Withaferin-A kills neuronal cells: An off-putting facet of Withania somnifera as a neuroprotectant.

Withania somnifera, commonly known as Ashwagandha, is widely utilized for treating neurological disorders in both traditional and modern medicine. While its neuroprotective effects are linked to several bioactive compounds, the individual safety profiles and potential cytotoxic effects of these compounds remain poorly understood. Previous studies from our research group have identified Withanone and Withanolide-A as neuroprotective agents. This study specifically evaluated the neurotoxic potential of Withaferin-A (WFA), a key steroidal lactone found in Withania somnifera, on differentiated SH-SY5Y cells. For six days, the SH-SY5Y cells were differentiated using 10 µM retinoic acid (RA). The neuronal phenotype was confirmed through morphological changes and increased expression of NeuN. Cytotoxicity assays demonstrated that WFA induces potent, dose-dependent cytotoxicity, resulting in approximately 50 %, 80 %, and 90 % cell death at concentrations of 0.6 µM, 1.2 µM, and 2.4 µM, respectively (p < 0.001). Treatment with WFA at 1.2 µM significantly increased both intracellular and mitochondrial reactive oxygen species (ROS), as shown by fluorescence imaging (p < 0.001). The loss of mitochondrial membrane potential was confirmed by JC-1 staining, indicating mitochondrial dysfunction. Western blot analysis indicated a dose-dependent increase in pro-apoptotic proteins Bax and Bid (p < 0.05 at 1.2 µM), an elevated Bax/Bcl-2 ratio (p < 0.001 at 1.2 µM), and enhanced activation of caspase-3, caspase-9, and cleavage of PARP-1 (p < 0.001 at 1.2 µM), indicating activation of the intrinsic apoptotic pathway. Molecular docking analysis revealed a strong binding affinity between WFA and PARP-1 (-8.2 kcal/mol), involving key residues Gly863, Ser904, and Tyr907, thereby supporting its role in PARP-1-mediated apoptosis. The computational findings were consistent with experimental observations of increased PARP-1 cleavage. This study concludes that WFA induces ROS-mediated mitochondrial dysfunction and caspase-dependent apoptosis in neuron-like cells. Consequently, future research should focus on critically assessing the safety and mechanistic effects of individual bioactive constituents of Withania somnifera, despite the well-established therapeutic potential of the plant.