Tetracycline antibiotics (TETs) are widely used in veterinary practice but can induce acute hepatotoxicity upon overdose, compromising animal health and productive performance. This study evaluated whether the natural isoflavone formononetin (FMN) can alleviate TET-induced liver injury in animals via coordinated regulation of the gut microbiota–metabolite–transcriptome axis. Using network toxicology and pharmacology, we predicted key targets and pathways involved. A mouse model of of acute, overdose‑induced TET‑associated liver injury (150 mg/kg, once daily for 3 days) was treated with FMN (50 or 100 mg/kg) for 14 days. Multi-omics analyses revealed that FMN attenuated hepatic steatosis, reduced ALT/AST levels, enhanced gut microbial diversity (enriched Limosilactobacillus) and restored intestinal barrier integrity. Mechanistically, FMN activated the Nrf2/HO-1 antioxidant pathway, modulated PPARα/CYP2E1-dependent lipid metabolism, and may be associated with ferroptosis inhibition. In summary, FMN exerts multi-target hepatoprotective effects by coordinately regulating gut microbiota homeostasis, alleviating oxidative stress and remodeling hepatic metabolic networks, thereby providing a preliminary basis for further evaluation of formononetin for antibiotic-associated liver injury in veterinary settings.

To Cite This Article: Zhang X, Wang X, Sui J, Al-Shahari EA, Momenah MA, Yu Y^, Chen S, Jiang W, Luo Y, Hu Y, Wang N, Gu X, Sun J, Li N, Bai W and Qu W, 2026. Multi-omics uncovers formononetin's protection against antibiotic-induced liver injury via microbiota-metabolite-transcriptome coordination. Pak Vet J, 46(5): 1152-1166. http://dx.doi.org/10.29261/pakvetj/2026.103