Integrative network toxicology, molecular docking, and in vitro experiments identify SLC6A2 as a novel candidate target in nicotine-induced osteoporosis.

In recent years, the health problems attributable to cigarette smoke (CS) have attracted widespread attention. However, the pathogenic mechanisms underlying osteoporosis development induced by nicotine, a neuroactive component of CS, remain unclear. In this study, we examined a potential molecular mechanism underlying nicotine-induced osteoporosis by network toxicology, molecular docking, molecular dynamics (MD) simulation, and in vitro experiments. On screening multiple databases, we identified 47 overlapping targets for nicotine- and osteoporosis-related genes, and subsequently performed protein-protein interaction (PPI) analysis on the identified targets. KEGG analysis suggested that the potential targets were mainly involved in the calcium, PI3K-Akt, and MAPK pathways. We identified seven core targets using five algorithms, among which SLC6A2 exhibited the strongest predicted binding affinity. Molecular docking confirmed good binding affinities between nicotine and these core targets, and molecular dynamics simulation verified the stability of the nicotine-SLC6A2 complex. Moreover, in vitro experiments further confirmed that nicotine significantly inhibits osteogenic differentiation of MC3T3-E1 cells, and differentially regulates the transcriptional expression of all seven core targets, with SLC6A2 being one of the downregulated ones. This identifies a previously unrecognized neural-related target (SLC6A2) in nicotine-induced bone toxicity. Our study provides a theoretical basis for developing multi-target regulation strategy that could contribute to mitigating the bone toxicity of complex environmental toxins.