电针对慢性应激疲劳证候模型大鼠神经免疫网络调节机制研究

【作者】 孟宏；

【导师】 图娅；

【作者基本信息】 北京中医药大学 ， 针灸学， 2003， 博士

【摘要】 慢性疲劳综合征(Chronic Fatigue Syndrome，简称CFS)由美国疾病控制中心1988年正式命名；当今社会，CFS发病率较高，并有逐年升高的趋势，造成巨大的经济损失．目前对该病的病因、实质及发病机理的认识尚无定论，但研究重点越来越集中到心理-神经-内分泌网络上；西医学在临床上亦缺乏有效的治疗方法；尤其是由于CFS缺少诊断和治疗的特异性标准，现行的“排除式”诊断标准在实际操作中存在诸多亟待调整和探讨研究之处。中医学体系是建立在整体宏观基础之上的，对人体调控机制的认识有独特的理论体系和行之有效的调节手段。从中医学整体观念、辨证论治、心身兼顾、形神并调的视角，对CFS进行全方位和大视野的探索性研究将有重要意义。 研究目的 1、目前我国仍采用美国CDC的诊断标准进行CFS的临床诊断，需要排除一系列疾病；然后进行对症治疗，这不但费用昂贵，而且临床可操作性极差。希望通过本研究，能够正确认识中医在这一心身疾病认识过程中的重要地位，使我国能够经过中西医结合的机理与临床研究，制定出合乎我国国情的易于操作的一整套诊断标准和治疗方案。 2、CFS可能是由于慢性应激所致的心理-神经-免疫网络紊乱而出现的以疲劳为主症的临床症候群。本实验拟通过对慢性应激所致三种证型疲劳模型大鼠的电针干预，从心理-神经-免疫网络的不同层面选取指标，从整体入手，多方位的、多层面的把握其内涵，探讨CFS的发病机理及针灸调节机制。并为中医临床辨证选穴、随证加减提供有益的指导；为中医科研对此类多因素、多脏器复杂疾病的研究提供一定参考。 研究方法 Wister雄性大鼠，体重160-180g，随机分为正常组、肝郁模型组、肝郁电针治疗组、脾虚模型组、脾虚电针治疗组、肾虚模型组、肾虚电针治疗组。模型组与电针组动物制造慢性应激疲劳模型，电针组在造模的同时进行针刺治疗。肝郁电针组取百会、足三里、太冲；脾虚电针组取百会、足三里、三阴交；肾虚组取百会、足三里、太溪。共21天。 通过行为学检测对模型进行判定；用高效液相加电化学法检测大鼠不同脑组织中单胺类神经递质的含量；采用石蜡切片，免疫组织化学法和应用图像分析仪观察电针对大鼠海马组织中BDNF的影响、对大鼠下丘脑β-EP的影响；用酶联免疫吸附法检测电针对模型动物血清IL-1β、IL-6含量的影响。 结果： 1、各模型组造模后的动物在规定的时间内，水平活动和垂直活动次数均较正常组明显减少。鼠尾悬挂实验间断不动时间延长、力竭游泳时间缩短、体重增加缓慢。说明造模成功。而电针治疗组可显著增加动物的水平活动和垂直活动次数；鼠尾悬挂实验间断不动时间缩短；力竭游泳时间延长；体重增加。说明电针能改善应激所致的体力下降和躯体疲劳状态。三组间比较差异不显著。 2、慢性应激可引起各模型组大鼠不同脑组织NE、5-HT、DA及其代谢产物的改变，含量均较正常组显著异常改变。电针可调节不同脑组织中异常改变的单胺类神经递质水平。 3、各模型组大鼠海马CA3区BDNF免疫组织化学反应显示阳性细胞数量明显减少，且多数细胞呈空泡状态，胞浆着色浅。电针治疗组BDNF染色明显好于各相应的模型组，经图像分析处理与模型组有显著差异；且与正常组相类似。2 电针对馒性应激疲劳症候模型大鼠神经免疫网络调节机制研究 4、各模型组下丘脑中pEP含量增高；各电针组p －EP含量降低，与相应模型组比较差异显著．三组间比较差异不显著。 5、各模型组大鼠血清IL4、IL6含量均明显高于正常对照组，电针可降低血淆R＊p、IL－6水平。 结论 1、从CDC的诊断标准不断的修订和近几年的研究趋向可以看出，对CFS症状表现的深入分析研究更加集中到心理－神经－免疫网络这一层面上来。CFS与应激关系密切，过度的体力、脑力劳动、应激性生活事件的刺激等皆为CFS的重要发病因素。 2、电针调整了中枢神经系统单胺类神经递质NE、DA、5－HT等紊乱的状态，这有利于神经内分泌和神经递质正常的功能活动，以此调节脏腑经络，平衡阴阳，稳定机体内环境。说明单胺类神经递质与心理－神经－免疫网络的密切关系，以及电针对其的调节作用。 3、观察电针对海马组织中脑源性神经生长因子的影响可以看出海马神经元的可塑性，起到治疗CFS的作用，从而说明CFS与心理－神经－免疫网络的必然联系。 4、卜内啡肽属于内源性阿片肽，是机体的主要应激激素。本研究首次证实电针可能通过p＊P的调节保护脑神经元，对抗恶性应激引起的脑组织神经元结构可塑性的损伤，从而起到治疗CFS的作用。 5、细胞因子不仅可以激活免疫系统，而且对神经和内分泌系统也有重要的调节作用。本研究首次观察到电针可以调节不同证型疲劳模型大鼠血清中细胞因子ILI6、IL6水平，即经过电针治疗的疲劳模型各组IL上p、IL巧均趋于正常。 本课题的创新之处 1、提出从心理－神经－免疫网络层面对CFS进行全方位，新视角的研究。 2、应用慢性应激的方法制造中医不同证型的疲劳模型动物，对CFS进行分型论治研究。基于?更多还原

