The Synergistic Combination of Everolimus and Paroxetine Exerts Post-ischemic Neuroprotection In Vitro

Ischemic stroke is a debilitating multi-factorial cerebrovascular disorder, representing an area of tremendous unmet medical need. Combination treatment has been proposed as a promising therapeutic approach towards combating ischemic stroke. The present study employs in vitro oxygen glucose deprivation (OGD) model to evaluate the post-ischemic neuroprotective efficacy of Everolimus and Paroxetine, alone and in combination. Post-OGD treatment with Everolimus and Paroxetine, alone or in combination, significantly improved the cell survival (similar to 80%) when compared to the cells subjected to ischemic injury alone. The individual neuroprotective doses of Everolimus and Paroxetine were found to be at 6.25 and 25 nM, respectively. Whereas, the synergistic neuroprotective dose for Everolimus:Paroxetine was 2:10 nM, calculated using the Chou-Talalay combination index and other four mathematical models. The synergistic combination dose downregulated neuroinflammatory genes (Tnf-alpha, Il1b, Nf-kappa B, and iNos) and upregulated the neuroprotective genes (Bcl-2, Bcl-xl, Hif-1, and Epo). The mitochondrial functioning and ROS neutralizing ability increased with combination treatment. Further, the active role of nitric oxide synthase and calmodulin were revealed while exploring the bio-activity of Everolimus and Paroxetine through network pharmacology. The present study for the first time demonstrates the synergistic post-ischemic neuroprotective efficacy of combination treatment with Everolimus and Paroxetine in vitro. Taken together, these findings clearly suggest that Everolimus in combination with Paroxetine may represent a promising therapeutic strategy for the treatment of ischemic stroke, further supporting the combination treatment strategy for this debilitating disorder.