针刺研究

2019, v.44(06) 424-429

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以生物信息学工具探讨“三焦”针法治疗老年性痴呆的细胞生物学机制
Using bioinformatics tools to explore cellular biological mechanisms of “Triple Energizer Acupuncture Method” in treating senile dementia

刘涛;张雪竹;韩景献;聂坤;
LIU Tao;ZHANG Xue-zhu;HAN Jing-xian;NIE Kun;The First Teaching Hospital of Tianjin University of Traditional Chinese Medicine;

摘要(Abstract):

目的:观察"三焦"针法对快速老化痴呆小鼠(SAMP8)痴呆状况的影响,探讨"三焦"针法治疗老年性痴呆的细胞生物学机制。方法:将7月龄SAMP8随机分为对照组、穴位组和非穴组,每组20只。穴位组取"膻中""中脘""气海""血海""足三里"穴针刺治疗,每穴针刺30s,每周针刺6d,连续4周。以Morris水迷宫隐蔽平台实验、空间探索实验检测SAMP8学习记忆能力的变化;提取小鼠海马组织,以基因芯片方法获得针刺相关的差异表达基因,再通过生物信息数据库工具分析差异表达基因的信息,利用等重同位素定量蛋白质组学技术(iTRAQ)和免疫荧光技术观察海马神经细胞及突触形态验证基因芯片分析结果。结果:与对照组和非穴组相比,穴位组小鼠第4、5天的逃避潜伏期显著缩短,停留在原象限平台的时间显著增加(P<0.05)。与非穴组比较,穴位组上调差异基因的细胞定位主要集中于神经元突起、细胞骨架。穴位组286个表达上调的蛋白中,与神经突起、细胞骨架相关的蛋白组分共有47个,多于非穴组的11个。免疫荧光结果显示,与对照组和非穴组比较,穴位组海马组织神经元突起结构更完整、更密集。穴位组神经元突起高亮区高度显著大于非穴组和对照组(P<0.05)。结论:调节神经元突起和细胞骨架相关基因和蛋白表达及相关功能,可能是"三焦"针法治疗老年性痴呆的细胞学机制之一。
Objective To observe the effect of needling "Danzhong"(CV17),"Zhongwan"(CV12),"Qihai"(CV6),"Xuehai"(SP10)and"Zusanli"(ST36)(Triple Energizer Acupuncture Method)on the learning-memory ability and profile of hippocampal differentially-expressed genes and proteins of SAMP8 mice(rapid aging mice),so as to explore its underlying mechanisms in improving Alzheimer's disease(AD).Methods A total of 60 SAMP8 were used as the dementia model and randomly divided into blank control,acupoint and non-acupoint groups(n=20 per group).The 5 acupuncture points and non-acupoints(subcostal region)on the bilateral sides were needled with filiform needles and manipulated manually for 30 sat each one,once daily,6 times a week for 4 weeks.The Morris water maze tests(location navigation tests and space probing trials)were used to evaluate the mouse's learning-memory ability.The hippocampal tissue was extracted to detect differentially expressed genes and proteins related to acupuncture intervention by gene chip and isobaric tags for relative and absolute quantitation(iTRAQ)techniques as well as bioinformatic analysis,separately.The information is analyzed through bioinformatics database tools.Finally,immunofluorescence staining was used to verify the results of microarray analysis.Results Compared with mice of the control and non-acupoint groups,the escape latency of location navigation task of Morris water maze tests on 4 th and 5 th day of training was significantly shortened in mice of the acupoint group,and the duration of stay in the original safe-platform quadrant was significantly increased in the acupoint group(P <0.05).Gene microarray displayed that in comparison with the control group,898 differentially expressed genes were up-regulated,418 genes were down-regulated in the hippocampus of acupoint group.The iTRAQ analysis indicated that in the acupoint and non-acupoint groups,286 and 299 differentially expressed proteins were up-regulated,319 and 179 proteins down-regulated,respectively.Of the 34 terms containing 47 proteins up-regulated by acupoint needling,including intermediate filament,keratin filament,myelin sheath,postsynaptic density,neuron projection were related with neurite and cytoskeleton.While in the non-acupoint group,of the 24 terms were listed by the system,only the myelin sheath involving 11 differentially expressed proteins functions in activities of neurite and cytoskeleton.Immunofluorescence staining of the hippocampal tissue showed that the high-density distribution areas of neurons and neurite fibers were characterized by decentralization and disordering,with the highlighted areas being mainly near the cell body parts in control mice,but in mice of the acupoint group,the highlighted areas at the neurite were relatively dense,the morphology of hippocampal cells was complete,the fiber structure was clear,dense and orderly,and the neurites were closely arranged and in order,indicating an improvement of the distribution and arrangement of nerve fibers after acupuncture.The height of neurite highlight area of the acupoint group was significantly higher than that of the non-acupoint group(P<0.05).Conclusion The "Triple Energizer Acupuncture"of acupoints is able to improve the learning-memory ability in SAMP8,which may be related to its effects in regulating the expression and function of hippocampal genes and proteins related to neurite and cytoskeleton.

关键词(KeyWords): 阿尔茨海默病;快速老化痴呆小鼠;生物信息学;神经突起;细胞骨架
Alzheimer's disease;Senescence accelerated mice prone 8;Bioinformatics;Neurite protuberance;Cytoskeleton

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金面上项目(No.81574049、81473766)

作者(Author): 刘涛;张雪竹;韩景献;聂坤;
LIU Tao;ZHANG Xue-zhu;HAN Jing-xian;NIE Kun;The First Teaching Hospital of Tianjin University of Traditional Chinese Medicine;

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DOI: 10.13702/j.1000-0607.180412

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