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
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摘要
目的研究栝楼桂枝颗粒对N-甲基-D-天冬氨酸(N-methyl-D-asparticacid,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(200,300μg·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。
OBJECTIVE To study the effect of Gualou Guizhi granule on the calcium signal pathway in N-methyl-Daspartic 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.
OBJECTIVE To study the effect of Gualou Guizhi granule on the calcium signal pathway in N-methyl-Daspartic 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.
引文
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[3]ZHANG C,DU F,SHI M,et al.Ginsenoside Rd protects neurons against glutamate-induced excitotoxicity by inhibiting Ca2+influx[J].Cell Mol Neurobiol,2012,32(1):121-128.
[4]REDMOND L,KASHANI A H,GHOSH A.Calcium regulation of dendritic growth via Ca M kinase IV and CREB-mediated transcription[J].Neuron,2002,34(6):999-1010.
[5]杨春梅,陈立典,陶静.古方今用瓜蒌桂枝汤[J].辽宁中医杂志,2012,39(8):1599-1600.
[6]CHEN Y L,CHEN L D,TAO J.Clinical research on treating limbs spasm from cerebral apoplexy with the Gualou Guizhi decoction[J].Clin J Chin Med(中医临床研究),2013,5(4):7-9.
[7]ZHU Y P.Clinical effect analysis of Trichosanthes kirilowii Cassia twig decoction for treatment of lower limb spasm after cerebral apoplexy patients[J].Guid China Med(中国医药指南),2015,13(29):27-28.
[8]战美玲.脑卒中后下肢痉挛应用栝楼桂枝汤治疗的效果分析[J].双足与保健,2017(11):160-161.
[9]CHEN X W,LI H,HUANG M Q,et al.Effect of Gua Lou Gui Zhi decoction on focal cerebral ischemia-reperfusion injury through regulating the expression of excitatory amino acids and their receptors[J].Mol Med Rep,2014,10(1):248-254.
[10]HUANG J,TAO J,XUE X H,et al.Gua Lou Gui Zhi decoction exerts neuroprotective effects on post-stroke spasticity via the modulation of glutamate levels and AMPAreceptor expression[J].Int J Mol Med,2013,31(4):841-848.
[11]LI Z F,LIN R H,MAO J J,et al.Protective effect of Gualou Guizhi decoction on BV-2 cells injuried by glutamate[J].JFujian Univ Tradit Chin Med(福建中医药大学学报),2013,23(5):14-17.
[12]ZENG J,ZHANG Y Q,WANG H Y,et al.Mechanism of Gualou Guizhi granule inhibits neuronal cell apoptosis in MCAO rats[J].Chin J Mod Appl Pharm(中国现代应用药学),2018,35(7):1007-1011.
[13]LI Z F,HU H X,LIN R H,et al.Neuroprotective effects of Gua Lou Gui Zhi decoction against glutamate-induced apoptosis in BV-2 cells[J].Int J Mol Med,2014,33(3):597-604.
[14]WANG H Y,ZHANG Y Q,LI H,et al.Neuroprotective effect of Gualou Guizhi granule on NMDA-induced hippocampal neuron excitotoxicity toxicity model[J].China J Tradit Chin Med Pharm(中华中医药杂志),2017,32(9):4115-4120.
[15]CORBYN Z.Statistics:a growing global burden[J].Nature,2014,510(7506):S2-S3.
[16]WU Y M,TAO R Y,MENG X B,et al.Research progress on mechanism of ischemic stroke injury[J].Jilin J Tradit Chin Med(吉林中医药),2018,38(3):368-372.
[17]PAN J Y,CAI R X,CHEN Y,et al.Analysis the effect of hyperbaric oxygen preconditioning on neuronal apoptosis,Ca2+concentration and caspases expression after spinal cord injury in rats[J].Eur Rev Med Pharm Sci,2018,22(11):3467-3473.
[18]POON A D,MCGILL S H,BHUPANAPADU SUNKESULA S R,et al.Ca2+/calmodulin-dependent protein kinase II and Dimethyl Sulfoxide affect the sealing frequencies of transected hippocampal neurons[J].J Neurosci Res,2018,96(7):1208-1222.
[19]RUIZ A,ALBERDI E,MATUTE C.Mitochondrial division inhibitor 1(mdivi-1)protects neurons against excitotoxicity through the modulation of mitochondrial function and intracellular Ca2+signaling[J].Front Mol Neurosci,2018(11):3.