Abstract

Lonicerae Japonicae Flos (LJF), a traditional Chinese medicine with strong anti-inflammatory and antioxidant effects, was previously shown to suppress ferroptosis in acute lung injury (ALI). This study further aimed to identify its anti-ferroptotic quality markers (Q-markers) and elucidate their mechanisms for ALI therapy. Chemical fingerprints of 22 LJF batches were established, and absorbed constituents were identified through serum pharmacochemistry. Principal component analysis (PCA) and gray correlation analysis (GCA) were applied to link chemical composition with pharmacological effects. Ferroptosis-related indicators were measured in an LPS-induced ALI mouse model after treatment with different extracts. The anti-ferroptotic activity of individual constituents was validated in BEAS-2B cells, leading to the identification of Q-markers. To confirm target engagement, transcriptomic sequencing, protein–protein interaction (PPI) network construction, ferroptosis-target enrichment, real-time quantitative polymerase chain reaction (RT-qPCR), molecular docking, and molecular dynamics (MD) simulations were conducted. 17 candidate constituents with potential anti-ferroptotic and anti-inflammatory activities were identified, of which 9 showed strong correlations with efficacy. Four compounds—protocatechuic acid, loganin, cynaroside, and isochlorogenic acid C—demonstrated significant ferroptosis-inhibitory activity and were designated as Q-markers. Transcriptomic and PPI analyses indicated that these constituents target key ferroptosis-related proteins, including IL-6, Stat3, and Mapk3. Their regulatory effects and binding stability were further validated by RT-qPCR, molecular docking, and MD simulations. LJF exerts anti-inflammatory protection through the synergistic inhibition of ferroptosis by multiple constituents. The identification of protocatechuic acid, loganin, cynaroside, and isochlorogenic acid C as Q-markers provides scientific evidence supporting the therap