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广东凉茶及针刺穴位对运动性疲劳大鼠影响的实验研究以及机制探讨

Experimental Study of the Effect of Guangdong Herbal Tea and Acupuncture Points on the Rat of Fatigue Induced by Exercise and Its Mechanism

【作者】 汶希

【导师】 徐志伟;

【作者基本信息】 广州中医药大学 , 中医基础理论, 2012, 博士

【摘要】 一、文献综述疲劳是机体承受负荷足够大时的一种保护性反应,是一种生理现象。对运动性疲劳的定义为“机体生理过程不能持续其机体在特定水平上或不能维持预定的运动强度”。疲劳产生的机制有衰竭学说、堵塞学说、突变学说、自由基学说等。运动性疲劳属于中医的劳倦之类的范畴。根据藏象学说,运动性疲劳的发生发展大致与脏腑有关。五脏方面,中医认为“肾藏精”,肾主骨生髓,乃是先天之本,是体力产生和维持的基础动力和源泉。肾精不足,骨髓耗损,则会引起先天之气虚亏,骨骼系统发育不良,容易产生运动性疲劳。脾为“后天之本,气血生化之源”,脾主四肢肌肉,与运动的关系息息相关。脾受损,肌肉发育不良,力量不足。肝藏血,当运动时大量血液涌向骨骼肌,这时就需要肝脏调节血量,用以供应肢体的运动。肝主筋,如果肝功能正常,肝血丰富,可以使筋脉得养,运动有力,肌腱关节滑利,灵活而持久。若肝的气血不够充盈,则筋失濡养,动作迟缓,达不到预定的强度就出现了运动性疲劳。心主血脉,心藏神,心与运动员运动时的血液循环和意志品质密切相关。我们也认为,从中医的角度,运动性疲劳,既有实证,也有虚证,也有虚实夹杂证。二、研究目标与内容研究目标:广东凉茶是广东地区居民根据当地气候、水土特性,在长期预防疾病与保健过程中以中医养生理论为指导,以天然中草药为原料,煎熬而成的一种汤剂,具有清毒、生津止渴、防治疾病等功效。目前,作为饮品越来越受到人们的喜爱,但凉茶的功效“清热解毒,防止上火”是个相对模糊的概念,缺乏严谨的定义和科学依据。而针灸一直是专家比较推荐的祛除运动性疲劳的方法,本研究以中医药理论为指导,依据广东凉茶和针刺穴位的功效与应用,结合目前运动性疲劳产生机制等方面的研究进展,采用现代生物化学和病理学等研究方法,选取邓老凉茶和王老吉凉茶作为代表,观察凉茶和针刺穴位对大鼠生化,抗氧化、形态学和细胞凋亡指标的影响,以期为进一步解释凉茶的“清热解毒,防止上火”的功效提供了新的理论支持和科学数据。为使人们理解凉茶的功效,更好的推广和开发凉茶饮料奠定基础。希望能从中医的角度出发,在生物化学、细胞凋亡等层面,对防治运动性疲劳做出有益的研究和尝试。研究内容:1、运动和补充凉茶以及针刺对大鼠日常行为,症状的影响。2、补充不同种类的凉茶对实验大鼠生化,形态学和骨骼肌、肝肾功能等指标的影响。3、针刺穴位对实验大鼠生化,形态学和骨骼肌、肝肾功能等指标的影响。4、探讨凉茶和针刺延缓运动性疲劳的可能作用机制。三、研究方法与结论研究方法:SPF级雄性SD大鼠72只,随机分为6组,正常对照组、运动疲劳模型组、邓老凉茶组、王老吉凉茶组、宝矿力组、针灸组。