Neurotoxic mechanism of roxarsone based on network toxicology and molecular docking

【Objective】 The aim of the study was to explore the neurotoxic mechanism of roxarsone （ROX） and provide theoretical basis for elucidating its toxic effects in animals as a feed additive and for assessing the toxicological risks of ROX exposure to humans.【Method】 Network toxicology was employed to identify the potential targets of ROX and neurotoxicity-related targets. Subsequently， the intersection of these targets was used to construct a protein-protein interaction （PPI） network for screening core targets， followed by GO and KEGG signaling pathway enrichment analysis. A ROX-target-pathway network was then established. Molecular docking was performed to verify the binding ability of core targets， and molecular dynamics simulation was used to evaluate the binding stability of complexes formed by ROX and core targets. Finally， gavage administration experiments in mice were conducted to validate the neurotoxic mechanism of ROX on mice nervous system.【Result】 A total of 108 overlapping targets of ROX and neurotoxicity were obtained， and the PPI network identified caspase-3 （CASP3）， glycogen synthase kinase 3β （GSK3β）， cyclin-dependent kinase 2 （CDK2）， and estrogen receptor 1 （ESR1） as core targets. GO functional annotation analysis and KEGG signaling pathway enrichment analysis of the comprehensive core targets revealed that the NF-κB signaling pathway， which mediated inflammatory responses， played a key role in the neurotoxic mechanism induced by ROX. The targets such as GSK3β， CASP3 and MDM2 played important roles in ROX-target-pathway network， and might be the key targets through which ROX affected neurotoxicity. ROX produced spontaneously and stably only to the four core targets （CASP3， CDK2， ESR1， GSK3β） with binding energies all ≤ -5.0 kcal/mol， among which the binding to CDK2 was the most stable （bin-ding energy being -6.01 kcal/mol）. Molecular dynamics simulation results showed that the complex systems of ROX with ESR1， CDK2， CASP3， and GSK3β exhibited stable binding and favorable hydrogen bond interactions. ROX induced hippocampal neuron damage in mice brain tissues， significantly up-regulated inflammatory cytokine levels， and enhanced GSK3β activity （P<0.05）， thereby exerting neurotoxicity.【Conclusion】 CDK2， CASP3， ESR1， and GSK3β are the key toxic targets of ROX， among which GSK3β regulates the activity of other targets through an interaction network. ROX may exert neurotoxicity by inhibiting the Wnt signaling pathway， which enhances GSK3β activity. This activation further triggers Caspase-3 and NF-κB pathways， leading to neuronal apoptosis and inflammatory imbalance in mice brain tissues and impairing the self-repair capacity of neurons.