Integrated network toxicology and in vivo experiments reveal the mechanisms of Evodiae fructus-induced nephrotoxicity in mice

Evodiae Fructus (EF) is a traditional Chinese medicine with effects including promoting blood circulation, relieving pain, and exerting anti-tumor and anti-inflammatory activities, and is widely used in clinical practice. However, its toxicological characteristics and potential nephrotoxic mechanisms have not been fully elucidated. This study aimed to investigate the nephrotoxic constituents and targets of EF and verify its potential molecular mechanisms by integrating network toxicology, molecular docking, and in vivo experimental approaches. Network toxicology analysis identified 21 toxic constituents and 89 potential nephrotoxic targets. Protein–protein interaction (PPI) network analysis revealed AKT1, CASP3, STAT3, and JUN as core regulatory genes. KEGG pathway enrichment suggested significant involvement of apoptosis, PI3K-Akt, and JAK–STAT signaling pathways. Molecular docking results indicated favorable binding affinities between EF toxic constituents and these core targets. In vivo experiments demonstrated that EF dose-dependently increased the kidney index and serum blood urea nitrogen (BUN) and creatinine (Cr) levels in mice, accompanied by renal histopathological injury and increased expression of the renal tubular injury biomarkers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Furthermore, EF exposure downregulated AKT1 protein expression while upregulating STAT3, JUN, and CASP3 expression. Collectively, these findings suggest that EF-induced nephrotoxicity may involve multi-component and multi-target regulation of apoptosis-related processes and PI3K-Akt/JAK-STAT-associated signaling pathways. This study provides a mechanistic basis for the safety evaluation and rational clinical use of EF.