Abstract:

To elucidate the mechanisms by which licochalcone A (LA) attenuates influenza A virus (IAV) H3N2–induced macrophage injury through modulation of autophagy and ferroptosis, an established IAV-infected RAW264.7 macrophage model was employed. The optimal LA concentration (15 μmol/L) was determined using a Cell Counting Kit-8 (CCK-8) assay. Cells were assigned to control, model (IAV), and model + LA (IAV + 15 μmol/L LA) groups. Mitochondrial ultrastructure was examined by transmission electron microscopy; protein expression related to ferroptosis, autophagy, and the AKT/mTOR pathway was analyzed by Western blotting analysis; intracellular ROS levels were assessed by flow cytometry; cytokine secretion was quantified by ELISA; and levels of MDA, Fe²⁺, and GSH were synchronously measured. CCK-8 assays demonstrated a marked reduction in cell proliferation in the model group, which was effectively restored following LA treatment (P < 0.001). Electron microscopy revealed pronounced mitochondrial swelling, cristae disruption, and vacuolar degeneration in IAV-infected cells, whereas LA intervention substantially alleviated these structural abnormalities. Compared with the control group, the model group exhibited significant downregulation of GPX4 and FTH1 protein expression by 55% and 58% (P < 0.001 and P = 0.0088, respectively), accompanied by a 26% increase in TP53 expression (P = 0.011); these aberrant changes were reversed by LA treatment. In addition, IAV infection resulted in significantly elevated levels of ROS, MDA, and Fe²⁺, together with a reduction in GSH content. Following LA intervention, ROS, MDA, and Fe²⁺ levels decreased by 29%, 41%, and 36%, respectively, while GSH levels increased by 42% (P < 0.001, P = 0.005, P < 0.001, and P = 0.001, respectively). The concentrations of IL-6, IL-1β, and TNF-α were markedly increased in the model group (all P < 0.001), whereas LA treatment significantly suppressed their release. Furthermore, IAV infection was associated with reduced expression of p-AKT1, p-mTOR, and the LC3-II/LC3-I ratio (P = 0.004, P < 0.001, and P = 0.0097, respectively), along with increased P62 expression (P = 0.001); these alterations were significantly reversed by LA, indicating activation of the AKT/mTOR pathway and restoration of autophagic flux. Collectively, these findings demonstrated that LA activated AKT/mTOR signaling to enhance autophagic flux while inhibiting ferroptosis, thereby alleviating IAV-induced mitochondrial damage, oxidative stress, and inflammatory responses in macrophages and ultimately exerting potent anti-influenza effects.

Key words: Influenza A virus (H3N2), Autophagy, Ferroptosis, Licochalcone A, RAW264.7 macrophages