基于线粒体生物合成探讨中枢疲劳的中医证候及生物学机制

发布时间：2018-01-01 06:24

  本文关键词：基于线粒体生物合成探讨中枢疲劳的中医证候及生物学机制　出处：《北京中医药大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 中枢疲劳 肝郁脾虚 体复康 线粒体

【摘要】：目的本研究拟通过对古代文献的整理和分析,分别从概念、范畴、症状和证候特征等方面对中枢疲劳的中医理论进行分析和归纳。在此基础上,结合现代医学研究进展,探讨线粒体生物合成和中枢疲劳肝郁脾虚证候生物学机制的相关性。并从肝脾论治的角度探讨中药组方——体复康加减方的治疗依据。实验研究中,建立并评价中枢疲劳大鼠模型。在此基础上评价体复康加减方的药效,进而从物质能量代谢、氧化作用、线粒体功能及形态逐级深入探讨中枢疲劳肝郁脾虚证的生物学机制和中药药理机制。最后检测线粒体生物合成重要通路SIRT1-PGC1α-NRF1各分子指标基因和蛋白的表达变化,明确体复康加减方基于中枢线粒体生物合成的作用分子靶点,为临床干预提供研究思路和基础。方法理论研究:首先通过对中枢疲劳相关的名词术语进行整理,采用工具书查询对应含义。其次,以中枢疲劳相关基础核心词"疲"、"劳"、"倦"分别作为检索词,检索第五版《中华医典》,筛选相关古代文献对应条文进行整理,分析中枢疲劳在中医理论中的对应症、和证的相关记载。同时,通过现代国内外文献检索,探讨基于线粒体生物合成中枢疲劳和肝郁脾虚证的相关性。最后,提出从肝脾论治的角度归纳体复康加减方治疗中枢疲劳的理论依据。实验研究:中枢疲劳大鼠模型建立和评价中,将大鼠随机分为四组:正常组,5天模型组,14天模型组,21天模型组。实验结束后采用一般情况观察(神态、毛发等),行为学实验(旷场实验、高架十字迷宫实验、负重力竭游泳实验),和线粒体超微结构评价模型,筛选出最优模型。给药实验中,将大鼠随机分为六组:正常组,模型组,体复康加减方低、中、高剂量组,阳性对照组。首先通过一般情况和行为学结果评价药效,并通过药效反证中枢疲劳肝郁脾虚病证结合模型。其次通过对外周血血清CK、BUN、LDH以及肝脏MDA、SOD检测,探讨能量代谢和氧化作用机制。进而通过透射电镜观察海马CA1区线粒体形态变化,同时通过RT-PCR实验检测mtDNA拷贝量,进一步探讨中枢疲劳肝郁脾虚证大鼠模型线粒体功能变化。最后,通过免疫组化、Western blot和RT-PCR实验分别检测SIRT1、PGC-1α、以及NRF1蛋白和基因表达变化。由浅及深,逐层分析中枢疲劳肝郁脾虚证的生物学机制和体复康加减方的分子作用靶点。结果理论研究:(1)中枢疲劳在中医理论中与"神劳"、"志倦"所表达的含义最为接近。其症状表现为"神劳则魂魄散,志意乱",集中体现在精神意识活动的功能下降,本体感知下降,提示高级中枢的认知、主观控制和驾驭情绪的功能下降。这和中枢疲劳中枢神经系统功能下降涉及认知、情绪的临床表现相符合。中枢疲劳的病机与阴阳、气血、五脏都有着密切关联。临床上由于诱导因素和各体本身差异的不同,又可表现出不同的证候特点。肝脾两脏的功能互相影响,其二者的调摄、协同在中枢疲劳的发生过程中起到核心作用。肝脾本身脏腑功能的特点,加之现代人群的起居作息以及饮食特征,导致肝郁脾虚成为现代人群中枢疲劳的主要证候特征。(2)中枢疲劳和肝郁脾虚证的生物学机制都与线粒体生物合成密切相关。肝郁状态下,人体处于长期压力和应激刺激,线粒体生物合成系统的神经保护作用被减弱,生物合成效率降低,膜电位流动性下降,中枢神经细胞线粒体功能障碍,引起HPA轴递质释放失衡,则体现为情绪躁怒或压抑。同时,脾虚状态下,线粒体生物合成机制受损,功能下降的线粒体无法通过正常的生物合成,以新陈代谢的形式合成新的线粒体蛋白和基因,从而导致线粒体能量代谢功能降低,ATP合成和加工效率下降,无法将营养物质充分转化为能量,在机体则宏观表现为水谷精微运送失职,周身不荣。另一方面,线粒体生物合成调控线粒体自身的新陈代谢,维持着线粒体蛋白和基因的质量和数量正常。其工作效率下降,导致线粒体DNA数量减少,线粒体形态改变,出现线粒体肿胀,空泡样变化,这一反应与脾虚水湿内停的宏观表征也具有一致性。(3)在对中枢疲劳肝郁脾虚证候特点把握的基础上,提出了从肝脾论治中枢疲劳的治疗思路,并为疏肝健脾方一一体复康加减方治疗中枢疲劳肝郁脾虚证提供了理论依据,进一步证实了从肝脾论治中枢疲劳的科学性和可行性。实验研究:(1)模型评价研究结果显示,与正常组相比,旷场实验结果提示5天(p0.05)和14天(p0.01)造模均可造成大鼠中央格停留时间升高。高架十字迷宫结果显示21天模型组大鼠开放臂进入次数(p0.05)和时间(p0.001)较正常组显著降低。14天和21天模型组大鼠负重游泳力竭时间较正常组减少(p0.05);此外21天模型组大鼠海马线粒体出现明显肿胀、脊断裂并消失以及双层膜结构损坏的退行性改变。(2)体复康加减方干预后行为学实验结果显示,和模型组相比,旷场实验中,中药低、中、高剂量组和阳性对照组均降低了中央格停留时间(P＜0.001)、总穿格距离(P0.01,P0.05,p0.05,p0.05)、总穿格次数(P0.05,p0.05,p0.05,p0.05)、最大连续活动距离(P0.01,p0.05,P0.05,p0.05)以及平均速度(P0.01,p0.05,p0.05,p0.05);增加了垂直活动时间(p0.05,p0.05,P0.01,P0.01)、修饰时间(P0.05,P0.01,p0.05,p0.05)和修饰次数(P0.05,P0.01,p0.05,P0.05)。高架十字迷宫实验结果显示,和模型组对比,各治疗组均增高了高架开放臂进入次数(p0.01,P0.05,P0.01,P0.01)和时间(P0.01,p0.05,p0.05,P0.01)比例,以及中央格停留时间(P0.001,P0.05,P0.05,P0.05)。负重力竭游泳实验结果显示,各治疗组较模型组均延长了大鼠负重力竭游泳的力竭时间(P0.001)。(3)和模型组相比,中药低、中、高剂量组和阳性对照组显著降低了血清CK(P0.01,P0.05,P0.01,p0.05)和 BUN(P0.001);均可升高肝脏 SOD 的活性(P0.01,P0.001,P0.01,P0.001),并降低肝脏 MDA含量(P0.001,P0.001,P0.001,P0.01)。(4)透射电镜观察显示,中药低、中、高剂量组和阳性对照组均改善了线粒体超微结构的退行性变化,包括线粒体数量减少、线粒体肿胀、脊断裂或消失和双层膜结构破坏。