Multi-omics integration reveals that pterostilbene ameliorates cyclophosphamide-induced liver injury via the gut-liver axis by inhibiting inflammation and oxidative stress.

Cyclophosphamide (CTX) is a commonly used immunosuppressant and chemotherapeutic drug; however, its clinical application is often restricted by drug-induced liver injury (DILI). Pterostilbene (PTS), a dietary polyphenol abundant in blueberries and other berries, is known for its antioxidant and anti-inflammatory properties; however, its hepatoprotective effects and underlying mechanisms on CTX-induced liver injury remain largely unexplored. This study adopted an integrated approach combining network pharmacology, in vivo and in vitro models, untargeted metabolomics, and gut microbiota 16S rRNA sequencing to investigate the protective effects of PTS on CTX-induced liver injury and explore the underlying multi-dimensional mechanisms. PTS significantly ameliorated CTX-induced liver injury by regulating inflammation and oxidative stress. Network pharmacology corroborated these findings, revealing that the targets of PTS against DILI were enriched in pathways closely related to inflammation and oxidative stress, such as reactive oxygen species and MAPK signaling, with key molecules including EGFR, PIK3CA, and RELA. Experimental validation confirmed that PTS inhibited the overactivation of EGFR/MAPK, PI3K/AKT, and NF-κB pathways and activated the Nrf2/HO-1 antioxidant pathway. Metabolomics analysis revealed that PTS ameliorates CTX-induced hepatic metabolic perturbations by regulating key metabolites, including lipids (e.g., PGP), amino acids (e.g., taurine), vitamins (e.g., tocotrienol), and oxidative stress-related markers (e.g., ophthalmate). Gut microbiota profiling showed that PTS restores CTX-induced dysbiosis by increasing the abundance of beneficial genera (e.g., Bacteroides) and reducing that of pathogenic taxa (e.g., Proteobacteria). Multi-omics correlation analysis further uncovered a robust network linking gut microbial composition, hepatic metabolites, and markers of inflammation and oxidative stress. In conclusion, PTS protects against CTX-induced liver injury via multiple targets and pathways, involving the gut-liver axis, inflammatory-oxidative regulation, and metabolic homeostasis. This study provides a theoretical basis for the potential application of PTS in the prevention and treatment of DILI and offers novel insights into liver-protective strategies targeting intestinal microecology.