补益脾胃元气方药对Aβ诱导的海马神经元JNK及p38MAPK信号通路相关蛋白及其磷酸化表达的影响
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摘要
目的:通过研究补益脾胃元气类方药人参、大补元煎、洗心汤对β淀粉样蛋白(β-amyloid protein,Aβ)诱导的海马神经元JNK(c-Jun N-terminal kinase,JNK)及p38MAPK(p38mitogen-activated protein kinase,p38MAPK)信号通路的影响,探讨补益脾胃元气方对Aβ诱导的海马神经元损伤的保护作用及防治阿尔茨海默病(Alzheimer'sdisease,AD)的可能机制。方法:通过寡聚肽Aβ_(1～42)诱导海马神经元损伤模拟AD细胞模型;分别以蒸馏水、人参、洗心汤、大补元煎颗粒剂溶液对新西兰家兔灌胃给药,14天后抽取各组脑脊液,与终浓度为25μmol/L的寡聚肽Aβ_(1～42)单独或共同作用于原代海马神经元48小时,分别检测模型组、正常脑脊液对照组、安理申组、人参组、大补元煎组、洗心汤组海马神经元JNK和磷酸化JNK(phosphorylated JNK,p-JNK)及p-38MAPK和磷酸化p38MAPK(phosphorylated p38,p-p38MAPK)的表达水平。结果:对培养至7～8天的成熟原代海马神经元,进行神经元树突标志物微管相关蛋白-MAP2标记,结果显示原代海马神经元在所培养的总细胞中所占比例达到95%以上。免疫荧光实验显示,人参、大补元煎及洗心汤三组补益脾胃元气方药中p-JNK、p-p38MAPK的表达明显降低(P<0.05),蛋白印迹实验中三组补益脾胃元气方药中p-JNK、p-p38MAPK的表达同样降低(P<0.05)。结论:补益脾胃元气方药可通过抑制JNK和p38MAPK信号通路中关键信号分子p-JNK、p-p38MAPK蛋白的表达,有效拮抗Aβ_(1～42)神经毒性,抑制由Aβ_(1～42)诱导的JNK和p38MAPK信号通路激活所引发的炎症信号通路活性增强及细胞损伤凋亡发生,起到神经保护作用。
引文
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[2] Cerpa W,Dinamarca MC,Inestrosa C.Structure-function implications in Alzheimer's disease:effect of Aboligomers at central synapses[J].Curr Alzheimer Res,2008,5(3):233～243
[3] Kim W,Kim Y,Min J,et al.A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's pep-tide[J].ACS Chemical Biol,2006,1(7):461～469
[4] 曹方圆,罗德欣,黄玉雕.一种稳定阿尔茨海默病细胞模型的建立方法[J].中风与神经疾病杂志,2017,34(3):221～223
[5] 柴高尚,李宜赔,王黎.Humanin 对Aβ1_42所致阿尔茨海默病大鼠空间记忆的影响及其作用机制[J].中华神经医学杂志,2010,9(3):253～257
[6] Bogoyevitch MA,Boehm I,Oakley A,et al.Targeting the JNK MAPK cascade for inhibition:basic science and therapeutic potential[J].Biochim Biophys Acta,2004,1697:89～95
[7] 陶迎红,王学美,杨金霞.等.经皮穿刺延髓池抽取兔和大鼠脑脊液的方法富宏[J].中国比较医学杂志,2006,16(11):686～687
[8] 李国民,成海燕,于建春,等.快速老化鼠SAMP8认知障碍与海马神经元丢失相关性探讨[A].快速老化鼠SAMP8认知障碍与海马神经元丢失相关性探讨[C].中国河南欧阳,2010-08-20:539～540
[9] Thornton TM,Rincon M.Nos-classical p38map kinase functions:cell cycle ckeckpoints and survival[J].Int J Biol Sci,2009,5(1):44～51
[10] Ji RR,Gereau RW 4th,MalcangioM,et al.MAP kinase and pain [J].Brain Res Rev,2009,60(1):135～148
[11] Bogoyevitch MA,Boehm I,Oakley A,et al.Targeting the JNK MAPK cascade for inhibition:basic science and therapeutic potential[J].Biochim Biophys Acta,2004,1697:89～95
[12] Dhanasekaran D N,Reddy E P.JNK signaling in apoptosis[J].Oncogene,2008,27(48):6245～6251
[13] Liu J,Lin A.Role of JNK activation in apoptosis:a double-edged sword[J].Cell Res,2005,15(1):36～42
[14] Weston CR,Davis RJ.The JNK signal transduction pathway[J].Currop in Cell Biol,2007,19(2):142～149
[15] 黄德弘,刘孟渊,闫小峰.加味当归芍药散对纤丝状Aβ1～42诱导脑内炎症因子及磷酸化MAPK信号分子表达的影响[J]中国实验方剂学杂志,2011,17(2):143～148
[16] Morel C,Ibarz G,Oiry C,et al.Cross-interactions of two p38mit ogen activated protein (MAP) kinase inhibitors and two cholecystokin in(CCK) receptor antagonists with the CCK1receptor and p38MAP kinase[J].J Biol Chem,2005,280(22):21384～21393
[17] Ambrosino C,Nebreda AR.Cell cycle regulation by p38MAP kinase[J].Biol Cell,2001,93(1～2):47～51
[18] Hochedlinger K,Wagner EF,Sabapathy K.Differential effects of JNK1 and JNK2 on signal specific induction of apoptosis[J].Oncogene,2002,21(15):2441～2445
[19] Bogoyevitch MA,Kobe B.U ses for JNK:the many and varied substrates of the c-Jun N-terminal kinases[J].Microbiol Mol Biol Rev,2006,70(4):1061～1095
[20] 张冲.P38MAPK和JNK信号通路在阿尔茨海默病、帕金森病患者外周血淋巴细胞中的表达及意义[D].河北:河北医科大学,2012
[21] 粟广辉.加味洗心汤合多奈派齐治疗老年性痴呆的临床研究[J].中医临床研究,2013,5(11):26～28
[22] 王清峰,张丹,李柱.健脾化痰法治疗老年性痴呆痰浊阻窍证的疗效及机制研究[J].中医临床研究,2013,5(2):12～13
[23] 刘华.洗心汤对老年血管性痴呆中医智能综合评价及生活治疗的影响[J].四川中医,2016,34(11):101～103