發(fā)布時間：2018-01-01 06:24

  本文關(guān)鍵詞：基于線粒體生物合成探討中樞疲勞的中醫(yī)證候及生物學(xué)機制　出處：《北京中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 中樞疲勞 肝郁脾虛 體復(fù)康 線粒體

【摘要】：目的本研究擬通過對古代文獻(xiàn)的整理和分析,分別從概念、范疇、癥狀和證候特征等方面對中樞疲勞的中醫(yī)理論進(jìn)行分析和歸納。在此基礎(chǔ)上,結(jié)合現(xiàn)代醫(yī)學(xué)研究進(jìn)展,探討線粒體生物合成和中樞疲勞肝郁脾虛證候生物學(xué)機制的相關(guān)性。并從肝脾論治的角度探討中藥組方——體復(fù)康加減方的治療依據(jù)。實驗研究中,建立并評價中樞疲勞大鼠模型。在此基礎(chǔ)上評價體復(fù)康加減方的藥效,進(jìn)而從物質(zhì)能量代謝、氧化作用、線粒體功能及形態(tài)逐級深入探討中樞疲勞肝郁脾虛證的生物學(xué)機制和中藥藥理機制。最后檢測線粒體生物合成重要通路SIRT1-PGC1α-NRF1各分子指標(biāo)基因和蛋白的表達(dá)變化,明確體復(fù)康加減方基于中樞線粒體生物合成的作用分子靶點,為臨床干預(yù)提供研究思路和基礎(chǔ)。方法理論研究:首先通過對中樞疲勞相關(guān)的名詞術(shù)語進(jìn)行整理,采用工具書查詢對應(yīng)含義。其次,以中樞疲勞相關(guān)基礎(chǔ)核心詞"疲"、"勞"、"倦"分別作為檢索詞,檢索第五版《中華醫(yī)典》,篩選相關(guān)古代文獻(xiàn)對應(yīng)條文進(jìn)行整理,分析中樞疲勞在中醫(yī)理論中的對應(yīng)癥、和證的相關(guān)記載。同時,通過現(xiàn)代國內(nèi)外文獻(xiàn)檢索,探討基于線粒體生物合成中樞疲勞和肝郁脾虛證的相關(guān)性。最后,提出從肝脾論治的角度歸納體復(fù)康加減方治療中樞疲勞的理論依據(jù)。實驗研究:中樞疲勞大鼠模型建立和評價中,將大鼠隨機分為四組:正常組,5天模型組,14天模型組,21天模型組。實驗結(jié)束后采用一般情況觀察(神態(tài)、毛發(fā)等),行為學(xué)實驗(曠場實驗、高架十字迷宮實驗、負(fù)重力竭游泳實驗),和線粒體超微結(jié)構(gòu)評價模型,篩選出最優(yōu)模型。給藥實驗中,將大鼠隨機分為六組:正常組,模型組,體復(fù)康加減方低、中、高劑量組,陽性對照組。首先通過一般情況和行為學(xué)結(jié)果評價藥效,并通過藥效反證中樞疲勞肝郁脾虛病證結(jié)合模型。其次通過對外周血血清CK、BUN、LDH以及肝臟MDA、SOD檢測,探討能量代謝和氧化作用機制。進(jìn)而通過透射電鏡觀察海馬CA1區(qū)線粒體形態(tài)變化,同時通過RT-PCR實驗檢測mtDNA拷貝量,進(jìn)一步探討中樞疲勞肝郁脾虛證大鼠模型線粒體功能變化。最后,通過免疫組化、Western blot和RT-PCR實驗分別檢測SIRT1、PGC-1α、以及NRF1蛋白和基因表達(dá)變化。由淺及深,逐層分析中樞疲勞肝郁脾虛證的生物學(xué)機制和體復(fù)康加減方的分子作用靶點。結(jié)果理論研究:(1)中樞疲勞在中醫(yī)理論中與"神勞"、"志倦"所表達(dá)的含義最為接近。其癥狀表現(xiàn)為"神勞則魂魄散,志意亂",集中體現(xiàn)在精神意識活動的功能下降,本體感知下降,提示高級中樞的認(rèn)知、主觀控制和駕馭情緒的功能下降。這和中樞疲勞中樞神經(jīng)系統(tǒng)功能下降涉及認(rèn)知、情緒的臨床表現(xiàn)相符合。中樞疲勞的病機與陰陽、氣血、五臟都有著密切關(guān)聯(lián)。臨床上由于誘導(dǎo)因素和各體本身差異的不同,又可表現(xiàn)出不同的證候特點。肝脾兩臟的功能互相影響,其二者的調(diào)攝、協(xié)同在中樞疲勞的發(fā)生過程中起到核心作用。肝脾本身臟腑功能的特點,加之現(xiàn)代人群的起居作息以及飲食特征,導(dǎo)致肝郁脾虛成為現(xiàn)代人群中樞疲勞的主要證候特征。(2)中樞疲勞和肝郁脾虛證的生物學(xué)機制都與線粒體生物合成密切相關(guān)。肝郁狀態(tài)下,人體處于長期壓力和應(yīng)激刺激,線粒體生物合成系統(tǒng)的神經(jīng)保護(hù)作用被減弱,生物合成效率降低,膜電位流動性下降,中樞神經(jīng)細(xì)胞線粒體功能障礙,引起HPA軸遞質(zhì)釋放失衡,則體現(xiàn)為情緒躁怒或壓抑。同時,脾虛狀態(tài)下,線粒體生物合成機制受損,功能下降的線粒體無法通過正常的生物合成,以新陳代謝的形式合成新的線粒體蛋白和基因,從而導(dǎo)致線粒體能量代謝功能降低,ATP合成和加工效率下降,無法將營養(yǎng)物質(zhì)充分轉(zhuǎn)化為能量,在機體則宏觀表現(xiàn)為水谷精微運送失職,周身不榮。另一方面,線粒體生物合成調(diào)控線粒體自身的新陳代謝,維持著線粒體蛋白和基因的質(zhì)量和數(shù)量正常。