immune infiltrating cells in AS,and explore the possible involvement of biomarkers in atherosclerosis.Methods:Differential gene expression analyses based on the GSE100927 dataset in the Gene Expression Omnibus(GEO)and 389 oxidative stress(OS)genes based on gene set enrichment analysis(GSEA).We identified 74 differentially expressed genes related to oxidative stress(DEOSGs).“CIBERSORT” and “WGCNA” R Packages were used to compare the differences of immune infiltration levels between AS and control samples.The DEOSGs(N=74)were intersected with the key modules’ genes of WGCNA(N=972),and 27 differentially expressed immune-related oxidative stress genes(DEIOSGs)were obtained.To identify the pivotal genes,I constructed the protein-protein interaction(PPI)network using the STRING database,and Cytoscape software.Biomarkers with diagnostic value for AS were determined by ROC analysis.In addition,I verified their diagnostic value for atherosclerosis by the GSE57691 dataset.Correlation analysis and single cell analysis were used to further clarify which type of immune cells the biomarkers were mainly associated with.MMP9,ALOX5,NCF2,NCF and NCF4 were identified as diagnostic markers for AS.Correlation analysis and single-cell analysis showed that these five diagnostic genes were mainly associated with macrophages.Reverse transcription polymerase chain reaction(RT-PCR)confirmed that MMP9,ALOX5,NCF2,NCF and NCF4 genes were highly expressed in ox-LDL treated RAW264.7 macrophages.Immunohistochemistry(IHC)and western blotting confirmed the higher expression of ALOX5 in plaque tissues.ALOX5 si RNA was transfected to knockout ALOX5 in ox-LDL treated RAW264.7macrophage model.LDH was detected to reflect cell injury and reactive oxygen species(ROS)were detected to reflect oxidative stress.To further explore the mechanism of oxidative stress-related biomarkers involved in atherosclerosis,ferroptosis genes,necroptosis genes,and pyroptosis genes were intersections with DEGs of GSE100927 respectively.Results:MMP9,ALOX5,NCF2,NCF1 and NCF4 were finally identified diagnostic markers for AS.It was highly expressed in RAW264.7 macrophages induced by ox-LDL.The ALOX5 in serum of high-fat APOE-/-mice had a higher expression level than that in control group.It suggested that ALOX5 can be used as a serological biomarker in clinical atherosclerosis patients.Immunohistochemistry(IHC)and western blotting confirmed ALOX5 expression levels in arterial tissues of high-fat fed APOE-/-mice were higher than that of the control group.Single cell analysis showed that ALOX5 was mainly expressed in macrophages within plaques.By detecting LDH to reflect cell damage,ox-LDL can induce macrophages damage and reduce cell viability.In ox-LDL induced macrophages,ALOX5 expression was higher than that in normal controls.Transfection of ALOX5 si RNA could reduce the expression level of ALOX5 in RAW264.7 macrophages.The reactive oxygen species(ROS)changes in macrophages were detected to reflect oxidative stress.ALOX5 knockdown could decrease ROS levels in ox-LDL-induced macrophages.The results of intersections suggest that ALOX5 is the key molecule in regulating ferroptosis.Conclusion:MMP9,ALOX5,NCF2,NCF1 and NCF4 are diagnostic genes for AS,which are associated with oxidative stress and multiple immune infiltrating cells in plaques.ALOX5 may act as a ferroptosis regulator to mediate macrophage death and promote plaque necrotic core formation.