Abstract: Objective: This study aimed to comprehensively elucidate the toxic mechanisms of toxoflavin by identifying its critical molecular targets and signaling pathways, thereby providing a theoretical framework for toxicity prevention and therapeutic target identification. Methods: A network pharmacology strategy was applied to predict potential targets of toxoflavin and construct a protein–protein interaction (PPI) network. Cytotoxicity-related overlapping targets were screened and further subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. In human hepatic stellate cells LX2 and human renal epithelial cells 293T, cell viability and proliferation were assessed using the cell counting kit-8 (CCK-8) assay. Apoptosis was detected by flow cytometry. Western blot analysis was performed to determine the expression of pro-apoptotic proteins including Bim, Bak and cleaved-caspase 3. Phosphorylation levels of MAPK pathway key proteins (JNK, p38 and ERK) were also detected. Molecular docking simulations between toxoflavin and target proteins were performed using MGLTools 1.5.7 and AutoDock 4.2 software. Results: A total of 28 overlapping targets associated with toxoflavin-induced cytotoxicity were identified via network pharmacology. MAPK8 (JNK1) was recognized as the core target. KEGG enrichment analysis indicated that these core targets were significantly enriched in the PI3K/AKT, ErbB and MAPK signaling pathways. Toxoflavin inhibited the viability of LX2 and 293T cells in a concentration-dependent manner. It also markedly suppressed cell proliferation and promoted cell apoptosis. The expression of pro-apoptotic proteins Bim, Bak and cleaved-caspase 3 was upregulated after toxoflavin administration. Phosphorylation levels of JNK and p38 were elevated, whereas ERK phosphorylation showed no significant change. Molecular docking results confirmed that toxoflavin exhibits favorable binding affinity with JNK/p38 proteins. Conclusion: Toxoflavin may upregulate the phosphorylation levels of JNK/p38 in the MAPK signaling pathway. It then modulates the expression of downstream pro-apoptotic proteins.Ultimately, it suppresses proliferation and induces apoptosis in liver- and kidney-derived cells.

Key words: Toxoflavin, Network pharmacology, Mitogen-activated protein kinase (MAPK) signaling pathway, Cytotoxicity