【作者】 王斌；

【导师】 丁树哲；

【作者基本信息】 华东师范大学 ， 运动人体科学， 2008， 博士

【摘要】 研究目的:适宜运动使机体产生良好适应,根据应激适应理论,可以说应激是机体产生适应的基础。中枢神经系统的海马脑区是应激的敏感部位,应激则可能引致海马的损伤发生。应激过程中以肾上腺皮质激素增多为主要特征,海马中含有丰富的糖皮质激素受体,在神经元兴奋性双向调节、细胞凋亡、突触长时程增强及学习和记忆形成中均有重要作用,应激引起海马糖皮质激素受体变化,通过激素反应元件影响蛋白质合成、海马神经元兴奋性的改变等。海马内有丰富的谷氨酸能神经元,神经元释放兴奋性氨基酸,通过NMDA受体影响细胞膜对离子的通透性,尤其是钙离子的通透性,正常的NMDA受体活性和海马神经内的钙离子水平是海马学习和记忆的基础;在严重应激损伤时兴奋性氨基酸或NMOA受体活性加大,引起细胞内及线粒体内的钙离子超载,钙离子超载则引起氧化磷酸化脱偶联等效应,产生自由基,而自由基又会进一步引起组织的氧化损伤,即氧化应激。钙离子、活性氧则迅速启动神经的保护机制,如核因子kappa B、雌激素等。雌激素自身即具有抗氧化作用,是一种抗氧化剂;同时,雌激素通过非基因组(快速)和基因组机制(慢速)起到神经保护作用,并可能对HPA轴起到调节作用,应对机体的应激状态。本研究旨在通过游泳这一运动模式,以水环境为参照,观察环境及运动对海马功能相关指标及应激反应的HPA轴激素的影响,探讨应激对海马功能的影响,以及海马神经保护和对HPA轴的可能调节机制,为运动性中枢疲劳理论的发展提供依据。研究方法:研究选用雄性SD大鼠进行实验,分别观察急性(一次)运动、经过耐力训练过的大鼠进行急性运动的效应。实验设计几个不同的时间观测点,即运动15min、运动至力竭后即刻、运动至力竭后1hr、运动至力竭后24hr。相应的参照组大鼠以水环境暴露,也类似设立急性(一次)水环境及长期水环境,取样也设为相同的4个点。实验进行中使用两个独立的水池,耐力训练大鼠进行游泳的同时,参照组大鼠则同时浸于水中(自由),以尽量减少非实验施加因素的影响,如水温、激素的生物节律等,但参照组的水深仅为大鼠直立后达耳下缘左右(约15-20cm)。以本实验室创立的大鼠负重游泳乳酸阈强度模型建立的强度进行耐力训练和力竭性运动强度设定。测试大鼠的血红蛋白、力竭游泳能力及乳酸阈强度的提高为模型建立成功的考察指标。实验采用放射免疫法测定了大鼠部分激素含量,包括下丘脑中的促肾上腺皮质激素释放激素(CRH)、皮质酮、雌二醇;血清中的促肾上腺皮质激素、皮质酮、睾酮。观察了海马CA1区神经元的细胞体的电镜超微结构。采用顺磁共振自旋捕获技术测定了海马CA1区羟自由基信号强度。采用western blot法测定了海马糖皮质激素受体(GR)、雌激素受体alpha(ERα)、核因子kappa B(NF-κB)、N-甲基-D-天门冬氨酸受体2A亚型(NMDAR2A)、钙调蛋白激酶Ⅱalpha亚型(CaMKⅡα)。研究结果:1、动物运动模型。每周6次,每次30min以乳酸阈强度90%负重游泳6周后,大鼠游泳到达力竭的时间显著延长(训练过的大鼠44.42±32.29min,未训练过的大鼠21.32±20.12min,p＜0.05);训练大鼠血红蛋白含量显著性高于未训练大鼠(训练过的大鼠146.08±7.35g/L,未训练过的大鼠134.50±8.33g/L,p＜0.05)。2、海马CA1区锥体细胞形态。急性、慢性水环境及急性、慢性运动作用均可能引起其超微结构的变化。其变化程度有所不同,均未见有明显的核固缩、核碎裂及核溶解等细胞凋亡的典型表现。急性水环境因素作用下,大鼠海马CA1区锥体细胞形态发性改变,如细胞外膜不清,细胞内细胞器也发生损伤,如核膜消失、线粒体空泡化现象等。从急性水环境作用时间看,急性水环境作用15min和作用后1hr,细胞的受损状况更为严重,而在急性环境因素后即刻和24hr受损则小于15min和1hr大鼠的情况。慢性水环境与急性影响结果类似。急性运动因素作用下,大鼠海马CA1区锥体细胞形态发性改变,细胞外膜和核膜的变形,内质网和线粒体肿胀等。急性运动作用后即刻和之后的1hr,细胞受损情况最为严重,之后的24hr,细胞形态已经基本正常化。慢性运动因素作用下,大鼠海马CA1区锥体细胞结构变形最大的是运动15min和运动后24hr,在运动后即刻及1hr,其受损状况反而相对来说较小。3、海马羟自由基。长期水环境影响的大鼠再施加急性水环境因素与未进行水预适应的大鼠施加急性水环境因素,以及耐力训练大鼠和未进行过耐力训练的大鼠进行急性力竭运动时,从各因素作用的总体看海马羟自由基信号强度无显著性差异,水环境和运动对海马羟自由基的影响具有同一性;但耐力训练大鼠力竭运动后即刻海马羟自由基强度显著性高于未训练大鼠运动后即刻大鼠,与之相对应的长期水环境大鼠在水环境作用后即刻与水预适应大鼠水环境后即刻比没有差异。4、激素。各组大鼠下丘脑CRH无显著性差异,但仍可见耐力训练过的大鼠在力竭后即刻下丘脑CRH均数高于未训练大鼠力竭运动后即刻(无显著性差异)。耐力运动大鼠下丘脑皮质酮整体上显著性高于长期水环境大鼠;训练和未训练过的大鼠在力竭性运动后即刻下丘脑皮质酮均显著性高于各自的运动15min大鼠;未训练过的大鼠运动后1hr下丘脑皮质酮就显著性低于运动后即刻,而耐力训练大鼠则依然保持较高的下丘脑皮质酮水平。各组大鼠血清ACTH无显著性差别。血清皮质酮,各组大鼠在水或运动因素作用后即刻显著低于15分时,因素作用后1hr显著高于即刻值。以所有大鼠下丘脑皮质酮含量与血清皮质酮浓度作相关分析发现,下丘脑皮质酮含量与血清皮质酮呈弱的负相关关系,R=-0.228,p＜0.05。下丘脑雌二醇,耐力训练大鼠和长期水环境大鼠均较一次水环境或一次运动大鼠高,但只有长期水环境大鼠与一次水环境大鼠比有显著性差异;一次水环境、长期水环境后急性水环境、一次运动和耐力训练大鼠急性运动后即刻、1hr和24hr均显著性高于各自15min,运动后直至24hr均处于高水平。一次水环境组大鼠血清睾酮显著性高于长期水环境、急性运动和耐力训练大鼠急性运动组。各组大鼠运动后24hr血清睾酮达最高值,差异具有显著性。5、海马相关信号蛋白含量。海马雌激素受体α水平,一次水环境、长期水环境和一次运动大鼠在相应作用因素后即刻均显著性高于15min时、以及之后的1hr及24hr,而耐力训练大鼠在运动后即刻是下降趋势,运动后24hr升高。耐力训练大鼠海马糖皮质激素受体在相同的采样时间点时均有低于其它各处理因素大鼠的趋势,但无显著性差异。