葛根素对氧糖剥夺大鼠海马神经细胞Ca^(2+)和NO的影响

Effects of Puerarin on the Levels of Calcium and Nitric Oxide in Cultured Hippocampal Neurons during Oxygen-Glucose Deprivation

目的研究葛根素(puerarin,Pur)对缺氧缺糖状态下海马神经细胞内Ca2+和NO水平的影响。方法选用新生大鼠体外培养8 d的海马神经细胞,进行3 h的氧糖剥夺(oxygen/glucose deprivation,OGD)或30 min的L-谷氨酸(0.5mmol·L-1)处理后再正常培养24 h,Pur处理组在OGD或谷氨酸处理的同时和以后24 h给予不同剂量(40,100μmol·L-1)Pur,Annexin V联合流式细胞仪检测细胞的凋亡率和坏死率;以Fluo-3或DAF-2为荧光标记,用激光共聚焦显微镜观测30 min的OGD过程中海马神经细胞Ca2+和NO的动态变化以及Pur的影响。结果OGD诱导海马神经细胞Ca2+和NO水平快速升高,其最高值分别达正常对照组的2.9和1.9倍;Pur明显减缓OGD期间神经细胞Ca2+内流和NO合成,与OGD组相比,40和100μmol·L-1的Pur分别使细胞Ca2+峰值降低29.2%和37.1%(P<0.05和P<0.01),NO峰值降低23%和33%(P<0.05和P<0.01),显著抑制细胞内Ca2+和NO水平的升高;同时Pur可有效抑制OGD和谷氨酸引起的神经细胞死亡。结论Pur可通过抑制神经细胞谷氨酸/Ca2+/NO通路的活动而有效保护缺氧缺糖对神经细胞的损伤作用。

OBJECTIVE To investigate the effects of puerarin（Pur） on the Levels of calcium and nitric oxide in cultured hippocampal neurons during oxygen-glucose deprivation.METHODS Primary hippocampal cultures were prepared from 2 day-old Sprague-Dawley rats.After 8 d in vitro,the cultures subjected to 3 h of oxygen/glucose deprivation（OGD） or 30 min of glutamate treatment.Pur at 40 or 100 μmol·L^-1 were added into the cultures during OGD or treatment of glutamate and for the following 24 h respectively.Apoptosis and necrosis were detected using flow cytometric analysis of Annexin-V and PI labeling cells 24 h after OGD or treatment of glutamate.Futhermore,the changes in the levels of intracellular Ca^2＋ or nitric oxide（NO） were measured by confocal laser scanning microscopy with Fluo-3,Ca^2＋ probe,or DAF-2,NO probe respectively,during 30 min OGD.RESULTS OGD induced a rapid Ca^2＋ influx and NO synthesis,and the peaks of the intracellular Ca^2＋ or NO reached the levels of 2.9 or 1.9 times of the control.However,Pur（40,100 μmol·L^-1） markedly slowed down OGD-induced Ca^2＋ influx and lowered the intracellular Ca^2＋ peak by 29.2% and 37.1%（P〈0.05 and P〈0.01）,and significantly inhibited OGD-induced NO synthesis by 23% and 33%（P〈0.05 and P〈0.01）.Meanwhile,apoptosis and necrosis resulted from OGD or glutamate excitotoxicity were significantly reduced in the cultured hippocampal neurons by addition of Pur.CONCLUSION 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.