The pyranocoumarin-enriched extract from the leaves of Calophyllum membranaceum alleviates LPS-induced acute lung injury in mice by inhibiting the TLR4/MD2 interaction.

Acute lung injury (ALI) is a pulmonary inflammatory response caused by various factors, with higher morbidity and mortality. Pyranochromone derivatives isolated from Calophyllum membranaceum exhibit significant anti-inflammatory effects; however, their molecular targets and in vivo therapeutic potential for ALI remain unexplored. In this study, we obtained a pyranochromone-enriched extract (PEC) from C. membranaceum leaves and evaluated its potential therapeutic effects and mechanisms in lipopolysaccharide (LPS)-induced ALI using RAW264.7 cells and C57BL/6 J mice models. In vitro, PEC inhibits inflammatory responses and M1 polarization in RAW264.7 cells in a dose-dependent manner. In vivo, PEC could ameliorate LPS-induced lung injury pulmonary edema, and inflammatory cell infiltration. Co-immunoprecipitation (Co-IP) assays demonstrated that PEC can simultaneously bind to both TLR4 and MD2 proteins. Furthermore, molecular docking analysis revealed that PEC may act at the interface between TLR4 and MD2. Moreover, PEC can markedly decrease the levels of IL-1β, IL-6 and TNF-α, and decrease the protein expressions of TLR4/NF-κB pathway and NLRP3 pathway both in vitro and in vivo. In summary, this study demonstrates that PEC can simultaneously bind to both TLR4 and MD2 proteins, thereby inhibiting the formation of the TLR4/MD2 complex and reducing the levels of inflammatory factors. Compounds like PEC may represent a novel class of TLR4 inhibitors with a unique mode of action, offering a new therapeutic strategy for the treatment of ALI and other inflammatory diseases.