Kaempferol alleviates copper-induced nephrotoxicity by targeting MAPK8 and CASP3 in broiler: Insights from network toxicology, network pharmacology, molecular docking and dynamics simulation.

Copper (Cu) exposure poses significant threats to renal health yet effective mitigation strategies remain scarce. Kaempferol (Kae) is a naturally occurring flavonoid with well-documented antioxidant and anti-apoptosis activities. Nevertheless, its protective effects in renal copper poisoning and the underlying molecular mechanisms have not been fully elucidated. This study integrated network toxicology, pharmacology, molecular docking, molecular dynamics simulation and a broiler model preliminarily revealed the mechanism of Kae alleviates Cu-induced nephrotoxicity (CuN). The potential targets of CuN scanned from Comparative Toxicogenomics Database (CTD), GeneCards database (GC) and the targets of Kae scanned from SwissTargetPrediction, PharmMapper database were used to perform GO and KEGG enrichment analysis. STRING and Cytoscape were employed to constructed PPI networks and locked on core targets. Molecular docking and dynamics simulations were used to investigate the binding affinity and stability of Kae with the core targets. A broiler model to further validate the impact of Kae on key signaling pathways. Notably, Kae effectively ameliorated histopathological damage caused by excess Cu and restored renal function (P < 0.05). Network results revealed the involvement of apoptosis and MAPK signaling pathway, with Caspase-3 (CASP3) and Mitogen-activated protein kinase 8 (MAPK8) identified as core targets. In vivo experiments demonstrated that Cu significantly up-regulated the levels of Cleaved-CASP3 and phosphorylated MAPK8 and the expression of genes and proteins involved in promoting endogenous apoptosis (P < 0.05), while Kae markedly reversed these effects and reduced TUNEL staining positive rate from 43.83% to approximately 12.84% (P < 0.05). In summary, our findings demonstrate that Kae alleviates CuN by targeting the apoptotic process and the MAPK/JNK signaling axis, highlighting its potential as a natural therapeutic agent against heavy metal-induced nephrotoxicity in agricultural settings.