除了正常对照组以外、其他五组均进行运动跑台训练。训练方案是根据Bedford理论改造的,每周7次,连续2周。针灸组取足三里穴进行针刺,其余组饮料灌胃。运动结束后,实验大鼠取血、骨骼肌、海马等。检测血糖、血氨、血尿素氮(BUN)、血肌酐(Cr),骨骼肌Ca2+浓度、骨骼肌和海马的MDA含量、T-AOC,乙酰胆碱酯酶活性,以及caspase-3在骨骼肌和海马的表达,并对骨骼肌、肝脏、海马光镜下观察结构。结论:1.本实验采用2周大强度跑台运动造模,实验大鼠血乳酸上升、MDA上升、总抗氧化能力下降、骨骼肌钙离子浓度增加,血氨上升,骨骼肌和海马capase-3表达增强外周疲劳和中枢疲劳的指标都出现了变化。说明本实验建立的运动性疲劳动物模型是成功的。而模型组的症状表明,运动性疲劳在中医范畴,并不仅仅是虚证,也有实证,会出现虚实夹杂证。2.我们的实验证明,王老吉凉茶和邓老凉茶能够有效降低降低骨骼肌中的MDA含量,增加骨骼肌的总抗氧化能力。增加大鼠海马的总抗氧化能力,减少MDA含量。我们推断,广东凉茶“清热解毒,防止上火”的功效,与其良好的抗氧化能力密切相关。3.王老吉凉茶和邓老凉茶能够补充运动中消耗的能量物质,维持血糖水平,降低肝脏、心脏细胞膜损伤,降低AST水平。4.针刺“足三里”穴,可以在多个环节和层面防治运动性疲劳。骨骼肌层面:使骨骼肌中的MDA含量减少,增加骨骼肌的总抗氧化能力,降低钙离子浓度;肝肾功能层面:能够增加肝糖原,肌糖原含量,降低血尿素氮。中枢神经层而,针灸对运动疲劳大鼠神经细胞保护机制的良性调节作用,可能是通过下调caspase-3的表达,阻止caspase-3的活化,最终达到对抗细胞凋亡的目的,对剧烈运动后的脑组织有保护作用,但相关机制不清,需进一步研究。四、创新点、存在问题与展望创新点:1.在理论上,通过了了查阅文献进行了文献综述,结合实验的症状观察,提出运动性疲劳属于虚实夹杂证这一新假说。2.在应用方而,从各个侧面较全面的反映了广东凉茶和针灸两种不同中医药手段对运动性疲劳的影响,为运动性疲劳的防治提供了具中医药特色的新思路,也为临床和科研提供了评价,研究运动性疲劳及干预措施评价的指标和工具。3.在成果方面,应用广东凉茶来干预运动性疲劳,目前国内外无人进行相关研究。同时,将凉茶“清热解毒,防止上火”的功效与抗氧化联系起来,为进一步,用现代医学的技术和方法研究广东凉茶奠定了一定的基础。存在的问题与展望:1.大鼠模型的塑造与评价仍需进一步完善,跑台是否完全模拟了运动员的训练状态,中枢性疲劳还需要哪些指标来评价,是否能用中医的诊断方法来建立运动性疲劳大鼠的标准化指标等都需要进一步研究。2.因受时间、经费的限制,本实验仅仅初步证实了运动和针灸对细胞凋亡caspase-3表达的影响,其作用机制与通路仍需进一步研究。特别是运动对骨骼肌与大脑细胞凋亡的作用机制与通路。3.本研究初步证实了针刺足三里穴对运动性疲劳多个环节的作用,但是其机制,以及选择其他穴位、其他方法的功效是否有差异尚不清。4.本研究初步表明,邓老凉茶和王老吉凉茶能够改善运动性疲劳状态,将其“清热解毒,防止上火”的功效与抗氧化联系起来。但是作为饮品凉茶当中有效的成分是哪些,也是我们今后研究的方向。

