IJCEM Copyright © 2008-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711
Int J Clin Exp Med 2013;6(7):546-551

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

Hong Zhang, Hongzhuan Li, Xueqiu Liu, Jianzhong Bi

Department of Neurology, The Second Hospital of Shandong University, Jinan, Shandong Province, 250033, China; Fourth People’s
Hospital of Jinan City, Jinan Shandong Province, 250031, China

Received June 19, 2013; Accepted July 16, 2013; Epub August 1, 2013; Published August 15, 2013

Abstract: 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. (IJCEM1306020).

Keywords: Cortical neuron, hypoxia/reperfusion, endoplasmic reticulum stress, caspase, apoptosis, immunoblotting

Address correspondence to: Dr. Hong Zhang, Department of Neurology, The Second Hospital of Shandong University, Jinan,
Shandong Province, 250033, China. E-mail: zhang002233@sina.com