In-silico novel thioredoxin-interacting protein inhibitors from Syzygium aromaticum and Terminalia chebula and their validations to combat arsenic-Induced toxicity in Vero cells

Arsenic is a toxic metalloid that can lead to oxidative stress in cells by inducing reactive oxygen species (ROS) and interfering with cellular antioxidant defenses. Thioredoxin interacting protein (TXNIP) is an important regulator of redox processes. Activation and upregulation of TXNIP may play a central role in the mechanisms underlying arsenic-induced oxidative stress, inflammation, and cytotoxicity. Arsenic exposure has been shown to upregulate TXNIP expression, further amplifying oxidative stress, causes cellular damage. In the present study, the antioxidant potential of 50 phytochemicals from S. aromaticum and T. chebula, were screened using molecular docking studies against TXNIP, followed by MD simulations studies. The study revealed stigmasterol to exhibit the highest negative dock score and hence best binding affinity towards the target protein (TXNIP). The minimum binding energy of -10.14 Kcal/mol as compared to the fisetin with a binding energy of -7.15 Kcal/mol, the latter being selected as one of the standard drugs for our study. The MD simulation study of the stigmasterol-TXNIP complex for 100 Nanoseconds exhibited a stable interaction between protein-ligand thus validating our docking studies. The study also involved in vitro analysis of the best inhibitor of TXNIP uncovered in molecular docking studies. The in-vitro analysis demonstrated that stigmasterol pre-treatment conferred significant protection against Sodium Arsenite-mediated cytotoxicity in cultured Vero cells (African green monkey kidney cells). Furthermore, we employed 2'-7'-Dichlorodihydrofluorescein diacetate staining and captured fluorescent images, fluorescence images provided visual evidence supporting the cytoprotective role of stigmasterol, as evidenced by a reduction in oxidative stress compared to arsenic-treated cells.