Xu Xiaohong, Zheng Xiaoxiang
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
J Ethnopharmacol. 2007 Sep 25;113(3):421-6. doi: 10.1016/j.jep.2007.06.012. Epub 2007 Jul 3.
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.
本研究旨在阐明葛根素（Pur）对脑缺氧缺血的神经保护机制。从2日龄的Sprague-Dawley大鼠制备原代海马培养物。体外培养8天后，对培养物进行3小时氧/葡萄糖剥夺（OGD）。通过膜联蛋白-V和碘化丙啶（PI）标记细胞的流式细胞术分析发现，在3小时OGD期间及随后的24小时内添加Pur可使培养的海马神经元中的凋亡和坏死显著减少。Pur（40和100 microM）还减轻了谷氨酸（Glu）诱导的神经元损伤，抑制了0.5mM Glu诱导的凋亡和坏死。此外，分别用Ca(2+)探针Fluo-3和NO探针二氨基荧光素二乙酸酯（DAF DA）通过共聚焦激光扫描显微镜测量细胞内Ca(2+)的变化和一氧化氮（NO）的生成。与流式细胞术分析结果一致，Pur（40和100 microM）显著减缓了OGD诱导的Ca(2+)内流并降低了细胞内Ca(2+)峰值。同时，Pur显著抑制了OGD诱导的NO合成。我们的研究结果表明，Pur可改善体外OGD诱导的海马神经元死亡。Pur的保护作用与抑制谷氨酰胺能递质的作用、细胞内Ca(2+)升高和神经元NO合成有关。
