Involvement of Alox5 and Alox15 in doxorubicin-induced ferroptosis of cardiomyocytes

AimTo investigate the role of lipoxygenases in doxorubicin-induced ferroptosis in cardiomyocytes based on transcriptomics.MethodsRNA sequencing (RNA-seq) was performed on RNA samples from doxorubicin (DOX)-treated H9c2 cardiomyocytes, with untreated H9c2 cells serving as the control group. Differential gene expression analysis was conducted using the edgeR package, and protein-protein interaction (PPI) analysis of differentially expressed genes (DEGs) was performed using the STRING database. Key genes Alox5 and Alox15 were further validated at both mRNA and protein levels. Cell viability was assessed using the CCK-8 assay to determine the optimal drug concentration. The experiment was divided into five groups: Control, DOX, DOX + Fer-1 (ferroptosis inhibitor), DOX + Zileuton (Alox5 inhibitor), and DOX + PD146176 (Alox15 inhibitor). Apoptosis was quantified by flow cytometry. Reactive oxygen species (ROS) levels were measured via fluorescence microscopy and FITC-channel flow cytometry. Intracellular Fe2+, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were analyzed using assay kits. The protein expression of Alox5, Alox15, ACSL4, Ptgs2, and GPX4 was assessed by Western blot, while their mRNA levels were evaluated via RT-qPCR. Secreted IL-6 and TNF-α were detected by ELISA.ResultsCompared with the control group, the DOX-induced injury model showed 3 603 significantly upregulated genes and 1 378 significantly downregulated genes. Alox5 and Alox15, which exhibited extensive PPI interactions, were selected as target genes. Western blot and RT-qPCR confirmed that DOX treatment significantly upregulated Alox5 and Alox15 expression in H9c2 cells, consistent with RNA-seq results. Inhibitor intervention demonstrated that both Alox5 and Alox15 inhibitors reduced DOXinduced H9c2 cell death, decreased ROS, Fe2+ and MDA levels, suppressed the upregulation of ACSL4, Ptgs2, IL-6, and TNF-α, and restored GPX4, GSH, and SOD levels.ConclusionsDOX-treated H9c2 cardiomyocytes exhibit overexpression of Alox5 and Alox15. Inhibitors of Alox5 and Alox15 can mitigate DOX-induced ferroptosis and inflammatory responses in cardiomyocytes.