Integrated network pharmacology and experimental validation to elucidate the mechanism of WIN55,212-2 in mitigating sepsis-induced pulmonary fibrosis in mice.

WIN55,212-2, a non-selective cannabinoid receptor agonist, was investigated for its protective effects and underlying mechanisms in a sepsis-induced pulmonary fibrosis (SIPF) mouse model. In vivo experiments demonstrated that WIN55,212-2 effectively attenuates pulmonary inflammation and fibrosis. Integration with network pharmacology analyses further revealed a multi-target, multi-pathway mechanism of action, providing a theoretical framework for its potential clinical application and targeted therapeutic strategies. In this study, a cecal ligation and puncture (CLP)-induced SIPF model was established in mice. WIN55,212-2 markedly alleviated CLP-induced lung injury, as evidenced by improved alveolar architecture, reduced inflammatory cell infiltration, and decreased collagen deposition. Histological analyses confirmed restoration of lung morphology, while immunofluorescence demonstrated reduced collagen I expression. At the molecular level, WIN55,212-2 suppressed pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and enhanced anti-inflammatory and reparative mediators (IL-10 and TGF-β). Furthermore, it downregulated fibrosis markers (α-SMA, vimentin, and collagen I) and inhibited activation of the RAGE signaling axis and its downstream effectors, including phosphorylated JAK2, STAT1, PI3K, and JNK. CB1 or CB2 antagonists effectively reverse the effects of WIN55,212-2. Network pharmacology analysis indicated that WIN55,212-2 modulates multiple inflammation- and fibrosis-associated pathways, with key targets such as TP53, TLR4, MAPK1, and PIK3R1, supporting a synergistic, multi-target mode of action. Collectively, these findings suggest that WIN55,212-2 mitigates SIPF progression through coordinated regulation of multiple molecular targets and pathways, highlighting its potential as a therapeutic candidate and providing a basis for future translational and targeted intervention strategies.