Therapeutic potential of inosine in acute lung injury: mechanistic insights into TLR4 suppression and macrophage polarization.

BACKGROUND: As severe inflammatory condition, acute lung injury (ALI) can progress to acute respiratory distress syndrome (ARDS), for which no effective therapy has been reported. Inosine, a bioactive nucleoside derived from Dioscorea opposita thunb., exhibits anti-inflammatory and immunoregulatory performances, while its therapeutic potential and mechanisms in ALI remain unclear. PURPOSE: To clarify the protective effects of inosine in LPS-induced ALI and the underlying molecular mechanisms by metabolomics, transcriptomics, and in vitro and in vivo tests. METHODS: A murine ALI model was induced via intratracheal LPS administration, followed by inosine treatment. Pulmonary function, inflammatory cytokines, macrophage polarization, and metabolic and transcriptomic changes were evaluated. Clodronate liposomes were used to deplete pulmonary macrophages and assess their role in the functioning of inosine. LPS-treated 16HBE cells and immune cell co-cultures were employed to examine apoptosis, cytokine levels, and TLR4 signaling. RESULTS: Treatment with inosine led to significantly attenuated lung injury, improved pulmonary function, and reduced levels of IL-1ß, IL-6, IL-18 and TNF-α. Specifically, inosine promoted M1-to-M2 macrophage polarization, thereby enhancing anti-inflammatory responses. Clodronate liposome depletion led to degradation of protective effects of inosine, indicating a key role of macrophages. Metabolomics analysis revealed that treatment with inosine restored glycolysis, lipid metabolism, and amino acid homeostasis, while transcriptomics analysis showed downregulation of TLR4/NF-κB, PI3K-Akt, and NOD-like receptor pathways, with upregulation of anti-inflammatory genes.In vitro tests indicated that treatment with inosine led to decreased apoptosis rate of LPS-induced 16HBE cells, decreased levels of inflammatory cytokines, and inhibited TLR4 activation, and such effects were enhanced by TLR4 inhibitor TAK-242. SPR analysis confirmed that inosine can inhibit signaling of TLR4 via direct binding. CONCLUSION: Inosine shows protective effects against ALI as it may regulate TLR4 signaling to modulate macrophage polarization, metabolic homeostasis, and inflammatory responses. This study comprehensively integrate multi-omics analysis and functional assays to reveal the direct interaction between inosine and TLR4 for the first time, and provides insights into its anti-inflammatory and immunoregulatory mechanisms.