In silico and in vitro insights into stigmasterol targeting Keap1/Nrf2, Bcl-2/Bax and IKKβ/IκBα protein-protein...

Inorganic arsenic is a known neurotoxin that contributes to neurodevelopmental and neurodegenerative disorders by promoting oxidative stress, mitochondrial dysfunction and inflammation. It crosses the blood-brain barrier and accumulates in brain tissue. Arsenic-induced neurotoxicity poses a significant health challenge, driving the need for multi-targeted therapeutic agents. This study investigated the neuroprotective potential of stigmasterol, a plant-derived phytosterol, against arsenic-induced damage in mouse HT-22 hippocampal cells through integrated computational and experimental approaches. Molecular docking and dynamics simulations revealed that stigmasterol disrupts the Keap1-Nrf2 complex (E-total, - 346 kJ/mol), and may promote Nrf2 activation and antioxidant gene expression. It stabilizes the Bcl-2/Bax complex (E-total, - 540 kJ/mol), enhancing anti-apoptotic signaling, and strengthens the IKKβ/IκBα interaction (E-total, - 687 kJ/mol), inhibiting NF-κB-mediated inflammation. In vitro assays confirmed that stigmasterol protected the cells in dose-dependent manner, restoring cell viability up to ~ 81%, and significantly reduced ROS levels compared to arsenic-treated cells. Neuronal morphology analysis further confirmed stigmasterol's ability to preserve neurite integrity and cellular connectivity against arsenic-induced damage. These findings highlight the multi-faceted neuroprotective mechanisms of stigmasterol in an in vitro neuronal model and suggest its potential role in mitigating arsenic-induced neuronal damage, which requires further validation through in vivo and pharmacological studies.