【摘要】 【目的】观察补阳还五汤预干预对全脑缺血大鼠皮层神经元L型Ca2+通道的影响,探讨Ca2+信号异常参与缺血性神经元损伤的机制及补阳还五汤抗脑缺血的分子机制。【方法】72只SD大鼠随机分为12组,即假手术组(2组)、模型组和补阳还五汤组(此2组各分为缺血再灌注后2、12、24、48、72 h 5个时间点组);补阳还五汤各组按0.64 g/kg剂量灌胃,每天2次,连续5 d。除假手术组外,各组均参照改良的Pulsinelli 4血管闭塞法复制全脑缺血大鼠模型,缺血后的大鼠分别在存活2、12、24、48、72 h后进行皮层神经细胞急性分离,单通道电流经EPC-9膜片钳放大器放大,采用Pulse&Pulsefit采集入计算机,检测各组大鼠血流再灌注后不同时间点的L型Ca2+通道的平均开放时间和开放概率。【结果】模型大鼠皮层神经元L型Ca2+通道因缺血激活而开放,其开放时间在再灌后各时间点均比假手术组延长,开放概率分别在再灌注2 h和24h时出现高峰;而补阳还五汤组的皮层神经元L型Ca2+通道的开放时间在再灌72 h时比模型组降低(P<0.01),其开放概率在模型组出现第1个高峰时(再灌2 h)被显著性地降低至与假手术组相仿水平(与模型组比较P<0.01,与假手术组比较P>0.05)。【结论】补阳还五汤在缺血再灌早期(再灌2 h),主要通过降低L型Ca2+通道开放概率,即影响L型Ca2+通道的可利用性以减少Ca2+内流。而在缺血再灌后期(再灌72 h),主要通过降低L型Ca2+通道开放时间,即影响L型Ca2+通道开放特性以减少Ca2+内流。更多还原

【Abstract】 【Objective】 The effect of pretreatment with Buyang Huanwu Decoction(BHD) on L-type calcium channels of cortical neurons in rats with global cerebral ischemia was observed,thus to explore the molecular therapeutic mechanism of BHD for cerebral ischemia as well as the pathogenic mechanism of Ca2+ message in ischemic injury of neurons.【Methods】SD rats were randomized into pseudo-operation group,model groups and BHD groups.And the model groups and BHD groups were divided into six groups according to the time points after reperfusion(2nd,12th,24th,48th and 72nd hour after reperfusion respectively).BHD groups received 0.64g/kg BHD by gastric infusion,bid,for 5 continuous days.Except the rats in pseudo-operation groups,the rats in other groups were induced global cerebral ischemia by modified Pulsinelli’s four-vessel occlusion.Cerebral cortical neurons in rats were isolated promptly in 2nd,12th,24th,48th and 72nd hour after reperfusion.The recorded single-channel current was amplified by the EPC-9 patch-clamping amplifier,and then input into the computer by Pulse+Pulsefit.The analytical software TAC was used to detect the opening time and opening probability of the L-type Ca2+ channels in rats at different time points after reperfusion.【Results】In the model groups,the opening time of the L-type Ca2+ channels was prolonged after reperfusion as compared with that in the pseudo-operation group,and the opening probability of the L-type Ca2+ channels arrived at the peaks in 2nd and 24th hour after reperfusion.In BHD groups,the opening time in 72nd hour after reperfusion was decreased(P<0.01 as compared with that in the model group),and the first peak of the opening probability was lowered,similar to that of the pseudo-operation group(P<0.01 as compared with that in the model group).【Conclusion】At the early stage of ischemia/reperfusion(in 2nd hour after reperfusion),the molecular therapeutic mechanism of BHD for cerebral ischemia is related to the decrease of opening probability of the L-type Ca2+ channels,i.e.,having an effect on the availability of the L-type Ca2+ channels thus to decrease the internal influx of Ca2+;At the late stage of ischemia/reperfusion(in 72nd hour after reperfusion),the molecular therapeutic mechanism of BHD for cerebral ischemia is related to the decrease of opening time of the L-type Ca2+ channels,i.e.,having an effect on the opening characteristics of the L-type Ca2+ channels thus to decrease the internal influx of Ca2+.更多还原