【Abstract】 Chronic Fatigue Syndrome (CFS) was officially named by Centers for Disease Control and Prevention (CDC) of the U.S government in 1988. The incidence of CFS rises year by year, which causes enormous economic losses. At present, there are no effective treatment methods because of its unclear etiology and pathogenesis. And more and more researches concentrate on the psycho-neuro-immunology network. The system of TCM set up on holism has unique theories and effectual measures to understand and regulate the human body. So it is from the angle of TCM theories: the concept of the organism as a whole, determination of treatment based on differentiation of symptoms and signs and consideration of body and sole, that there will be great meanings in the study of CFS.Objective: 1. At present, we still adopt the diagnostic criterion of CDC of U.S. government to diagnose CFS on clinic in China. After a series of disease being excluded, CFS can be diagnosed, and then the expectant treatment can be given. It is not only expensive, but also poorly maneuverable. From the research, it is expected to make out diagnostic criterion and therapeutic project easy to operate conforming to the national conditions of our country. 2. The symptom of CFS is mainly fatigue probably caused by disorder of the psycho-neuro-immunology network. The experiments use chronic stresses to induce three types of CFS in rats as models. Choose indexes from different aspects of psycho-neuro-immunology network to observe the intervention of acupuncture. This paper explains the pathogenesis of CFS and the regulation mechanism of acupuncture. Provides helpful guide for determination of treatment based on differentiation of symptoms and signs. Offers consultant to study this type of complicated disease related to multifactor and multi-organs.Methods: Wistar rats (male, weight 160-180g) were randomly distributed to control group, model groups and EA groups. Model groups and EA groups included three syndromes: depressed liver syndrome, spleen deficiency syndrome and kidney deficiency syndrome. All groups except the control were exposed for 21 days to different chronic stressors, and EA group accepted electro-acupuncture treatment at the same time.Three groups of points were selected for electro-acupuncture. Depressed liver group: Baihui (GV 20), Zusanli (ST 36), Taichong (Liv 3). Spleen deficiency group: Baihui (GV 20), Zusanli (ST 36), Sanyinjiao (SP 6). Kidney deficiency group: Baihui (GV 20), Zusanli (ST 36), Taixi (KI3).Results: 1. Compared with control group, the slowly increased weight, the sucrose preference and activities of rats in open-field test, tail hanging test and exhausting swimming in different model groups manifested a series of abnormal variations, which indicated that rats in the model groups suffered from chronic fatigue syndrome. EA can improve behavioral disorder of different groups’ rats. It shows that EA can improve the state of physical and mental fatigue. There is no difference among the three groups. 2. Chronic stress decreased the levels of NE, 5-HT, 5HIAA, DA, DOPAC, HVA in different brain tissues; EA can increase the levels of monoamine neurotransmitter in different brain tissues. 3. Immunohistochemical reaction showed that the content of positive BDNF neurons in hippocampus of three group fatigue animals was much less than that of control animals. The EA increased the content and improved the appearance of BDNF neurons in the fatigue animals. The EA groups had significant difference compared with the model groups and were similar with the control group.4. (3 -EP in hypothalamus of three fatigue model groups, were much higher than that of control groups. Electro-acupuncture decreased 3 -EP in the fatigue animals. There is no difference among the three EA groups. 5, The levels of serum IL-1 |3, IL-6 of each model group were significantly higher than those of control group. Electro-acupuncture reduced the levels of serum IL-1 P, EL-6 stimulated by different stresses.Conclusion: 1. We can find out that more and more更多还原