Effect of caspase-9 inhibition on endoplasmic reticulum stress induced cortical neuronal injury in rats.

Our study investigated the apoptotic mechanism of rat cortical neurons following hypoxia/reperfusion induced endoplasmic reticulum stress (ERS) in vitro and to explore the effect of caspase-9 inhibition on ERS induced apoptosis. Cortical neurons were collected from neonatal rats and cultured in vitro. Immunohistochemistry and immunofluorescence staining for neuron-specific enolase (NSE) were performed to determine the purity of neurons. AnnexinV/PI staining followed by flow cytometry was employed to detect apoptosis rate. Fluorescein isothiocyanate (FITC) staining was done to measure the expression of caspase-3 and -9. Western blot assay was carried out to measure the protein expression of caspase-12, glucose-regulated protein (GRP) 78 and Cytochrome C. The cortical neurons from neonatal rats could be purified and cultured in vitro. In the in vitro hypoxia/reperfusion of cortical neurons (hypoxia for 6 h and reperfusion for 24 h and 48 h), the protein expression of GRP78, caspase-3, 9 and 12 was markedly increased (P < 0.01). Following pre-treatment with caspase-9 inhibitor, the number of apoptotic cells was significantly reduced following hypoxia for 6 and reperfusion for 24 h or 48 h (P < 0.05). Moreover, the expression of caspse-3 and 12 and GRP78 was also significantly reduced in the presence of caspase-9 inhibitor treatment (P < 0.05), but the release of Cytochrome C remained unchanged (P > 0.05). These results demonstrated that ERS is involved in the neuronal apoptosis following in vitro hypoxia/reperfusion, and caspase-9 inhibition can depress the ERS induced apoptosis of neurons.