Shengjiang San suppresses C5a/C5aR1-mediated neutrophil activation and NET formation to alleviate acute lung injury
TL;DR
SJS attenuated LPS-induced ALI and restrained neutrophil activation and NET formation mediated by C5a/C5aR1 signaling, and Ferulic acid was identified as a candidate circulating constituent that reduced C5a-activated NET formation.
Abstract
Excessive neutrophil activation and neutrophil extracellular trap (NET) formation contribute to hyperinflammation and disruption of the alveolar capillary barrier in acute lung injury (ALI). C5a/C5aR1 signaling links complement activation to neutrophil migration, Ca 2+ mobilization, ROS production, and NET formation. This study investigated whether Shengjiang San (SJS) regulates these responses in ALI and explored candidate circulating constituents. ALI was induced in mice by intratracheal LPS administration, and SJS treatment was initiated before LPS exposure. Lung injury, barrier integrity, neutrophil responses, NET formation, C5a/C5aR1 changes, and lung transcriptomic profiles were assessed. Purified murine neutrophils were stimulated with C5a or PMA to evaluate the effects of SJS-containing serum. Serum pharmacochemistry, plasma exposure analysis, biolayer interferometry, molecular docking, and neutrophil assays were used to characterize ferulic acid. SJS reduced pulmonary inflammation, edema, vascular leakage, neutrophil accumulation and activation, and NET formation, while preserving the integrity of the alveolar capillary barrier. Transcriptomic profiling showed enrichment of complement activation, neutrophil activation, and NET formation among genes differentially expressed between the SJS and Model groups. SJS also lowered C5a concentrations in plasma and lung homogenates and reduced pulmonary C5aR1 expression. In neutrophils, SJS-containing serum significantly suppressed C5a-stimulated neutrophil functions, including migration, Ca 2+ flux, oxidative burst, and NET release, while PMX205 produced a similar inhibitory pattern. SJS-containing serum also inhibited PMA-induced NET formation. Ferulic acid was detected in serum as a prototype constituent with measurable plasma exposure, showed concentration-dependent binding to recombinant human C5a with an apparent K D of 21.65 μM, and reduced histone H3 citrullination and NET formation in C5a-stimulated neutrophils. SJS attenuated LPS-induced ALI and restrained neutrophil activation and NET formation mediated by C5a/C5aR1 signaling. Ferulic acid was identified as a candidate circulating constituent that reduced C5a-activated NET formation.