Neuroprotective effects of estradiol and progesterone against hypoxia-induced apoptosis in mouse primary cortical neurons

Hypoxia leads to neuronal damage and 17β-estradiol (E2) and progesterone (P4) exhibit neuroprotective properties in such conditions. However, the precise mechanisms behind these effects remain unexplored. This study evaluates the neuroprotective effects and underlying mechanisms of E2, P4, and their combination (EP) against hypoxic injury. We created a cerebral hypoxia injury model using the hypoxia/reoxygenation (H/R) technique. Primary cortical neurons from 15-day-old mouse embryos were cultured for 7 days. They were then treated with E2, P4, or EP and subsequently subjected to hypoxia. Neurons were identified using MAP2; and neurites and cell bodies were immunolabeled with SMI-311 and SMI-312. Cell viability and damage were assessed by MTS and lactate dehydrogenase assays, apoptosis by PI/Hoechst staining, and cleaved caspase-3 expression by Western blotting. The results showed that most cultured cells were neurons with well-developed neurites. Hypoxia significantly reduced cell viability and increased neuronal death, with elevated caspase-3 expression. Hormone treatments enhanced cell viability and reduced neuronal death. To investigate the potential mechanism, we analyzed the cleaved caspase 3 expression but no significant differences in caspase-3 expression were observed between groups. Overall, hormone therapy alleviated hypoxic injury but did not affect caspase activity. This study provides insights into the neuroprotective mechanisms of hormone therapy.