红景天苷对大鼠体外培养海马神经元物理缺氧损伤钙离子含量、钙激活中性蛋白酶及钙通道蛋白表达的影响

Expression influence of salidroside on calcium ion content,calcium activated neutral protease and calcium channel protein of rat hippocampal neurons cultured in vitro by physical hypoxic injury

目的研究红景天苷对大鼠海马神经元物理缺氧损伤的保护作用及机制。方法体外原代培养新生SD大鼠海马神经元并鉴定。以尼莫地平为阳性对照药,通过苏木素-伊红染色、激光共聚焦显微镜技术观察各实验组神经元形态,通过荧光分光光度法检测各实验组神经元细胞内游离钙离子浓度([Ca2+]i),实时定量聚合酶链反应检测各实验组钙通道蛋白NMDAR1、Cav1.3 mRNA的表达差异,免疫细胞化学染色法检测各实验组钙激活中性蛋白酶Calpain 1蛋白质的表达差异,分析红景天苷对大鼠海马神经元物理缺氧损伤的保护作用。结果红景天苷各剂量组均可提高缺氧神经元存活率。与模型组比较,红景天苷各剂量组神经元[Ca2+]i显著降低(P<0.01,P<0.01,P<0.05);NMDAR1和Cav1.3 mRNA的表达率均显著降低(P<0.05或P<0.01);Calpain 1阳性细胞百分率均显著降低(P<0.01或P<0.05)。结论红景天苷通过下调物理缺氧神经元L-型钙通道和NMDAR亚基的表达抑制钙超载,对神经元起保护作用。

Objective To study the effects and mechanisms of salidroside against hypoxia injury on hippocampus neuron of rats. Methods Hippocampus neurons of Sprague-Dawley neonatal rats（24 h） were cultured in vitro,and then they were identified.The positive control group of physical hypoxia was nimodipine.Forms on each experimental unit of hippocampus neurons were examined by hematoxylin and eosin stain and laser confocal microscopy（LSCM）.Intracellular free calcium ion concentration（i） of neurons in each group was detected by fluorospectrophotometry.Changes in expression of NMDAR1,Cav1.3 mRNA in each group were detected by real-time polymerase chain reaction.Changes in expression of calcium activated neutral protease 1（Calpain 1）were detected by immunocytochemistry stain.And the protective effect of salidroside against hypoxia injury on hippocampus neuron of rats was analysed. Results The high,intermediate and low dose of salidroside increased the survival rate of neuron which was subjected to hypoxia.Compared with model group,in the salidroside group with different dosage,the intracellular free calcium ion concentration（i） of neurons decreased significantly（P0.01,P0.01,P0.05）;the expression levels of NMDAR1 and Cav1.3 mRNA decreased significantly（P0.05 or P0.01）,while the positive cell percentage of Calpain 1 decreased too（P0.01 or P0.05）. Conclusion Salidroside play protective effect on rat neuron by attenuating the calcium overload induced by physical hypoxia by depressing the expression level of NMDAR1 and Cav1.3 mRNA.