【Abstract】 Part one:Literature SummaryFatigue is a protective reaction when the body’s exercise load is enough, fatigue is a physiological phenomenon. Exercise-induced fatigue are defined as a phenomenon of reduced motor ability caused by failure of maintaining the normal function of the organism at a certain level and/or at the projected intensity during the physiological process of the organism. The study on occurrence mechanism of exercise-induced fatigue including exhaustion theory, blocking-up hypothesis、mutation theory、free radical theory, etcExercise-induced fatigue pertains to the category of excessive physical labor in the rangeof overstrain according to traditional Chinese medicine. According to the visceral manifestation theory, the occurrence and development of exercise-induced fatigue approximately is related to viscera. In the aspects of five viscera, Traditional Chinese Medicine considers that "the kidney stores the essence", kidney in charge of bone and Marrow, we considerer kidney as the congenital foundationg, the origination source of power. The deficiency of kidney essence can influence the bone and muscle development which could easy cause the exercise-induced fatigue. Traditional Chinese Medicine considers that "The spleen provides the materiel basis of the acquired constitution, is the source of growth and development", and occupies an important position in the course of life activity.Spleen controls the muscle and four limbs which are related to Sports. If the spleen get hurts, the muscle became dysplasia, then the deficiency of strength, liver storing blood, when people doing sports, large blood stream to skeletal muscle, so we need liver regulate the flow of blood, to support the movement of limbs, liver controls the activity of the tendon, if the liver function is normal, the tendon and joints get nourished by the abundant liver blood, liver could make the sports powerful, smoothly, and persistently, the deficiency of liver blood induce the malnurition of tendon, so the movement will get slow and will to be exercise-induced fatigue quickly. From the theory of "heart governs the blood, the heart storing spirit", heart is closely related to the athletes’blood circulation will and character. We also considered that from the view of TCM, exercise-induced fatigue is not only asthenia syndrome but also excess syndrome, meanwhile exercise-induced fatigue is a disease with combination of excess and asthenia syndromes.Part two:Study target and ContentGuangdong herbal tea is widely consumed in Guangdong as traditional health drinks, with the function "heat-clearing and detoxifying, preventing suffer from excessive internal heat".With the improvement of living standards in recent years, more and more people are concerned about health care and health, as a class of herbal-based drinks tea not only more and more of the Guangdong residents love, but also herbal tea has been developed throughout all China. But the relatively ambiguous conceptof heat-clearing and detoxifying, preventing suffer from excessive internal heat is lack of precise definition.Based on Specialists-recommending, acupuncture is a good method to dispel exercise-induced fatigue.According to Traditional Chinese Medicine theory, this research select Wang Laoji herbal tea and Deng Lao herbal tea as the represents of Guangdong herbal, to compare the effect of Guangdong herbal and acupuncture on exercise-induced fatigue, from a brand-new perspective of pathology,antioxidation, biochemistry which has provided further theoritcal support and scientific data to elucidate this concept, as well as to promote and develop herbal tea. By the research, we want to make beneficial study and attempt of preventing exercise-induced fatigue.Content1. The effect of exercise^Guangdong herbal tea、acupuncture on the daily behavior and symptoms of rat.2. The influence of different kinds herbal tea supplement on the index of skeletal muscle, the function of kidney and liver, biochemorphology, biochemistry of rats. 3. The effect of acupuncture points on the index of skeletal muscle,the function of kidney and liver, biochemorphology, biochemistry of rats.4. To investigate the possible mechanism of herbal tea and acupuncture on exercise-induced fatigue.Part three:Research method and ConclusionMethodThere are all together72male SD rats, they are randomly divided into six groups:control group, fatigue treadmill training group, Wang Laoji herbal tea group, Deng Lao herbal tea group, pocari group, acupuncture group, After one week of natural feeding, except for the control, the other five group received2weeks training on treadmill, the training formula was based on the theory of Bedford, six times one week, last for two weeks, the acupuncture group accepted acupuncture treatment at the same time, Zhongwan and Zusanli were selected for acupuncture. The three beverage group accepted gastric perfusion with beverage. Draw material like hippocampus、skeletal muscle、 kidney、liver、serum after exhausting exercise. Related indexes were detected after training; content of MDA in hippocampus and skeletal muscle, the T-AOC activity of hippocampus and skeletal muscle、content of glucose、content of blood ammonia、BUN、blood lactate、free calmodulin of skeletal muscles、 