[Protective effects of breviscapine against cultured rat hippocampal neuronal toxicity induced by glutamate].
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.
Xu
Xiao-Hong
XH
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004. xuxh63@zjnu.cn
Chen
Yu
Y
Zheng
Xiao-Xiang
XX
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005419:Flavonoids / Q000494:pharmacology*; D018698:Glutamic Acid / Q000633:toxicity*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics
D005419:Flavonoids; D018696:Neuroprotective Agents; D012333:RNA, Messenger; D051636:X-Linked Inhibitor of Apoptosis Protein; C061097:breviscapine; D018698:Glutamic Acid; D002118:Calcium