摘要
目的:通过研究大补元煎对淀粉样前体蛋白/早老素1(APP/PS1)小鼠海马Wnt/β-连环蛋白(β-catenin)信号通路的作用,探讨大补元煎促进神经发生的可能机制。方法:5月龄APP/PS1小鼠30只和野生鼠10只,分为正常组、模型组、奈哌齐组(6.5×10^-4g·kg^-1·d^-1)和大补元煎组(13.2 g·kg^-1·d^-1),灌胃30 d,正常组和模型组给予等体积生理盐水。应用水迷宫评价小鼠学习记忆力;应用苏木素-伊红(HE)染色观察小鼠海马神经元病理形态变化;应用免疫组化(IHC)检测5-溴脱氧尿苷(Brdu),肾上腺皮质激素(Dcx)和神经元核抗原(Neu N)的阳性细胞数;应用实时荧光定量聚合酶链式反应(Realtime PCR)和蛋白免疫印迹法(Western blot)检测小鼠海马中β-catenin,糖原合成酶激酶-3β(GSK-3β)mRNA和蛋白表达水平。结果:与正常组比较,模型组小鼠平台潜伏期和游泳总路程显著升高(P<0.01),而穿越平台次数和目标象限时间显著降低(P<0.01);与模型组比较,多奈哌齐组和大补元煎组小鼠平台潜伏期和游泳总路程降低(P<0.05,P<0.01),而穿越平台次数和目标象限时间升高(P<0.05)。与正常组比较,模型组小鼠海马齿状回(DG)区神经元层次和数量减少,可见明显坏死的神经元;与模型组比较,多奈哌齐组和大补元煎组小鼠海马DG区神经元层次和数量增多,坏死神经元数量减少。与正常组比较,模型组小鼠海马DG区Brdu,Dcx和Neu N标记的阳性细胞数明显降低(P<0.01);与模型组比较,多奈哌齐组和大补元煎组小鼠海马DG区Brdu,Dcx和Neu N标记的阳性细胞数增加(P<0.05,P<0.01)。与正常组比较,模型组小鼠海马中β-catenin mRNA和蛋白表达水平显著下降(P<0.01),而GSK-3β的基因和蛋白表达水平显著升高(P<0.01);与模型组比较,多奈哌齐组和大补元煎组小鼠海马β-catenin mRNA和蛋白表达水平升高(P<0.05,P<0.01),而GSK-3βmRNA和蛋白表达水平下降(P<0.05,P<0.01)。结论:大补元煎通过调节Wnt/β-catenin信号通路促进APP/PS1双转基因小鼠海马神经发生。
Objective:To investigate the possible mechanism of Dabuyuan Jian to promote neurogenesis by studying the effect of Dabuyuan Jian on Wnt/β-catenin signaling pathway in hippocampus of amyloid precursor protein/presenilil(APP/PS1)mice.Method:Totally 305-month-old APP/PS1 mice and 10 wild mice were divided into control group,model group,donepezil group(6.5×10^-4g·kg^-1·d^-1)and Dabuyuan Jian group(13.2 g·kg^-1·d-^1),and given drugs by gavage for 30 days.The control group and the model group were given an equal volume of saline.The learning and memory of mice were evaluated by water maze.The pathological changes of hippocampal neurons were observed by hematoxylin-eosin(HE)staining.The immunohistochemistry(IHC)was used to detect label positive cells of 5-bromodeoxyuridine(Brdu),adrenocortical hormone(Dcx)and neuronal nuclear antigen(NeuN).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western bolt were used to detect the mRNA and protein expression levels ofβ-catenin and glycogen synthase kinase-3β(GSK-3β)in hippocampus of mice.Result:Compared with the control group,the latency and swimming total distance of the model group were significantly increased(P<0.01),while the number of crossing platforms and the target quadrant time were significantly reduced(P<0.01).Compared with the model group,the platform latency and the total swimming distance of the donepezil group and the Dabuyuan Jian group were decreased(P<0.05,P<0.01),while the number of crossing platforms and the target quadrant time were increased(P<0.05).Compared with the control group,the number of neurons in the hippocampal dentate gyrus(DG)area of the model group was decreased,and necrotic neurons were observed.Compared with the model group,the number of neurons in the hippocampal DG area of the mice in the donepezil group and the Dabuyuan Jian group was increased,while the number of necrotic neurons was decreased.Compared with the control group,the number of positive cells labeled with Brdu,Dcx and NeuN in the hippocampal DG area of the model group was significantly decreased(P<0.01).Compared with the model group,the number of positive cells labeled with Brdu,Dcx and NeuN in the hippocampal DG area of the donepezil group and the Dabuyuan Jian group was increased(P<0.05,P<0.01).Compared with the control group,gene and protein expression levels ofβ-catenin in the hippocampus of the model group were significantly decreased(P<0.01),whereas gene and protein expression levels of GSK-3βwere significantly increased(P<0.01).Compared with the model group,gene and protein expression levels ofβ-catenin in hippocampus of donepezil group and Dabuyuan Jian group were increased(P<0.05,P<0.01),while gene and protein expression levels of GSK-3βwere increased(P<0.05,P<0.01).Conclusion:Dabuyuan Jian could promote hippocampal neurogenesis in APP/PS1 double transgenic mice by regulating Wnt/β-catenin signaling pathway.
作者
何丽玲
龙清华
胡慧
王平
石和元
HE Li-ling;LONG Qing-hua;HU Hui;WANG Ping;SHI He-yuan(School of Basic Medicine,Hubei University of Chinese Medicine,Wuhan 430065,China;School of Nursing,Hubei University of Chinese Medicine,Wuhan 430065,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第7期8-14,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金面上项目(81573865).
作者简介
第一作者:何丽玲,在读博士,从事中医药防治老年病研究,E-mail:642755743@qq.com;通信作者:石和元,博士,副教授,硕士生导师,从事中医衰老理论及老年病证治疗规律研究,E-mail:84037493@qq.com
