Post-treatment approach for hypoxia and ischemia: Selective targeting of non-nuclear estrogen receptors signaling pathway inhibits neurotoxicity and apoptosis

The aim of this study was to address the urgent global need for an effective therapeutic strategy against acute stroke and perinatal asphyxia. A promising answer to limitations of currently existing therapies might be the newly designed Pathway Preferential Estrogen-1 (PaPE-1). Estrogen receptor-mediated signaling is pivotal for neuroprotection, and its impairment during hypoxia/ischemia may increase the risk of cerebral infarction. Our goal was to selectively activate non-nuclear estrogen receptors signaling, providing a wide therapeutic window and minimizing adverse hormonal effects associated with signaling through nuclear estrogen receptors. Our cellular model of post-treatment in perinatal asphyxia and stroke was constituted by primary neocortical cultures undergoing 6 hours of hypoxia and/or ischemia conditions followed by PaPE-1 being administered for subsequent 18 hours of reoxygenation period. The effectiveness of the treatment was validated by a variety of biochemical and molecular assays, e.g.: AlamarBlue viability assay, LDH release, Fluoro-Jade C staining, mitochondrial membrane potential measurements, neutral red uptake, ROS formation, and quantifying the expression of neurotoxicity- and apoptosis-related genes and proteins using qPCR and ELISAs. Our research proved that post-treatment with PaPE-1 protects brain neurons against hypoxia/ischemia by inhibiting neurotoxicity, neurodegeneration, along with enhancing cellular metabolic activity, reducing oxidative stress and expression of apoptosis-related factors such as FAS, FASL, BAX, BCL-2 and GSK3 β. These findings substantiated that targeting non-nuclear estrogen receptors with PaPE-1 serves as an effective therapeutic intervention against stroke and perinatal asphyxia. Funding: National Science Centre of Poland, grant number 2021/43/D/NZ7/00633.