【作者】 郭冠华；

【导师】 王华；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的阳虚证是中医临床常见的证候之一,其病症特点以机体组织器官功能减退，机体反应性降低，代谢活动减弱，热量不足等为主。老年阳虚机体学习与记忆能力减退是导致老年机体生活质量降低和进一步发展为严重智力障碍疾病的早期病症阶段。基于“双固一通”针灸治疗思想指导，本课题以老年阳虚模型大鼠为研究对象，选取关元、足三里、百会实施电针刺激为干预，借助现代研究有关行为学、神经电生理、电镜、蛋白免疫印迹（Western blot）和反转录酶-聚合酶链锁反应（RT-PCR）等科学方法，观察“双固一通”电针对老年阳虚证模型大鼠学习记忆能力改善疗效，以及“双固一通”电针对海马突触长时程增强（LTP）调控改善的相关机制，为“双固一通”电针疗法改善学习记忆能力疗效机制提供科学依据。方法1动物分组与处理清洁级3月龄雄性SD大鼠40只，体重180±20g。随机分为正常对照组（Young group,Y）、老年阳虚模型组（Model group，M）、电针对照组（Electropuncture treatment Control group，EAC）、“双固一通”电针组（Electropuncture treatment group，EA）,每组10只。正常对照组每日皮下注射等量0.9%生理盐水1次，连续47d。其余三组每日颈背部皮下注射125mg/kg D-半乳糖连续40d，再予后腿肌肉注射1.5mg/100g氢化可的松连续7d，制作老年阳虚大鼠模型。正常对照组（Y）组与老年阳虚模型组（M）组不予电针治疗。“双固一通”电针组（EA）取“关元”、“后三里”和“百会”穴，“关元”直刺2～3mm；双侧“后三里”直刺3～5mm，“百会”向后方平刺5mm。其中“关元”与“后三里”隔日交替接一对电极，接通HANS LH202H型电针仪，选用连续波，频率2Hz，强度1mA，通电15min,每周6次，共治疗4周。电针对照组（EAC）取“中极”、“阴陵泉”、“印堂”穴，“中极”、“阴陵泉”隔日交替接一对电极，治疗方法同EA组。各组大鼠于治疗第4周进行学习记忆行为学试验，治疗并试验结束后，禁食24h取材、进行相应指标检测。2检测指标通过Morris水迷宫开展定位航行试验、空间探索试验检测大鼠行为学空间学习记忆能力；酶联免疫吸附测定法（ELISA）检测大鼠血清cAMP、cGMP含量；以及海马BDNF含量；BL420型电生理仪检测海马长时程增强（LTP）海马兴奋性突触后电位(fEPSP)；电镜观察大鼠海马CA1区突触结构,并采用MoticImages Advanced3.0图像分析系统测量分析突触界面曲率、突触活性区长度、突触后致密物厚度和突触间隙宽度；Western blot检测海马NMDA受体蛋白表达；RT-PCR检测海马PKCmRNA的表达。3统计分析全部数据均采用SPSS19.0统计学软件进行分析，数据以均数±标准差（x±s）的形式表示，两样本均数的比较采用独立样本的t检验，多样本均数的比较采用单因素方差分析。结果1大鼠血清中cAMP、cGMP含量及cAMP/cGMP比值比较酶联免疫吸附法检测大鼠血清中cAMP、cGMP含量，与Y组比较，M组、EA组、EAC组造模大鼠血清cAMP水平降低、cGMP升高，cAMP/cGMP比值降低，组间比较均有显著差异（P＜0.01），M组、EA组、EAC组大鼠自实验处理第48天时（即造模完成时）观察出现肢体蜷曲、体重下降、反应迟钝、消瘦、少食、畏寒肢冷、毛松、竖毛、失去光泽及脱毛等明显虚损症状,提示造模成功。2大鼠定位航行逃避潜伏期比较经过5天的Morris水迷宫检测实验，各组大鼠寻找平台的平均潜伏期（s）从第三天开始各组大鼠寻找平台的平均潜伏期有明显的变化，并且随着训练次数的增加，至第五天各组潜伏期均有不同程度的缩短。与Y组比较，M组大鼠第1、3、5天潜伏期均明显增长，组间比较有显著差异(P＜0.01)；与M组比较，EA组大鼠第3、5天潜伏期时间均明显缩短，组间比较有显著差异(P＜0.01)；与EA组比较，EAC组大鼠第3、5天潜伏期明显增加，组间比较有显著差异(P＜0.01)。3大鼠空间探索首次跨越时间和跨越次数比较各组大鼠第一次跨越原平台象限时间比较：与Y组相比，M组所需时间明显延长，组间有显著差异(P＜0.01)；与M组相比，EA组大鼠所需时间有所缩短，组间有显著差异(P＜0.01)；与EA组相比，EAC组大鼠第一次跨越原平台象限时间明显延长，组间有显著差异(P＜0.01)。各组大鼠跨越次数比较：与Y组相比，M组大鼠跨越次数明显少，组间有显著差异(P＜0.01)；与M组相比，EA组大鼠跨越次数明显增加，组间有显著差异(P＜0.01)；与EA组相比， EAC组大鼠跨越次数少，组间有显著差异(P＜0.01)。Y组、EA组、EAC组大鼠的游泳轨迹主要集中在平台象限，而M组大鼠的游泳轨迹呈随机分布。4大鼠脑CA1区海马长时程增强（LTP）检测比较以高频刺激（high frequency stimulation HFS）前的基础性fEPSP平均值为基准值(100%)，给予HFS刺激后，30min、60min和90min时fEPSP平均幅度，Y组为(217.1±8.7)%、(192.2±5.2)%和(186.6±6.1)%，M组为(181.3±7.8)%、(128.5±6.2)%和(110.1±6.2)%，与Y组比较海马LTP显著受到抑制，具有显著性差异(P＜0.01)。EA组fEPSP相对幅度值为(212.4±5.9)%、(174.6±6.1)%和(161.4±2.4)%;EAC组分别为(193.1±5.8)%、(151.9±4.9)%和(146.9±3.8)%。与M组比较，EA组和EAC组fEPSP相对幅度值均明显升高，海马LTP均得到明显改善(P＜0.01)。