Multi-Target Mechanisms of Whey Protein Against NAFLD: Integrating Bile Acid Metabolism, Gut Microbiota and Hepatic Inflammation.

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 < 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 < 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.