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.