Multi-target Synergistic Regulatory Mechanisms of Aristolochic Acid-Induced Urothelial Carcinoma: Evidence from Network Toxicology and Molecular Dynamics

Urothelial carcinoma (UC) is characterized by high incidence and recurrence rates, with aristolochic acid (AA) acting as a crucial pathogenic inducer that is closely associated with upper urinary tract UC. However, the molecular regulatory mechanisms underlying AA-UC interactions remain unclear, and traditional toxicological studies have inherent limitations. This study systematically explored AA-induced UC mechanisms to identify core targets and pathways, providing insights for UC early warning, targeted intervention and establishing a novel technical paradigm for safety evaluation of toxic components in traditional Chinese medicines. A multi-technique strategy was employed: AA-UC intersection targets were screened via multiple databases, followed by protein-protein interaction (PPI) network construction for core target identification; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to clarify biological functions and pathways; molecular docking predicted AA-core target binding modes, and molecular dynamics simulations verified binding stability. A total of 100 intersection targets were identified, with core targets including CYP2E1, CYP2A4 and EGFR. KEGG analysis revealed 82 significantly enriched pathways, dominated by MAPK, PI3K-Akt and cytochrome P450 metabolic pathways. AA-core target binding energies were all < -7.0 kcal/mol, and CYP3A4-AA/CYP2E1-AA complexes illustrated stable binding in simulations. In conclusion, AA stably binds core targets (e.g., CYP2E1/CYP2A4) as well as activates key pathways, synergistically promoting UC via multi-target/pathway interactions, thus providing crucial molecular evidence and technical support for precise UC control and traditional Chinese medicine safety evaluation.