Xuebijing injection ameliorates LPS-mediated acute lung injury via multi-target synergy by alleviating inflammation and ferroptosis

Acute lung injury (ALI) is a diffuse alveolar injury caused by infections and other predisposing factors, with associated alveolar dysfunction, pulmonary oedema, and acute respiratory failure. Currently, no specific therapeutic agents are clinically available for ALI. Xuebijing injection (XBJ) is a widely used clinical traditional Chinese medicine formulation, primarily used for respiratory infections. However, the effects and mechanisms of XBJ in ALI are not fully understood. This study demonstrated that XBJ treatment ameliorated ALI progression by heightening alveolar barrier integrity and reducing histopathological lung damage. Mechanistically, XBJ inhibited the activation of the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome pathways, resulting in reduced production of key pro-inflammatory cytokines (e.g., IL-6, IL-1β, and TNF-α). It also restored immune balance by regulating Treg/Th17 cells and inhibited ferroptosis. Using integrated chemical biology approaches, pyruvate kinase M2 (PKM2), enolase 1 (ENO1), PDZ binding kinase (PBK), eukaryotic translation initiation factor 3i (EIF3I), and kelch-like ECH-associated protein 1 (Keap1) were identified as direct intracellular targets of XBJ, which was further confirmed through various chemical and biological methods. Moreover, compounds from XBJ, which is entered into the blood and lungs, such as palmitic acid, ethyl 4-hydroxy-3-methoxycinnamate, sugiol, oleic acid, and 10,12-octadecadiynoic acid, could bind to these targets, respectively. In summary, XBJ protected against lipopolysaccharide (LPS)-induced ALI by multi-targets, thereby modulasting inflammatory and immune responses, while inhibiting the MAPK/NF-κB/NLRP3 pathways and ferroptosis. These findings offered mechanistic evidence of the application value of XBJ in the treatment of ALI.