Network toxicology, machine learning combined with experimental validation: Rutin ameliorates NAFLD and prevents BHA-induced hepatotoxicity via Casp3 suppression

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease globally. Butylated hydroxyanisole (BHA), a widely used synthetic antioxidant, can induce hepatotoxicity upon high-dose or long-term exposure. The impact of BHA on pre-existing NAFLD remains unclear, representing a potential environmental health risk. Rutin, a natural flavonoid beneficial for NAFLD, may protect against this BHA-aggravated injury, but its mechanism is unknown. Potential targets of rutin, BHA, hepatotoxicity, and NAFLD were retrieved from public databases. Overlapping targets were identified and used to construct a protein-protein interaction (PPI) network. Functional enrichment analyses, including Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, were performed. Hub genes were screened using the Least Absolute Shrinkage and Selection Operator (LASSO) and Random Forest (RF) machine learning algorithms. Based on the bioinformatic findings, Casp3 was selected for further investigation. Molecular docking was conducted to evaluate the binding affinity between rutin and Casp3. An in vitro NAFLD model was established. The effects of rutin on Casp3 expression (assessed by Western blot and qRT-PCR), lipid accumulation (evaluated by TG and TC levels), liver function (determined by ALT and AST activities), and cell apoptosis (analyzed by flow cytometry) were measured. Venn analysis identified 20 common targets, with PPI networks, core modules, KEGG, and GO enrichment collectively highlighting critical signaling pathways. LASSO and RF screened 8 and 6 genes, respectively, with 4 genes overlapping; Casp3 was identified as the pivotal target. Rutin exhibited a binding affinity of −7.7 kcal/mol for Casp3, compared to −6.2 kcal/mol for BHA. In vitro , rutin attenuated BHA-induced increases in Casp3 expression, lipid levels (TG/TC), liver enzymes (ALT/AST), and apoptosis. These protective effects were abrogated by Casp3 overexpression. Rutin alleviated BHA-induced hepatotoxicity in NAFLD by suppressing Casp3, providing a molecular foundation for the potential therapeutic application of rutin in this context. • Network toxicology and machine learning identify Casp3 as a core target in rutin, BHA, and NAFLD-related toxicity. • Rutin ameliorates NAFLD and BHA-induced toxicity by regulating metabolism and inhibiting apoptosis. • Rutin prevents BHA-induced hepatotoxicity via Casp3 suppression.