A型流感病毒通过激活HIF-1α/iNOS/VEGF信号通路诱导肺上皮细胞铁死亡的机制

Mechanism of influenza A virus inducing ferroptosis in lung epithelial cells by activating the HIF-1α/iNOS/VEGF signaling pathway

摘要:

【背景】已发现A型流感病毒（influenza A virus，IAV）可激活多种程序性细胞死亡途径，这些途径在宿主细胞防御系统中起着重要作用。铁死亡是一种新型的非凋亡细胞死亡，主要由铁依赖性脂质过氧化介导。【目的】探讨HIF-1α/iNOS/VEGF信号通路在IAV感染诱导的细胞铁死亡中的作用机制。【方法】使用IAV感染小鼠肺上皮细胞（MLE-12）构建细胞损伤模型后检测细胞病毒滴度和炎性因子分泌；使用荧光探针法和比色法检测细胞活性氧（reactive oxygen species，ROS）、超氧化物歧化酶（superoxide dismutase，SOD）、总铁离子和亚铁离子；透射电镜观察细胞超微结构；检测细胞铁死亡标志物mRNA和蛋白表达；生物信息学预测流感病毒诱导铁死亡潜在作用机制；激光共聚焦观察IAV对细胞缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）表达影响，并检测其信号通路相关元件mRNA和蛋白表达；构建HIF-1α敲低模型，探讨HIF-1α/iNOS/VEGF信号通路在IAV诱导细胞铁死亡中的作用。【结果】铁死亡抑制剂能降低IAV感染细胞的病毒载量和炎性因子的分泌，并能抑制细胞ROS、总铁离子和亚铁离子含量，促进细胞SOD活性，修复细胞线粒体损伤，逆转铁死亡标志物mRNA和蛋白表达；生物信息学预测发现HIF-1α/iNOS/VEGF信号通路在IAV诱导的铁死亡中具有重要的相关性；试验验证IAV感染能促进细胞HIF-1α的激活和易位入核，并激活HIF-1α、血管内皮生长因子（vascular endothelial growth factor，VEGF）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的mRNA和蛋白表达。【结论】IAV感染可以诱导细胞发生铁死亡，其作用机制可能是通过激活HIF-1α/iNOS/VEGF信号通路发挥作用。

Abstract:

[Background] Influenza A virus (IAV) can activate multiple programmed cell death pathways, which play important roles in the host defense system. Ferroptosis is a novel non-apoptotic type of cell death and mainly mediated by iron-dependent lipid peroxidation. [Objective] To explore the mechanism of IAV-induced ferroptosis via the HIF-1α/iNOS/VEGF signaling pathway. [Methods] Mouse lung epithelial (MLE-12) cells were infected with IAV to establish a cell injury model, and the virus titer and inflammatory cytokine secretion were examined. The fluorescence probe and colorimetric methods were used to measure the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), total iron ions, and ferrous ions. Transmission electron microscopy was employed to observe the cell ultrastructure. The mRNA and protein levels of cell ferroptosis markers were determined. Bioinformatics tools were used to predict the potential mechanism of IAV-induced ferroptosis. Laser confocal microscopy was employed to observe the effect of IAV on the expression of hypoxia-inducible factor-1α (HIF-1α) in cells, and the mRNA and protein levels of the proteins involved in the HIF-1α/iNOS/VEGF signaling pathway were determined. A HIF-1α knockdown model was established to explore the role of the HIF-1α/iNOS/VEGF signaling pathway in IAV-induced cell ferroptosis. [Results] The ferroptosis inhibitor reduced the virus titer and inflammatory cytokine secretion of IAV-infected cells. Moreover, in lowered the levels of ROS, total iron ions, and ferrous ion, increased the SOD activity, repaired mitochondrial damage, and reversed the mRNA and protein levels of ferroptosis markers. Bioinformatics predicted that the HIF-1α/iNOS/VEGF signaling pathway was closely associated with IAV-induced ferroptosis. IAV infection promoted the activation and nuclear translocation of HIF-1α in cells and up-regulated the mRNA and protein levels of HIF-1α, vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS). [Conclusion] IAV infection can induce cell ferroptosis by activating the HIF-1α/iNOS/VEGF signaling pathway.