海马NF-κB整体上各处理因素和采样时间点间无显著性差异,但一次水环境后即刻显著性低于一次水环境后1hr和24hr,并且显著性低于同样是即刻状态的长期水环境大鼠、一次运动大鼠和耐力训练大鼠;运动后24hr,耐力训练大鼠海马NF-κB显著性高于未训练大鼠;同时,未训练大鼠运动后即刻最高,而耐力训练大鼠则是运动后24hr最高。海马NMDAR2A含量,耐力训练大鼠整体上显著性高于一次运动大鼠,耐力训练大鼠力竭运动后即刻显著性高于长期水环境大鼠;耐力训练和未训练大鼠运动后即刻均高于各自的后1hr和24hr水平。CaMKⅡα水平,从整体看,一次急性运动与一次水环境大鼠存在显著性差异,长期水环境大鼠与一次水环境大鼠存在显著性差异,因素后24hr与因素作用后即刻和之后1hr有显著性差异;耐力训练大鼠运动后24hr水平显著性高于长期水环境24hr组和未训练大鼠运动力竭后24hr组。6、相关性分析。大鼠力竭游泳时间与下丘脑CRH和海马ERα相关,偏相关系数分别为0.573,p＜0.001:-0.584,p＜0.001。其它具有显著性的两变量pearson相关有:NF-κB与CaMKⅡα相关(r=0.253,p＜0.05),ERα与GR、下丘脑雌二醇、下丘脑皮质酮(r=0.428,p＜0.001;r=-0.250,p＜0.05;r=-0.219,p＜0.05),海马羟自由基信号强度与血清睾酮、血清皮质酮(r=0.232,p＜0.05;r=-0.443,p＜0.001),血清皮质酮与下丘脑皮质酮(r=-0.220,p＜0.05),下丘脑内皮质酮与CRH(r=0.214.p＜0.05)。结论:1)HPA轴激素相互调节中CRH和下丘脑皮质酮起重要作用,而非血清皮质酮;下丘脑应激增高(皮质酮增高),伴随雌二醇生成增多。下丘脑皮质酮和血清皮质酮含量呈负相关关系,尚需进一步研究。2)海马超微结构可因环境和游泳运动刺激发生改变,其变化可在不长的时间内恢复,可能不是损伤性变化,更可能是学习和记忆或适应过程中的一个阶段表现。3)海马对HPA轴的负反馈调节与海马的整体机能状况有关,海马ERα和羟自由基与HPA的负反馈调节有关。CaMKⅡα可能与HPA轴下游激素有关,并影响海马在应激时的机能。4)海马NF-κB可能起到的神经保护作用,在运动性疲劳发生时(本研究的力竭运动)并不具重要意义,其意义在于修复和恢复过程,这对于运动训练的适应机制的研究十分重要。5)海马ERα直接与运动能力有关,ERα含量越高,运动能力越低(可能是提供此直接证据的首次报道)。下丘脑CRH与运动能力正相关,CRH含量越高,运动能力越强。更多还原

【Abstract】 Objectives:Exercise enables the adaptation of the body,and stress can be said to be the basis of body adaptation,according to the stress reaction stages,i.e.,the alert stage,the resistance stage and the failure stage.On the other hand,the hippocampus of the central nervous system is a sensitive part of stress;stress may lead to hippocampal damage.The process of stress is characterized by increased adrenal hormones.The hippocampus is rich in glucocorticoid receptors,and plays an important role in the two-way adjustment of excitatory neurons,in apoptosis,in long-term synaptic potentiation,and in learning and memory formation.Stress induces changes in hippocampal glucocorticoid receptors,and affects protein synthesis through hormone response elements and excitatory neurons in the hippocampus.The hippocampus,rich in glutamate neurons,releases excitatory amino acids,and,through NMDA receptors,affects the permeability of cell membranes on ions,calcium ions in particular.Normal NMDA receptors activity and the calcium levels of hippoeampal neurons is the basis of hippoeampal learning and memory;in severe stress injury,the activity of excitatory amino acids or NMDA receptors increases,which causes calcium ion overload in cells and mitochondria,which,in turn,witnesses de-coupling effects of oxidative phosphorylation and generates free radicals,which further leads to oxidative damage of tissues(i.e.,oxidative stress). Calcium ions and reactive oxygen species rapidly initiate the mechanism for neural protection,such as both nuclear factor kappa B and estrogen.Estrogen itself is an antioxidant;at the same time,through non-genomic(fast) and genomic(slow) mechanisms,estrogen plays a role in neural protection