Platycodin D3 protects against sepsis-associated acute lung injury through direct inhibition of the PKM2-NLRP3 axis.

BACKGROUND

Sepsis-induced acute lung injury (SI-ALI) is a leading cause of morbidity and mortality in critically ill patients and is characterized by excessive inflammation, oxidative stress, and metabolic dysregulation of macrophages. Pyruvate kinase M2 (PKM2) has emerged as a key metabolic regulator that links glycolysis to NLRP3 inflammasome activation; however, effective therapeutic strategies targeting this axis remain limited.

METHODS

Integrated bioinformatics analyses, including differential expression profiling, protein-protein interaction network analysis, and single-cell RNA sequencing, were used to identify PKM2 as a macrophage-associated target in SI-ALI. Hierarchical virtual screening of a natural product library identified platycodin D3 (PD3) as a potential PKM2-targeting compound. The interaction between PD3 and PKM2 was validated using molecular docking, microscale thermophoresis, surface plasmon resonance, and cellular thermal shift assays. The therapeutic effects and underlying mechanisms of PD3 were evaluated in LPS-induced SI-ALI mouse models and RAW264.7 macrophages using histopathological analysis, biochemical assays, Seahorse metabolic profiling, and genetic overexpression and mutagenesis approaches.

RESULTS

PKM2 was significantly upregulated in SI-ALI patients and LPS-stimulated macrophages, with particularly high expression in monocyte/macrophage populations. PD3 directly bound to and stabilized PKM2, resulting in the suppression of PKM2-NLRP3 inflammasome activation. In vivo, PD3 markedly alleviated lung injury, fibrosis, oxidative stress, and inflammatory cytokine production in SI-ALI mice. In vitro, PD3 restored mitochondrial respiration and glycolytic capacity while reducing oxidative damage and inflammatory responses in macrophages. These protective effects were abolished by overexpression of PKM2 or NLRP3, as well as by mutation of the PKM2 Arg319 residue, demonstrating that PD3 exerts its effects through direct targeting of PKM2 to disrupt the PKM2-NLRP3 axis.

CONCLUSION

This study identifies PD3 as a direct PKM2-binding compound that mitigates SI-ALI by inhibiting PKM2-dependent NLRP3 inflammasome activation and associated metabolic dysfunction. Targeting the PKM2-NLRP3 axis represents a promising therapeutic strategy for treating SI-ALI.