CR、activities of acetylcholinesterase、expression of the caspase-3in hippocampus and skeletal muscle. Kidney、live and hippocampus were obtained from n microscopic examination.Conclusion1. Compared with the control group, by the two weeks treadmill training, the exercise-induced fatigue rat’s contents of MDA in skeletal muscles、blood ammonia、blood lactate、free calmodulin of skeletal muscles、expression of the caspase-3in hippocampus and skeletal muscle increased significantly, the T-AOC markedly decreased. The index of peripheral and central have changed.It suggest that the exercise-induced model was successed. The symptom of fatigue treadmill training group suggest that, exercise-induced fatigue is not only asthenia syndrome but also excess syndrome, meanwhile is a disease with combination of excess and asthenia syndromes.2. Our experiment suggest that, Wang Laoji herbal tea and Deng Lao herbal tea could effectly reduce the content of MDA in skeletal muscle、increase in skeletal muscle of the total antioxidant capacity. They could increase the total antioxidant capacity of hippocampus in rat, reduce the content of MDA in skeletal muscle. We deduced that the function " heat-clearing and detoxifying, preventing suffer from excessive internal heat" of Guangdong herbal tea is closely related to the antioxidant capacity.3. Wang Laoji herbal tea and Deng Lao herbal tea could supply the energy material during exercise, maintain the level of blood glucose, ruduce the injury of liver and the membrane of hearts, reduce the level of AST.4. Acupuncture Zusanli, could prevent and treat exercise-induced fatigue in many steps and aspects. In the aspect of skeletal muscle:It could reduce the content of MDA inskeletal muscle, increase the total antioxidant capacity of skeletal muscle, reduced the content of calcium. In the aspect of the function of liver and kidney.-It could increase the content of liver glycogen, muscle glycogen, reduced the BUN. In the aspect of central nervous, the benign regulation effect of the protect mechanism of acupuncture on the neuron of exercise-induced rat might be reduce the expression of the caspase-3, prevent the activation of caspase-3, finally reach the purpose of anti-apoptosis, have protect the brain tissue after intensive exercise,but the related mechanism is not clear, it still need further study.Part four:Innovation point、Deficiency and Prospect Innovation Point1. In theory, by reviewing the related literatures for the review, combined with the observation of the symptoms through experiments, the author raised this noval hypothesis that:exercise-induced fatigue is a disease with combination of excess and asthenia syndromes.2. In terms of application, this research made a very comprehensive comparison of the impact of two different means of Traditional Chinese Medicine: Guangdong herbal tea and acupuncture on exercise-induced fatigue from all different perspectives.So this not only gives us inspiration on how to make the prevention and treatment of sports fatigue with Chinese medicine, but also would have profound influence for clinical and research evaluation, intervention indicators and tools.3. In terms of achivements, until now, no relevant research had conducted before neither in China nor abroad. This study firstly made application of Guangdong herbal tea to interfere with the exercise-induced fatigue. And, this research find the clues under the herbal tea "heat-clearing and detoxifying, preventing suffer from excessive internal heat " effect and antioxidant. By using the update techniques and methods of modern medicine, this research laid a certain foundation for the further study of Guangdong herbal tea.Deficiency and Prospect1. It is essential to further perfect the Establishment and assessment of rats model, and further study should made on:whether this treadmill has completely imitate the traning state of the atheletes. What kind of index shoude be abtained to evaluate the central nervous fatigue. Whether it is reasonable to use the "TCM diagnostic methods" as the standardizedi ndex for exercise-induced fatigue.2. Limited by time and funds, this experiment preliminary confirmed the effects of exercise and acupuncture on the expression of apoptosis Caspase-3, its mechanisms and pathways still needs further research, the mechanisms and pathways of the movements to exercise on apoptosis in skel etal muscle and brain cells apoptosis.3. In the aspect of central nervous, acupuncture havethe benign regulation effect of the protect mechanism on the neuron of exercise-induced rat might be reduce the expression of the caspase-3, prevent the activation of caspase-3, finally reach the purpose of anti-apoptosis, have protect the brain tissue after intensive exercise, but the related mechanism is not clear, it still need further study.4. This preliminary results indicated that Wang Laoji herbal tea and D eng Lao herbal tea could improve the state of exercise-induced fatigue, the function "heat-clearing and detoxifying, preventing suffer from exces sive internal heat "of Guangdong herbal tea is closely related to the anti oxidant capacity. We should discuss which effective components in the her bal tea in our further research

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