and may regulate the HPA axis against body stress.The objective of the present study,which observes the effects of environment and exercise on indicators of hippocampal functions and HPA axis hormones under stress through the swimming exercise model in terms of water environment, investigates the effects of stress on hippocampal functions,and the mechanism for neural protection and HPA axis regulation to provide the basis for exercise-induced central fatigue theory.Methods:To test the above hypothesis,male SD rats were selected,and the effects of acute exercise were observed for the acute exercise and endurance training conditions.The points of observation in the experimental design were 15min exercise, immediately after exhaustive exercise,lhr after exhaustive exercise,and 24hr after exhaustive exercise.The rats in the control group were exposed to the water environment,and the effects observed for the acute water environment and long-term water environment,with four points of observation designated.Two separate pools were used,and while the endurance training rats were swimming,the control group rats were immersed in water(free) to minimize the interference of non-experimental factors like water temperature and the biological rhythm of hormones.However,the water depth of the control group was only the level that reached the lower ear edges of the control rats(about 15-20cm).The intensity on the weight-bearing swimming lactate threshold intensity model was designated as the intensity of endurance training and exhaustive exercise.Increased hemoglobin levels,exhaustive swimming ability and lactate threshold intensity were designated as indicators of a successful model.Radioimmunoassay was used for determination of the levels of some hormones in the rats,including corticotropin-releasing hormone(CRH),corticosterone and estradiol in the hypothalamus,and adrenocorticotropin,corticosterone and testosterone in the serum.The electron microscope ultrastructures of the cells of the hippocampal CAI neurons were observed.Paramagnetic resonance techniques were used for determination of the intensity of hydroxyl radical signals in the hippocampus CA1 area.Western blot was used for determination of hippocampal glucocorticoid receptors(GR),estrogen receptor alpha(ERα),nuclear factor kappa B(NF-κB), N-methyl-D-aspartate receptor subtype 2A(NMDAR2A) and calmodulin kinaseⅡalpha subtypes(CaMKⅡα).To enrich the lactic acid threshold intensity exercise model,high-density lipoprotein cholesterol levels were tested.Results:Animal exercise model.6 weeks after weight-bearing swimming at 90%of the lactate threshold intensity for 30min,6 times a week,the time that the rats reached exhaustion was delayed(trained rats 44.42±32.29min,and untrained rats 21.32±20.12 min,p＜0.05),hemoglobin