Multi‐Target Mechanisms of Whey Protein Against <scp>NAFLD</scp> : Integrating Bile Acid Metabolism, Gut Microbiota and Hepatic Inflammation

ABSTRACT This study elucidates the protective mechanisms of whey protein (WP) in treating high‐fat diet (HFD)‐induced nonalcoholic fatty liver disease (NAFLD) in mice, emphasizing its role in bile acid regulation, intestinal flora homeostasis, and inflammatory suppression. ICR mice were subjected to a 12‐week HFD to establish NAFLD, followed by WP intervention (200 g/kg). Comprehensive analyses included histopathological assessment (HE staining), serum biomarkers, hepatic gene expression (qPCR), gut microbial profiling (16S rRNA sequencing), quantitative bile acid and short‐chain fatty acid (SCFA) analysis, and serum metabolomics. Core targets were predicted via network pharmacology and validated through molecular docking. WP administration markedly alleviated NAFLD progression by targeting multiple pathways: (1) It suppressed hepatic lipid deposition and inflammatory injury, downregulating NLRP3, NF‐κB, and TNF‐α ( p &lt; 0.05) while enhancing Nrf2/HO‐1‐mediated antioxidant defenses; (2) Network pharmacology prioritized IL‐1β, STAT3, and MMP9 as pivotal targets, with β‐lactoglobulin exhibiting high binding potentials (STAT3: −1.42 kcal/mol); (3) WP restored gut microbial balance, enriching beneficial taxa (e.g., Lactobacillus ) and fecal SCFAs; (4) It reprogrammed bile acid metabolism, elevating cholesterol‐cleaving enzymes (CYP7A1/CYP27A1) but inhibiting FXR/SHP ( p &lt; 0.05), alongside increased hepatoprotective bile acids (TDCA/TUDCA). Metabolomics identified WP‐induced anti‐inflammatory mediators (e.g., eicosapentaenoic acid) and perturbations in arginine and unsaturated fatty acid pathways, synergistically attenuating steatosis and fibrosis. WP counters NAFLD via a tripartite mechanism: gut microbiome‐directed SCFA synthesis, bile acid‐driven cholesterol disposal, and dual modulation of inflammation (NLRP3/NF‐κB) and oxidative stress (Nrf2/HO‐1). These insights position WP as a promising dietary strategy targeting the gut‐liver axis.