和模型组对比,中药低、中、高和阳性对照组均增高了海马mtDNA拷贝量(P0.01,P0.05,P0.01,p0.05)。(5)免疫组化结果显示,中药低、中、高剂量和阳性对照组海马中SIRT1(P0.05,P0.05,P0.05,P0.01)、PGC-1α(p0.05)、NRF1(P0.01,P0.05,P0.001,P0.001)免疫阳性物表达均高于模型组。Western blot实验结果显示,中药低、中、高剂量组和阳性对照组大鼠海马 SIRT1(P0.05,P0.01,P0.01,P0.01)、PGC-1α(P0.05,P0.001,P0.001,P0.001)、NRF1(P0.05,P0.001,P0.001,P0.01)蛋白表达均高于模型组。RT-PCR结果显示,中药低、中、高剂量组和阳性对照组大鼠海马 SIRT1(p0.05)、PGC-1α(P ＜ 0.01,p0.05,p0.05,P0.01)、NRF1(p0.05,P0.05,P0.01,P0.01)mRNA表达均高于模型组。结论中医理论中没有明确的概念和定义对应中枢疲劳,然而"神劳"(以及"志倦")的表达与之较为接近。中枢疲劳的证候特点集中体现为肝郁脾虚,中医临床可从疏肝健脾的角度治疗。中枢疲劳肝郁脾虚证的生物学基础和中枢神经系统细胞内线粒体生物合成机制密切相关。21天水环境小平台造模方法较符合中枢疲劳模型的表观效度、构建效度和预测效度。经行为学实验、线粒体超微结构观察、以及分子生物学实验证实,以疏肝健脾为治则的体复康加减方可以有效改善中枢疲劳大鼠模型的相关症状,其改善作用涉及加速能量代谢产物消除和提高抗氧化酶活性,分子生物学机制与上调海马SIRT1-PGC-1α-NRF1通路蛋白和基因表达,从而促进线粒体生物合成密切相关。
[Abstract]:The purpose of this study through the collation and analysis of ancient literature, from the concept, category, analyzes and summarizes the theory of TCM central fatigue symptoms and syndrome characteristics. On this basis, combined with the progress of modern medical research, to investigate the correlation between mitochondrial biogenesis and central fatigue syndrome of liver stagnation and spleen deficiency. And the biological mechanism to explore the prescription of traditional Chinese medicine according to treatment of Tifukang decoction from the liver and spleen. The angle of the experimental research, the establishment and evaluation of rat model of central fatigue. To evaluate the efficacy of Tifukang Decoction on the basis of this, and then from the material and energy metabolism, oxidative stress, biological mechanism and mechanism of mitochondrial function and pharmacology of traditional Chinese medicine sequential study the central fatigue syndrome of liver stagnation and spleen deficiency. The expression changes of the molecular markers of mitochondrial biogenesis pathway of SIRT1-PGC1 alpha -NRF1 gene and protein detection finally, Ming Indeed Tifukang Decoction effect molecular target based on central mitochondrial biogenesis, provide research ideas and basis for clinical intervention. Methods: theoretical research: firstly the terminology of central fatigue related collation, using books for corresponding meaning. Secondly, the central fatigue related basic core word "tired", "work", "tired" as key words, search the fifth edition of < > screening of traditional Chinese medicine, ancient literature corresponding provision of collation, analysis the corresponding disease in TCM theory of central fatigue, relevant records and certificate. At the same time, through the modern literature retrieval, to investigate the correlation between mitochondrial biogenesis in central fatigue and liver stagnation and spleen deficiency syndrome based on the theory of governance is proposed. Finally, from the perspective of