The aim of this study was to investigate the effects of breviscapine on cultured
rat hippocampal neuronal
toxicity induced by
glutamate. Primary hippocampal
neurons were prepared from 2 day-old SD
rats. After 8 days cultured
in vitro, the
cultures subjected to 30 min
treatment of 0.1, 0.5 and 1.0 mmol x L(-1)
L-glutamate, separately. Breviscapine (10, 20 and 40 micromol x L(-1)) was added into the
cultures during 30 min
treatment of
L-glutamate and for the following 24 h respectively. After 24 h of
L-glutamate treatment, flow cytometric
analysis of
Annexin V (marks
apoptosis) and PI (
propidium iodide, marks
necrosis) labeling
cells showed that
L-glutamate dose-dependently induced hippocampal neuronal
apoptosis and
necrosis. In agreement with these results, RT-PCR experiments indicated a biphasic
regulation of X-
chromosome-linked
inhibitor of apoptosis protein (XIAP)
mRNA after
L-glutamate treatment, i. e
up-regulation by 0.1 mmol x L(-1)
L-glutamate and
down-regulation by 0.5 and 1.0 mmol x L(-1)
L-glutamate. However, breviscapine markedly reduced
apoptosis and
necrosis due to
toxicity of 0.5 mmol L(-1)
L-glutamate. Compared with the vehicle-treated
L-glutamate group, the
apoptosis was reduced by 30.4% and 40.1%, and
necrosis was reduced by 32.5% and 38.8%,
after treatment by breviscapine of 20 and 40 micromol x L(-1). Meanwhile, breviscapine obviously reversed the
down-regulation of XIAP expression induced by
L-glutamate (
up-regulation by 45.1% and 54.9% when compared with that of the vehicle-treated
glutamate group). The results from the
detection of
confocal laser scanning microscopy with Fluo-3, a Ca2+ probe showed an obvious increase in intracellular Ca2+ during
L-glutamate treatment; and breviscapine of 20 or 40 micromol x L(-1) significantly slowed down
glutamate-induced Ca2+ influx and lowered the intracellular Ca2+ peak in hippocampal
neurons (P < 0.01). These results suggest that
neuroprotective effect of breviscapine against
glutamate excitotoxicity was associated with inhibition of the accumulation of intracellular Ca2+ and
up-regulation of XIAP expression in hippocampal
neurons.