Potential involvement of calcium and nitric oxide in protective effects of puerarin on oxygen-glucose deprivation in cultured hippocampal neurons.

The aim of this study was to clarify the mechanisms underlying neuroprotection of puerarin (Pur) against cerebral hypoxia-ischemia. Primary hippocampal cultures were prepared from 2-day-old Sprague-Dawley rats. After 8 days in vitro, the cultures subjected to 3h oxygen/glucose deprivation (OGD). Flow cytometric analysis of annexin-V and propidium iodide (PI) labeling cells found that apoptosis and necrosis were significantly reduced in the cultured hippocampal neurons by addition of Pur during 3h OGD and for the following 24h. Pur (40 and 100 microM) also attenuated glutamate (Glu) induced neuronal damage, suppressing apoptosis and necrosis induced by Glu of 0.5mM. Furthermore, the changes in intracellular Ca(2+) and generation of nitric oxide (NO) were measured by confocal laser scanning microscopy with Fluo-3, a Ca(2+) probe, and diaminofluorescein diacetate (DAF DA), a NO probe, respectively. In agreement with the results from flow cytometric analysis, Pur (40 and 100 microM) markedly slowed down OGD-induced Ca(2+) influx and lowered the intracellular Ca(2+) peak. Meanwhile, NO synthesis induced by OGD was significantly inhibited by Pur. Our findings suggest that Pur can ameliorate hippocampal neuronal death induced by OGD in vitro. The protective effects of Pur are associated with inhibiting the action of glutaminergic transmitter, intracellular Ca(2+) elevation and neuronal NO synthesis.