摘要Background:Influenza A virus(IAV)is a negative-sense RNA virus of the Orthomyxoviridae family and is the etiological agent of a highly contagious acute respiratory disease that can lead to acute lung injury.Objective:To elucidate the molecular mechanisms of IAV infection,an integrative research approach combining gene expression profiling,multinetwork analysis,and in vivo experimental validations was employed.Methods:First,a series of network-based analyses were performed,including protein-protein interaction network construction,weighted gene co-expression network analysis,and subsequent gene set enrichment analysis,to identify the major underlying mechanisms of IAV infection.Following gene expression analysis,core targets,both direct and indirect regulators,were screened.An IAV(H1N1)strain A/PR/8/34-induced acute lung injury mouse model was constructed for in vivo validations.Batch one included two groups to evaluate findings from the multi-network analysis:Mock(n=10;5 males and 5 females)and IAV(n=10;5 males and 5 females).Batch two included three groups to assess the role of toll-like receptor 3(TLR3)in IAV infection:Mock(n=6;3 males and 3 females),IAV(n=6;3 males and 3 females),and TLR3 inhibitor(n=6;3 males and 3 females).Body weight was measured on days 0,3,and 5 after infection.On day 5,lung tissues were collected to assess viral load and histopathological changes.Key targets were examined using enzyme-linked immunosorbent assay,Western blotting,and immunofluorescence staining,both in sera and lung tissues.Results:IAV infection was significantly associated with dysregulation of the immune-inflammation system,such as the LTR,nucle-otide-binding oligomerization domain-(NOD)like receptor,retinoic acid-inducible gene I-like receptor,and nuclear factor kappa-B signaling pathways.Gene set enrichment analysis further indicated that the TLR and necroptosis signaling pathways played crucial roles in the progression of IAV infection(TLR signaling pathway normalized enrichment score=2.3941,P=1.00×10?10;necroptosis normalized enrichment score=1.9421,P=6.21×10?7).Among the core targets,TLR3 and mixed lineage kinase domain-like protein(MLKL)may regulate gene expression at the transcriptional level(all P<0.05).In vivo validation using an IAV(PR8)infected acute lung injury mouse model demonstrated increased viral load and lung index,alveolar structural damage,and inflammatory cell infiltration.Immunofluorescence staining exhibited large gaps in Lamin B1 staining and breaches in Emerin signals following IAV-PR8 infec-tion.Expression levels of TLR3,p-receptor-interacting serine/threonine-protein kinase 3(RIPK3)/RIPK3,and p-mixed lineage kinase domain-like protein(MLKL)/MLKL proteins in lung tissues,as well as proinflammatory factors and mediators in sera,were significantly elevated after IAV infection.Moreover,enhanced neutrophil infiltration(myeloperoxidase)and citrullinated histone H3(a neutrophil extracellular trap-specific marker),both established indicators of neutrophil extracellular trap formation,were observed.Notably,treatment with a TLR3 inhibitor significantly ameliorated IAV-induced acute lung injury by regulating necroptosis-related targets.Conclusion:Our study provides network-based in vivo evidence that TLR3-receptor-interacting serine/threonine-protein kinase 3-MLKL-mediated necroptosis may underlie IAV-induced acute lung injury and could serve as a potential therapeutic target in severe influenza cases.