Rutin targeting NOX4 alleviates sepsis-induced acute lung injury via inhibition of NET formation through the ROS/MAPK/PAD4 pathway

Neutrophil extracellular traps (NETs) have been identified as pivotal contributors in driving excessive inflammation and organ damage during sepsis. Rutin has shown broad therapeutic potentials in inflammatory, infection, and cancer disease models. However, its role in sepsis-associated acute lung injury (ALI) has not been thoroughly elucidated. Here, we initially demonstrated that rutin (50-200 μM) significantly reduced the extracellular DNA level and NET formation in PMA-stimulated dHL-60 cells (P < 0.01). And this effect was not associated with rutin intercalation into DNA or DNA damage. Mechanistically, rutin directly bound to NOX4, which in turn reduced intracellular ROS and Ca²⁺ levels. This led to the subsequent downregulation of p-ERK, p-p38, p-JNK, PAD4, and CitH3, thereby suppressing NET formation through inhibition of the ROS-dependent MAPK pathway. In vivo, rutin markedly improved survival and alleviated lung inflammation by reducing the lung wet/dry weight ratio, total number of cells, total protein concentration, and the levels of inflammatory cytokines in bronchoalveolar lavage fluid. Rutin suppressed NET formation by reducing extracellular DNA level and MPO-DNA complex in serum and inhibiting the MAPK pathway. In conclusion, these findings identify rutin as a natural NOX4 inhibitor that suppresses NET formation via the ROS/MAPK/PAD4 pathway, supporting its therapeutic potential for sepsis.