其工作效率下降,導(dǎo)致線粒體DNA數(shù)量減少,線粒體形態(tài)改變,出現(xiàn)線粒體腫脹,空泡樣變化,這一反應(yīng)與脾虛水濕內(nèi)停的宏觀表征也具有一致性。(3)在對中樞疲勞肝郁脾虛證候特點把握的基礎(chǔ)上,提出了從肝脾論治中樞疲勞的治療思路,并為疏肝健脾方一一體復(fù)康加減方治療中樞疲勞肝郁脾虛證提供了理論依據(jù),進(jìn)一步證實了從肝脾論治中樞疲勞的科學(xué)性和可行性。實驗研究:(1)模型評價研究結(jié)果顯示,與正常組相比,曠場實驗結(jié)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時間升高。高架十字迷宮結(jié)果顯示21天模型組大鼠開放臂進(jìn)入次數(shù)(p0.05)和時間(p0.001)較正常組顯著降低。14天和21天模型組大鼠負(fù)重游泳力竭時間較正常組減少(p0.05);此外21天模型組大鼠海馬線粒體出現(xiàn)明顯腫脹、脊斷裂并消失以及雙層膜結(jié)構(gòu)損壞的退行性改變。(2)體復(fù)康加減方干預(yù)后行為學(xué)實驗結(jié)果顯示,和模型組相比,曠場實驗中,中藥低、中、高劑量組和陽性對照組均降低了中央格停留時間(P＜0.001)、總穿格距離(P0.01,P0.05,p0.05,p0.05)、總穿格次數(shù)(P0.05,p0.05,p0.05,p0.05)、最大連續(xù)活動距離(P0.01,p0.05,P0.05,p0.05)以及平均速度(P0.01,p0.05,p0.05,p0.05);增加了垂直活動時間(p0.05,p0.05,P0.01,P0.01)、修飾時間(P0.05,P0.01,p0.05,p0.05)和修飾次數(shù)(P0.05,P0.01,p0.05,P0.05)。高架十字迷宮實驗結(jié)果顯示,和模型組對比,各治療組均增高了高架開放臂進(jìn)入次數(shù)(p0.01,P0.05,P0.01,P0.01)和時間(P0.01,p0.05,p0.05,P0.01)比例,以及中央格停留時間(P0.001,P0.05,P0.05,P0.05)。負(fù)重力竭游泳實驗結(jié)果顯示,各治療組較模型組均延長了大鼠負(fù)重力竭游泳的力竭時間(P0.001)。(3)和模型組相比,中藥低、中、高劑量組和陽性對照組顯著降低了血清CK(P0.01,P0.05,P0.01,p0.05)和 BUN(P0.001);均可升高肝臟 SOD 的活性(P0.01,P0.001,P0.01,P0.001),并降低肝臟 MDA含量(P0.001,P0.001,P0.001,P0.01)。(4)透射電鏡觀察顯示,中藥低、中、高劑量組和陽性對照組均改善了線粒體超微結(jié)構(gòu)的退行性變化,包括線粒體數(shù)量減少、線粒體腫脹、脊斷裂或消失和雙層膜結(jié)構(gòu)破壞。和模型組對比,中藥低、中、高和陽性對照組均增高了海馬mtDNA拷貝量(P0.01,P0.05,P0.01,p0.05)。(5)免疫組化結(jié)果顯示,中藥低、中、高劑量和陽性對照組海馬中SIRT1(P0.05,P0.05,P0.05,P0.01)、PGC-1α(p0.05)、NRF1(P0.01,P0.05,P0.001,P0.001)免疫陽性物表達(dá)均高于模型組。Western blot實驗結(jié)果顯示,中藥低、中、高劑量組和陽性對照組大鼠海馬 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)蛋白表達(dá)均高于模型組。RT-PCR結(jié)果顯示,中藥低、中、高劑量組和陽性對照組大鼠海馬 SIRT1(p0.05)、PGC-1α(P ＜ 0.01,p0.05,p0.05,P0.01)、NRF1(p0.05,P0.05,P0.01,P0.01)mRNA表達(dá)均高于模型組。結(jié)論中醫(yī)理論中沒有明確的概念和定義對應(yīng)中樞疲勞,然而"神勞"(以及"志倦")的表達(dá)與之較為接近。中樞疲勞的證候特點集中體現(xiàn)為肝郁脾虛,中醫(yī)臨床可從疏肝健脾的角度治療。中樞疲勞肝郁脾虛證的生物學(xué)基礎(chǔ)和中樞神經(jīng)系統(tǒng)細(xì)胞內(nèi)線粒體生物合成機制密切相關(guān)。21天水環(huán)境小平臺造模方法較符合中樞疲勞模型的表觀效度、構(gòu)建效度和預(yù)測效度。經(jīng)行為學(xué)實驗、線粒體超微結(jié)構(gòu)觀察、以及分子生物學(xué)實驗證實,以疏肝健脾為治則的體復(fù)康加減方可以有效改善中樞疲勞大鼠模型的相關(guān)癥狀,其改善作用涉及加速能量代謝產(chǎn)物消除和提高抗氧化酶活性,分子生物學(xué)機制與上調(diào)海馬SIRT1-PGC-1α-NRF1通路蛋白和基因表達(dá),從而促進(jìn)線粒體生物合成密切相關(guān)。
[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，

本文編號：1363317