Withanolide A Ameliorates Global Cerebral Ischemia by Inhibiting Necroptosis Mediators PARP-1 and RIPK1

Cerebral ischemia is the second leading cause of global mortality and lacks proper therapeutics, which necessitates designing of novel strategies. Necroptotic pathway is involved in cerebral ischemic brain damage and designing multi target inhibitors against necroptotic pathway can be a suitable strategy to combat cerebral ischemic pathophysiological cascade. RIPK1 and PARP1 are established molecular mediators of necroptosis pathway and their inhibition might arrest the necroptotic pathway, thereby promoting cell survival. The present study investigates the ability of the phytochemical Withanolide A (WA) in inhibiting both PARP-1 and RIPK1 in-silico and in-vivo. In-silico molecular docking simulations show that WA inhibits both PARP-1 and RIPK1 with an affinity higher than PJ34 and Nec-1, the respective potent inhibitors. In-vivo studies in male Swiss albino mice were performed to evaluate the neuroprotective and necroptosis inhibiting ability of WA. WA shows an elaborate hydrophobic interaction pattern and greater number of hydrogen bond formation with both the enzymes in comparison to that of the reported inhibitors. In-vivo studies revealed that WA successfully reduces cerebral infarction percentage (p < 0.001) and significantly restores BBB disruption (p < 0.001). Treatment with WA also demonstrates down regulation of neuro-inflammatory cytokines TNFα (p < 0.01) and IL-1β (p < 0.05) signifying inhibition of the necroptosis mediators by WA. WA treatment also enhances survival of brain cortical cells as evident from Annexin-FITC/PI staining. Hence, this study establishes WA as a potential dual target inhibitor of necroptosis pathway which can combat cerebral ischemia induced pathophysiology.