IRF8靶向铁死亡在急性肺损伤中的作用及机制研究

Role and mechanism of IRF8 targeting ferroptosis in the pathogenesis of acute lung injury

目的探讨干扰素调节因子8(interferon regulatory factor 8,IRF8)在急性肺损伤(acute lung injury,ALI)中的作用及其机制。方法采用实时荧光定量逆转录聚合酶链反应(RT-qPCR)和蛋白免疫印迹(WB)检测体内外ALI模型中IRF8 mRNA和蛋白的表达。体外构建稳定过表达和敲低IRF8的A549细胞系,采用RT-qPCR检测炎症相关基因mRNA的表达;WB检测凋亡相关基因的表达。分别选取12只同窝阴性对照小鼠和12只Irf8敲除品系小鼠,构建ALI模型,收集造模后的小鼠肺组织并称重;收集并检测肺泡灌洗液中总细胞数以及总蛋白含量。采用苏木精-伊红染色及免疫荧光分析肺组织炎症浸润情况;利用RT-qPCR检测Irf8敲除小鼠肺组织炎症相关基因mRNA的表达;TUNEL染色分析肺组织凋亡情况;采用WB分别在体内外ALI模型中检测铁死亡相关信号分子的蛋白表达。结果IRF8在体内体外ALI中表达上调;体外敲低IRF8加剧细胞内炎症以及细胞凋亡,反之,过表达IRF8可在体外ALI模型中发挥保护作用;敲除Irf8加重小鼠肺损伤;进一步机制探索发现IRF8通过调控铁死亡相关信号分子表达从而减轻肺损伤。结论IRF8通过靶向铁死亡在肺损伤中发挥保护作用,为肺损伤的治疗提供潜在靶点。

Objective To explore the role and mechanisms of interferon regulatory factor 8(IRF8)in the pathogenesis of acute lung injury(ALI).Methods Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and western blots(WB)were used to detect the expression of IRF8 protein and mRNA in models of lung injury both in vivo and in vitro.IRF8 stable overexpression and knockdown A549 cell lines were established in vitro,and the expression levels of inflammation-related genes at the transcriptional level were detected using RT-qPCR.WB analysis was employed to assess the expression of apoptosis-related genes.Each group of 12 wild type(WT)mice and Irf8 knockout mice was selected to establish the model of ALI.Collect lung tissues from the modeled mice and weigh them.Bronchoalveolar lavage fluid(BALF)was collected and assessed for total cell count and total protein content.Hematoxylin-eosin(HE)staining and immunofluorescence(IF)analyses were performed to evaluate lung tissue inflammation infiltration.RT-qPCR was used to detect the mRNA expression of inflammation-related genes in the lung tissue of Irf8 knockout mice.The content of apoptosis in lung tissue was detected by TUNEL staining.In vivo and in vitro,the protein expression of ferroptosis-related signaling molecules was assessed by WB.Results The expression of IRF8 is upregulated both in mRNA and protein levels in ALI.IRF8 knockdown exacerbates intracellular inflammation and cell apoptosis in vitro.Conversely,IRF8 overexpression can exert a protective effect in vitro.Irf8 knockout further exacerbates lung injury in vivo.Mechanistically,IRF8 alleviates lung injury by regulating the expression of ferroptosis-related signaling molecules.Conclusion IRF8 exerts a protective role in lung injury by targeting ferroptosis,providing a potential target for the treatment of lung injury.