Tifukang Decoction in treatment of liver and spleen induced central fatigue theory. Experimental research: establishment and evaluation of rat model of central fatigue, the rats were randomly Divided into four groups: normal group, model group of 5 days, the 14 day group, 21 day model group. General observation by the end of the experiment (air, hair), behavioral experiment (open field test, the elevated plus maze test, weight exhaustive swimming test), and evaluate the mitochondrial ultrastructure model. Select the best delivery model. In the experiment, the rats were randomly divided into six groups: normal group, model group, Tifukang Decoction low, high dose group, positive control group. Firstly, the general situation and behavior evaluation of efficacy, and the efficacy of disproof of central fatigue model combined disease and syndrome of liver stagnation and spleen deficiency. Followed by peripheral blood serum CK, BUN, LDH and liver MDA, SOD detection of energy metabolism and oxidation mechanism. In order to observe morphologic changes of mitochondria in hippocampal CA1 region by transmission electron microscopy, and by the mtDNA RT-PCR copy detection experiments, to further explore the central fatigue of liver The changes of mitochondrial function in rat models of spleen deficiency disorder. Finally, by immunohistochemistry, Western blot and RT-PCR experiment were used to detect SIRT1, PGC-1 alpha, and NRF1 protein and gene expression changes. By the shallow and deep layers of molecular targets for central fatigue liver and spleen deficiency and biological mechanism of Tifukang decoction. The results of theoretical research: (1) the central fatigue in TCM theory and the "God" will work ", and" the meaning of the expression is most close. The symptoms of "God" soul scattered, ambition and chaos ", embodied in the spirit of consciousness function decline, body perception decreased, suggesting that senior center the subjective cognition, emotion control and manage function decline. This and the central fatigue of central nervous system dysfunction involving cognitive, emotional clinical manifestations consistent. Central pathogenesis and fatigue of yin and Yang, Qi and blood, internal organs are closely related to the clinical. Due to induction by And the body itself different, and can exhibit different syndrome characteristics. The liver and spleen two dirty functions influence each other, the regulation of the cooperative, played the key role in the process of occurrence of central fatigue. The characteristics of the liver and spleen viscera function itself, coupled with the modern people's living as income and diet characteristics, leading to Ganyupixu syndrome characteristics of modern people and become the main central fatigue. (2) the biological