Euphorbia humifusa Willd. ex Schltdl. Mitigates Liver Injury via KEAP1-NFE2L2-Mediated Ferroptosis Regulation: Network Pharmacology and Experimental Validation

Liver injury poses major health risks in livestock, necessitating effective therapeutic interventions. This study elucidates the hepatoprotective mechanisms of <i>Euphorbia humifusa</i> Willd. ex Schltdl. (EHW) by integrating network pharmacology, molecular docking, and experimental validation. Using a CCl₄-induced liver injury model mimicking veterinary clinical scenarios, EHW markedly alleviated hepatic damage, demonstrated by reduced liver index, serum ALT and AST levels, histopathological lesions, iron accumulation, inflammatory cytokines, and ferroptosis-associated gene expression. Network pharmacology identified EHW's core bioactive components (quercetin, kaempferol, and β-sitosterol) and critical targets (<i>IL-6</i>, <i>STAT3</i>, <i>HIF-1α</i>, <i>PTGS2</i>, <i>NFE2L2</i>, and <i>KEAP1</i>) which were linked to ferroptosis and oxidative stress. Molecular docking revealed robust binding affinities between these compounds and ferroptosis-related proteins. <i>In vivo</i> validation confirmed that EHW inhibited <i>KEAP1</i>, activated <i>NFE2L2</i>-mediated antioxidant defenses (upregulating <i>SOD1</i> and <i>NQO1</i>), restored iron homeostasis (lowering <i>TFR1</i>, elevating <i>FTH1</i>), and attenuated phospholipid peroxidation by suppressing <i>ACSL4</i> and <i>ALOX12</i>. These results indicate that EHW mitigates ferroptosis-driven liver injury via KEAP1-NFE2L2 signaling to restore iron homeostasis and reduce oxidative stress, offering a mechanistic foundation for its clinical application in veterinary hepatoprotection.