牛膝活性提取物对大鼠海马神经元生长的促进作用

Promotion effects of an active component isolated from Achyranthes bidentata polypeptides on rat hippocampal neuron growth

目的：研究牛膝活性提取物（ABPPk）对体外培养的大鼠海马神经元生长的促进作用.方法：以体外原代培养的胎鼠海马神经元为模型,通过微管蛋白（β-tubulinⅢ）免疫荧光染色,采用Leica Qwin软件测量不同浓度ABPPk处理24 h对海马神经元突起延伸和突起分支的影响;通过突触结合蛋白（SYN）和突触后致密蛋白（PSD95）双标免疫荧光染色,采用LeicaQwin软件测量ABPPk（250 ng/ml）处理7d对海马神经元突触形成的影响;通过免疫印迹定量分析不同浓度ABPPk作用24 h对海马神经元生长相关蛋白（GAP-43）表达水平的影响,作用7d对PSD-95表达水平的影响,以及ABPPk（250 ng/ml）作用不同时间对磷酸化胞外信号调节激酶（ERK1/2）水平的调节.分析ABPPk对海马神经元的作用与ERK通路的关系.结果：ABPPk可有效促进海马神经元的突起延伸和突触形成,免疫印迹结果显示,ABPPk能显著增加海马神经元GAP-43和PSD-95蛋白表达,作用5 min即能显著上调ERK磷酸化水平,作用15 min ERK磷酸化水平达到峰值,PD98059可抑制该过程.结论：ABPPk能促进体外培养的海马神经元突起生长和突触形成,上调GAP-43和PSD-95蛋白水平,其作用可能与ERK通路的激活有关.

Objective： To determine the effects of an active component isolated from Achyranthes bidentata polypeptides （ABPPk） on rat hippocampal neuron growth. Methods： The hippocampal neurons were photographed under a confocal microscope with fluorescent immunostaining with β-tubulin Ⅲ after treated with different concentrations （10 ng/ml, 50 ng/ml, 250 ng/ml） of ABPPk for 24 hours. The average neurite length, and the number of branching points were analyzed by Leica Qwin software. The synapse density of hippocarnpal neurons treated with 250 ng/ml ABPPk for 7 days was calculated by Leica Qwin software after double-labeled immunostaining with synaptotagmin （SYN） and postsynaptic density protein 95 （PSD-95 ）. Western blotting analysis was performed to examine the expression level of growth associated protein 43 （GAP-43） and PSI）-95 of the hippocampal neurons after treated with ABPPk （10 ng/ml, 50 ng/ml, 250 ng/ml） for 24 hours and 7 days, respectively. Western blotting analysis was also performed to examine the expression level of phosphorylated extracellular signal-related protein kinase 1/2 （ERK1/2） of the hippocampal neurons after treated with ABPPk （250 ng/ml） for different time （5 min, 15 min, 30 min, 1 h, 12 h）. PD98059, an antagonist of ERK was used to treat the hippocampal neurons with ABPPk to analyze the relationship between ABPPk effects and ERK signaling pathway. Results： ABPPk could effectively promote neurite elongation and synapse formation of cultured hippocampal neurons. Western blotting analysis showed that ABPPk up-regulated the expression level of GAP-43 and PSD-95 significantly. The level of phosphorylated ERK of hippocampal neurons was significantly up-regulated after treated with ABPPk for 5 min and reached to the peak value by 15 min treatment with ABPPk. Conclusion： ABPPk may promote the neurite growth and synapse formation of cultured hippocampal neurons, and up-regulate the expression level of GAP-43 and PSD-95. ERK signaling pathway might be involved in the mechanism.