mechanism of central fatigue and liver stagnation and spleen deficiency are closely related to mitochondrial biogenesis. Liver condition, human body is in constant pressure and stress. The neuroprotective effect of mitochondrial biosynthesis system has been weakened and the biosynthesis efficiency decreased, the membrane potential liquidity decreased central neuronal mitochondrial dysfunction, caused by the HPA axis of neurotransmitter release is imbalance, emotional irritability or depression. At the same time, the state of spleen deficiency, mitochondrial biogenesis mechanism Impaired function decline of mitochondria not through normal biosynthesis, in the form of new The new supersedes the old. mitochondrial protein synthesis and gene, resulting in mitochondrial energy metabolism decreased, the decrease of ATP synthesis and processing efficiency, can not be full of nutrients into energy, is manifested in the macro body for foodstuff transportation of dereliction of duty, the whole body is Rong. On the other hand, the regulation of mitochondrial biogenesis of mitochondria to maintain the quality and quantity of The new supersedes the old., mitochondrial protein and gene. The normal decline in its working efficiency, resulting in decreased amounts of mitochondrial DNA, mitochondrial morphological changes occur, mitochondrial swelling, vacuolar changes, macroscopic characterization of this reaction and spleen Dampness Retention is consistent with. (3) based on the grasp of the central fatigue syndrome of liver stagnation and spleen deficiency, put forward treatment treatment ideas of central fatigue from the liver and spleen, and liver Provide a theoretical basis for the development of a Jianpi Decoction in treatment of rehabilitation of central fatigue liver spleen deficiency, further confirmed the treatment of central fatigue is scientific and feasible from the liver and spleen. Experimental study: (1) the model evaluation results show that, compared with the normal group, the open field test results suggest that the 5 day (P0.05) and 14 days (P0.01) model in rats can be caused by the central cell residence time increased. The elevated plus maze test results showed that 21 days of rats in the model group the number of open arm entry (P0.05) and time (p0.001) than the normal group significantly decreased.14 and 21 days rats exhaustive swimming time less than the normal group (P0.05); in addition, 21 days in hippocampus of model group rats appeared obvious mitochondrial swelling, fracture and spinal degenerative damage disappeared and double membrane structure change. (2) Tifukang Decoction intervention study revealed that, compared with model group, the open field test, traditional Chinese Medicine 浣，

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