Andrographolide Suppresses Influenza A Virus-Induced Pyroptosis via PI3K/AKT-Mediated Caspase-3/GSDME Inactivation

Background/Objectives: Influenza A virus (IAV) infection triggers robust inflammation and acute lung injury. Andrographolide, a primary active compound from Andrographis paniculata, can mitigate IAV-induced inflammation; however, its precise mechanisms remain poorly elucidated. This study aimed to define its host-directed protective effects and molecular mechanisms. Methods: We used a lethal IAV (H1N1, PR8) model in BALB/c mice and infected A549 cells. Survival, lung pathology, cytokines, and viral titers were measured. Lung RNA sequencing identified dysregulated signaling pathways. PI3K/AKT and pyroptosis pro-teins were analyzed by Western blot. The PI3K/AKT axis was functionally validated with the AKT inhibitor in vivo and AKT1 siRNA in vitro. Results: Andrographolide improved survival, attenuated body weight loss, and reduced lung pathology and inflammatory cytokine levels in IAV-infected mice, without exhibiting direct antiviral activity. Consistent with the in vivo findings, andrographolide enhanced cell viability and suppressed cytokine secretion in infected cells. RNA sequencing revealed marked upregulation of the PI3K/AKT signaling pathway in the lungs of treated mice, as confirmed by increased PI3K and AKT phosphorylation. Furthermore, andrographolide downregulated the expression of key pyroptosis-executing proteins, including cleaved caspase-3 and the gasdermin E (GSDME) N-terminal fragment. These protective effects were substantially abrogated by an AKT inhibitor and AKT1 siRNA. Conclusions: These findings reveal a novel host-directed mechanism by which andrographolide alleviates IAV-induced immunopathology by activating the PI3K/AKT pathway, thereby suppressing caspase-3/GSDME-dependent pyroptosis. Thus, this axis represents a promising target for controlling excessive inflammation in severe influenza.