栝楼桂枝颗粒对神经元兴奋性毒性损伤后钙信号通路的影响

Effect of Gualou Guizhi Granule on Calcium Signaling Pathway after Excitotoxicity Injury of Neurons

关键词:  栝楼桂枝颗粒  N-甲基-D-天冬氨酸  CREB  CaMKII  CaM

Key words:  Gualou Guizhi granule  N-methyl-D-aspartic acid(NMDA)  CREB  CaMKII  CaM

摘要:

目的 研究栝楼桂枝颗粒对N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid，NMDA）诱导神经元兴奋性毒性损伤后钙信号通路的影响，并探讨其可能的机制。方法 建立NMDA诱导神经元兴奋性毒性损伤模型，栝楼桂枝颗粒干预后，采用MTT、LDH法检测神经元活性，免疫荧光染色法检测神经元特异性指标MAP-2蛋白的表达。Real-time PCR法检测神经元中CaMKⅡ、CREB mRNA的表达，Western blot法检测p-CREB、p-CaMKⅡ、CaMKⅡ、CREB及CaM蛋白的表达。结果 与NMDA组比较，栝楼桂枝颗粒（200，300 μg·mL^-1）组可显著提高细胞活力，降低LDH浓度（P<0.05或P<0.01），明显升高CREB、CaMKⅡ mRNA水平（P<0.05或P<0.01），明显升高MAP-2、p-CREB、p-CaMKⅡ及CREB水平（P<0.01），而显著降低CaM水平（P<0.01）。结论 栝楼桂枝颗粒对NMDA诱导的神经元兴奋性毒性损伤具有保护作用，此作用可能与促进CREB、CaMKⅡ的磷酸化，抑制CaM的表达有关。

Abstract:

OBJECTIVE To study the effect of Gualou Guizhi granule on the calcium signal pathway in N-methyl-D-aspartic acid(NMDA)-induced neuronal excitability toxicity, intend to illustrate its possible mechanism. METHODS The excitotoxicity damage model of neurons induced by NMDA was established. The activity of neuron was detected by MTT and LDH, and the expression of MAP-2 protein was detected by immunofluorescence staining after administering with the Gualou Guizhi granule. Real-time PCR was used to detect the expression of CaMKⅡ and CREB in neurons. And the expression of p-CREB, p-CaMKⅡ, CaMKⅡ, CREB and CaM protein were detected by Western blot. RESULTS Compared with NMDA group, Gualou Guizhi granule (200,300 μg·mL^-1) group significantly increased cell viability and decreased LDH concentration (P<0.05 or P<0.01), significantly increased the mRNA levels of CREB and CaMKⅡ(P<0.05 or P<0.01), significantly increased the levels of MAP-2, p-CREB, p-CaMKⅡ and CREB(P<0.01), and significantly decreased CaM level(P<0.01). CONCLUSION Gualou Guizhi granule have protective effect on the excitotoxicity damage induced by NMDA, which may be related to the phosphorylation of CREB and CaMKⅡ and the inhibition of the expression of CaM.