5各组大鼠海马CA1区神经细胞电镜观察比较Y组大鼠神经元核膜完整光滑，胞内细胞器丰富,神经毡有较多GrayI型突触，其突触前膜、突触后膜和突触间隙清晰，突触前终末有较多清亮的圆形囊泡；与Y组比较，M组大鼠突触界面曲率减小，突触活性区长度变短，突触后致密物厚度变薄，突触间隙宽度变大，组间比较均具有显著差异（P＜0.01）；与M组相比，EA组大鼠突触界面曲率增大,突触后致密物厚度变薄及突触间隙宽度变大，组间比较均具有显著差异（P＜0.01）；EAC组与M组比较无明显差异，但与EA组比较，各参数差异均具有统计学意义（P＜0.01）。6大鼠海马NMDA受体蛋白表达比较以β-ACTIN为内参照，计算NMDAR1／β-ACTIN光密度比值，反应NMDAR1相对含量变化。与Y组比较，M组大鼠海马中NMDAR蛋白的表达水平明显降低（P＜0.01）；与M组比较，EA组大鼠海马中NMDAR蛋白的表达水平升高（P＜0.05）；EA组与EAC组比较，大鼠海马中NMDAR蛋白表达差异有统计学意义（P＜0.05）。7大鼠海马BDNF含量比较与Y组比较，M组大鼠海马BDNF含量减少，组间差异具有统计学意义（P＜0.05）;与M组比较，EA组大鼠海马组织内BDNF含量明显提高，组间比较有显著性差异（P＜0.05）；与EAC组比较，EA组大鼠海马组织内BDNF含量表达较高，组间差异具有统计学意义（P＜0.05）。8大鼠海马中PKCγmRNA的表达比较将PCR扩增后的产物经1.6%琼脂糖凝胶电泳，结果显示PKCγmRNA扩增产物片段为105bp，符合设计要求。各组实验结果表明，与Y组比较，M组大鼠海马中PKCγmRNA的表达水平明显降低（P＜0.01）；与M组比较，EA组大鼠海马中PKCγmRNA的表达水平明显增高（P＜0.01）；组间比较有显著性差异（P＜0.05）；与EAC组比较，EA组大鼠海马中PKCγmRNA的表达较高，组间差异具有统计学意义（P＜0.05）。结论综上所述，本课题在成功诱导学习记忆减退模型基础上，根据中医辨证施治原则和“双固一通”针法指导，采用补益先天、后天和健脑益智之“关元”、“足三里”、“百会”组穴，实施电针法对大鼠进行处理，结果显示，“双固一通”电针能明显提高老年阳虚模型大鼠的学习记忆能力；该效应与“双固一通”电针能提高老年模型大鼠海马齿状回长时程增强的增幅、减轻大鼠海马组织病理学改变有明显相关性；研究同时发现，该效应的获得可能与“双固一通”电针能提高大鼠海马组织内BDNF和PKCmRNA含量及NMDAR蛋白的表达存在明显相关，并且“关元”、“足三里”、“百会”配伍电针疗效明显优于“中极”、“阴陵泉”、“印堂”配伍电针治疗。因此，通过本课题的研究，我们发现，“双固一通”电针具有改善老年阳虚模型大鼠学习记忆能力的效应，与电针对模型大鼠海马长时程增强可塑性变化的调控有相关性，其机制可能是通过电针能保护海马组织突触结构、提高海马组织BDNF、PKCmRNA含量及NMDAR蛋白的表达而实现的。更多还原

【Abstract】 ObjectiveAging is the result of the interaction of genetic, internal andexternal factors. It happens morphologically and functionally inthe law of life. The cognitive function will decline with aging inboth human beings and animals. What’s more, the Yang-deficiencysymptom will accompany with aging. This study focus on the learningand stduy decline in the aging model rats with Yang-deficiency, andhave a treatment with Shuanggu Yitong elEACtro-acupuncture to rats,it is to explore the efficacy Shuanggu Yitong acupuncture methodimproving the learning and study decline, and try to providescinetific evidence for the treatment how to control the Long-TermPotentiation in the Hippocampus.MethodsForty Sprague Dawley rats were randomly divided into fourgroups:Young Group(Y),Model Group,and electro-acupuncture controlGroup (EAC)，Shuanggu Yitong electro acupuncture Group (EA). Therats distributed in Model Group, EA Group, and EAC Group wereinjected with D-galactose subcutaneously for40days andhydrocortisone for7days in muscles of the rat’s left leg, thiscould induce the Yang-deficiency symptom in aging model rats. The rats in Young Group were injected of