Abstract: To elucidate the toxicological mechanism of toxoflavin, a network pharmacology strategy was used to predict potential targets of toxoflavin and construct a protein-protein interaction (PPI) network. The intersecting targets between toxoflavin and cytotoxicity were identified and subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The impact of toxoflavin on the viability and proliferation of human hepatic stellate LX2 cells and human embryonic kidney 293T cells 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 Bcl-2 interacting mediator of cell death (Bim), Bcl-2 homologous antagonist/killer (Bak) and cleaved cysteine-aspartic acid protease 3 (c-caspase 3). The phosphorylation levels of key proteins such as c-Jun N-terminal kinase (JNK), p38 and extracellular regulated protein kinases (ERK) in the mitogen-activated protein kinase (MAPK) signaling pathway were also detected. In addition, molecular docking simulations between toxoflavin and the target proteins were performed. The results revealed that a total of 28 overlapping targets associated with toxoflavin-induced cytotoxicity were identified via network pharmacology. MAPK8 (JNK1) was identified as the core target. KEGG enrichment analysis indicated that these core targets were significantly enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), epidermal growth factor receptor (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 induced cell apoptosis. The expression levels of Bim, Bak and c-caspase 3 were significantly upregulated after toxoflavin treatment. The phosphorylation levels of JNK and p38 were significantly elevated, whereas that of ERK showed no significant change. Molecular docking results confirmed the good binding affinity of toxoflavin exhibits for JNK and p38 proteins. In conclusion, toxoflavin may upregulate the phosphorylation levels of JNK and p38 in the MAPK signaling pathway, thereby modulating the expression of downstream pro-apoptotic proteins and ultimately suppressing proliferation and inducing apoptosis in liver and kidney cells.

Key words: toxoflavin; network pharmacology; mitogen-activated protein kinase signaling pathway; cytotoxicity