Integrated strategy of network pharmacology and in vitro screening to identify mechanism of diazinon-induced hippocampal neurotoxicity

Abstract

Diazinon (DZN) is a commonly used organophosphorus pesticide that was recently found to cause hippocampal degeneration in rodents. In this study, we elucidated the underlying molecular mechanisms through integrated network pharmacology and in vitro toxicity screening. 37 potential molecular targets of DZN-induced hippocampal neurotoxicity were predicted. Identified targets were then included in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A preliminary protein-protein network (PPI) was constructed using STRING, and the top 10 network hub target genes (Akt1, Mapk3, Tnf, Il6, Ptgs2, Il10, Il2, Il4, Creb1, and Fgf2) were screened for expression changes under DZN treatment. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assays revealed time- and dose-dependent toxicity of DZN against mouse hippocampus-derived HT22 cells. Acetylcholinesterase (AChE) activity assay suggested that DZN inhibited the AChE activity, and TUNEL staining revealed that DZN increased the apoptotic rate. The mRNA expression levels of 9 hub targets (all except Il10) showed significant changes during DZN treatment, and AChE activity inhibition correlated strongly with Akt1, Mapk3, Il6, Il2, and Fgf2. DZN-induced hippocampal neurotoxicity was associated with the altered activity of multiple signaling pathways (including PI3K-Akt, TNF, and apoptosis signaling). These results provided a theoretical basis for more precise elucidation of DZN neurotoxic mechanisms.