same amount of saline at sametime. Rats in EA Group were treated with Shuanggu Yitongelectro-acupuncture（Guanyuan,Zusanli,Baihui）, and EAC group werestimulated with compaired electro-acupuncture therapy(Zhongji,Yinlingquan,Yintang), each treatment were last for4weeks.Finally,Enzyme linked immunosorbent assay（ELISA）are used to detEACt thequality of cAMP and cGMP and the percentage of cAMP/cGMP, Morriswater-maze task was used as the judging critera for learning andmemory behavior; Synaptic structural parameters were analysised byelEACtron microscopy and biological image analysis system;Population spike was chEACked to detect the changes of potentialamplitude induced by the high frequency stimulation (HFS); Contentof BDNF was detected by the radioimmunity method;PKC mRNAexpression was examined through the RT-PCR;the expressiondifference of NMDA receptor also was checked through Western blotmethod.Results1.The quality of cAMP and percentage of cAMP/cGMP in Model ratswere lower than the Young group and EA group and EAC group, thequality of cGMP bEACame higher on the contrary.there weresignificant difference between them（P＜0.01）.The model rats showeda series of asthenic symptom such as: body curled up, weight loss,unresponsive, angular, eat less cold, and cold limbs, Mao Song,vertical hair, lose luster and hair removal.In the Morris water mazetask.2.the escape latency made by the model group was remarkablylonger than that of the Young Group (P<0.01); the escape latencyof the Young Group is shorter than EA Group and EAC group (P<0.05); there was differences between EA Group and EAC group (P<0.05).3.the firt acrossing time in model rats was longer than theyoung group rats, and it was shorten by the treatment of EA, therewere significant difference between them（P＜0.01）,the spaningtimes also increased through the treatment of EA, there weresignificant difference between them（P＜0.01）.4.The record after HFS indicated that the incidence of LTPinduction of rats and PS amplitude in the model group wassignificantly lower than that of the Young Group, EA Group and EACgroup (P<0.01); In comparison with the Young Group, there was nosignificant difference between Young Group， EA Group and EAC group(P>0.05); there was no significant difference between EA Group andEAC group (P>0.05).5.In comparison with the Young Group, the length of active zonesand the thickness of PSD are all decreased, the synaptic cleft widthenlarged significantly the model