Alox5、Alox15参与阿霉素诱导的心肌细胞铁死亡OA
Involvement of Alox5 and Alox15 in doxorubicin-induced ferroptosis of cardiomyocytes
目的 基于转录组学探究阿霉素(doxorubicin,DOX)所致心肌细胞铁死亡中脂氧合酶(lipoxygenase,LOX)的作用机制.方法 对阿霉素诱导的H9c2心肌细胞的RNA样本进行了二代测序(RNAseq),同时选取未处理H9c2细胞作为对照组.采用edgeR软件包对转录组数据进行差异表达分析,借助STRING数据库对差异基因进行蛋白互作分析(protein-protein interaction,PPI),并对差异基因Alox5、Alox15进行基因及蛋白水平验证.CCK-8法检测细胞凋亡率,用于筛选药物最佳浓度.应用Alox5抑制剂(Zileuton)、Alox15抑制剂(PD146176)对实验进行干预,将实验分为5组:Control、DOX、DOX+Fer-1、DOX+Zileuton、DOX+PD146176.流式细胞术检测各组凋亡情况.荧光显微镜及流式细胞仪FITC通道检测ROS水平.试剂盒法检测细胞内Fe2+、MDA、GSH、SOD含量.Western blot检测Alox5、Alox15、ACSL4、Ptgs2、GPX4 蛋白相对表达量.RT-qPCR 检测 Alox5、Alox15、ACSL4、Ptgs2、GPX4 mRNA相对表达量.ELISA检测培养上清液IL-6、TNF-α含量.结果 与空白对照组相比,阿霉素损伤模型组共有3 603个基因明显上调,1 378个基因明显下调.选取PPI互作关系较多的基因Alox5、Alox15做为目标基因.Western blot和RT-qPCR结果验证了阿霉素处理后,H9c2细胞内Alox5、Alox15表达水平明显上调,与RNAseq结果一致.抑制剂干预结果显示,Alox5、Alox15抑制剂可降低阿霉素引起的H9c2细胞死亡,降低阿霉素引起的H9c2细胞内ROS、Fe2+、MDA、ACSL4、Ptgs2、IL-6、TNF-α的表达升高并恢复细胞内GPX4、GSH、SOD水平.结论 阿霉素处理后的H9c2心肌细胞存在Alox5、Alox15过表达,Alox5、Alox15抑制剂可抑制阿霉素引起的心肌细胞铁死亡以及炎症反应.
Aim To investigate the role of lipoxygen-ases in doxorubicin-induced ferroptosis in cardiomyo-cytes based on transcriptomics.Methods RNA se-quencing(RNA-seq)was performed on RNA samples from doxorubicin(DOX)-treated H9c2 cardiomyo-cytes,with untreated H9c2 cells serving as the control group.Differential gene expression analysis was con-ducted using the edgeR package,and protein-protein interaction(PPI)analysis of differentially expressed genes(DEGs)was performed using the STRING data-base.Key genes Alox5 and Alox15 were further vali-dated at both mRNA and protein levels.Cell viability was assessed using the CCK-8 assay to determine the optimal drug concentration.The experiment was di-vided into five groups:Control,DOX,DOX+Fer-1(ferroptosis inhibitor),DOX+Zileuton(Alox5 inhibi-tor),and DOX+PD146176(Alox15 inhibitor).Apoptosis was quantified by flow cytometry.Reactive oxygen species(ROS)levels were measured via fluo-rescence microscopy and FITC-channel flow cytom-etry.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 as-sessed by Western blot,while their mRNA levels were evaluated via RT-qPCR.Secreted IL-6 and TNF-α were detected by ELISA.Results Compared with the control group,the DOX-induced injury model showed 3 603 significantly upregulated genes and 1 378 signifi-cantly 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 DOX-induced 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.Conclusions DOX-treated H9c2 cardiomyocytes exhibit overexpression of Alox5 and Alox15.Inhibitors of Alox5 and Alox15 can mitigate DOX-induced ferroptosis and inflammatory responses in cardiomyocytes.
沈明妹;张尚博;武凯鑫;赵慧;张志宏;章文;刘永超
北华大学医学技术学院,吉林 吉林 132013北华大学医学技术学院,吉林 吉林 132013北华大学医学技术学院,吉林 吉林 132013北华大学医学技术学院,吉林 吉林 132013北华大学药学院,吉林 吉林 132013北华大学临床医学院,吉林 吉林 132013北华大学医学技术学院,吉林 吉林 132013
医药卫生
阿霉素心脏毒性转录组学铁死亡脂质过氧化脂质代谢脂氧合酶
adriamycin-induced cardiotoxicitytran-scriptomicsferroptosislipid peroxidationlipid me-tabolismlipoxygenase
《中国药理学通报》 2026 (1)
39-48,10
吉林省科学技术厅科技发展计划项目(No 20220402072GH)
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