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

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.