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·L^-1 L-glutamate, separately. Breviscapine (10, 20 and 40 μmol·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·L^-1 L-glutamate and down-regulation by 0.5 and 1.0 mmol·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 μmol·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 μmol·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.