group（P＜0.01）; there was nodifferences between EA Group and EAC group（P>0.05）；In comparisonwith the model Group, the thickness of PSD are increased, and thesynaptic cleft width decreased remarkably（P＜0.01）; there was nodifferences between EA Group and EAC group（P>0.05）；the lengthof active zones enlarged in the EA Group（P＜0.01）; there was nodifferences in the EAC group（P>0.05）；In comparison with the YoungGroup, the synaptic interface curvature with no significantdifference in model Group, EA Group and EAC group（P>0.05）.6.compared with Young Group, the content of BDNF declinedremarkably in model Group（P＜0.05）; compared with model Group,the content of BDNF in EA Group increased（P＜0.05）；there was nodifferences in the EAC group（P>0.05）; in comparison with the EA Group, the content of BDNF in EA Group decreased significantly（P＜0.05）.7.compared with Young Group, the expression of PKC mRNAdeclined remarkably in model Group（P＜0.01）; in comparison withthe model Group, the expression of PKCmRNA raised remarkably in EAGroup and EAC group（P<0.01）；there was no differences between EAGroup and EAC group（P>0.05）； compared with Young Group, therewas no differences between EA Group and EAC group（P>0.05）.8.compared with Young Group, the expression of NMDAR declinedremarkably in model Group（P＜0.01）（P<0.01）；the expression ofNMDAR decreased in EAC group（P<0.05）；compared with EAC group，the expression of NMDAR raised remarkably in EA Group（P<0.05）；there was no differences between EA Group and Young Group（P>0.05）.ConclusionThe above results suggest that the Shuanggu Yitong acupuncturemethod can improve the spatial learning and memory in aging modelrats with Yang-deficiency;This efficency was obtained through thetreatment increased the incidence of LTP induction at thehippocampal dentate gyrus in the aging model rats; and the methodcan improve the hippocampal synaptic structural parameters; andadjust and regulate content of BDNF and the PKC mRNA expression，and the NMDA receptor expression in aging model rats withYang-deficiency.Therefor, the Shuanggu Yitong acupuncture methodcan improve the ability of learning and study in rats withYang-Defficiency, the mEAChanism was connEACted with ShuangguYitong acupuncture method can protEACt the instruct of Hippocampustissue and regulate the content of BDNF and PKCmRNA and NMDARaffEACted the Long-Term-Potentiation, but the more sEACret about them should be explore out by more researching in the future.更多还原