levels were significantly higher in the trained rats than in the untrained rats(trained 146.08±7.35g / L,and untrained 134.50±8.33g / L,p＜0.05).Form of pyramidal cells of the hippocampal CA1 area.Both the acute and chronic water environment conditions,and the acute and chronic exercise conditions led to changes in the ultrastructures.The extent of change differed;no obvious nuclear condensation,nuclear fragmentation or dissolution of nuclear apoptosis occurred.In the acute water environment condition,the form of pyramidal cells in the hippocampal CA1 area changed,such as unclear outer membranes of cell,and damage to intracellular organdies occurred,such as the disappearance of nuclear membranes and mitochondrial vacuolization.In terms of the effects of time,cell damage was more serious 15min and lhr,but less so immediately and 24hr after the acute environment condition.The results in the chronic water environment were similar.In the acute exercise condition,the form of pyramidal cells of the hippocampal CA1 area changed,such as deformation of the outer membranes of cells and that of nuclear membranes,and swelling of the endoplasmic reticula and mitochondria. Immediately and 1hr after acute exercise,damage to cells was the most serious,and the form of cells returned to normal 24hr after acute exercise.In the chronic exercise condition,the deformation of the structures of pyramidal cells of the hippocampal CA1 area was the most serious 15min and 24hr after exercise,while the damage proved small immediately and lhr after exercise.Hippocampus hydroxyl radicals.No significant differences in the intensity of hippocampus hydroxyl radicals occurred in the long-term water environment re-treated with acute,in the untreated condition re-treated with acute,and in the endurance training condition and the untrained re-treated with acute exhaustive exercise condition.The effects of water environment and exercise were parallel. However,the intensity was significantly higher in the endurance training rats than in the untrained rats immediately after exercise.No difference between the long-term water environment condition and and the untreated condition immediately after the factor.Hormones.No significant difference in hypothalamus CRH occurred under the conditions,but hypothalamus CRH averages were higher in the trained rats than in the untrained rats immediately after exhaustive training(with no significant difference). The overall significance of hypothalamus corticosterone levels was higher in the endurance exercise rats than in the long-term water environment rats;hypothalamus corticosterone levels were significantly higher in the rats immediately after than 15min after exercise;corticosterone in the hypothalamus was significantly lower in the untrained rats lhr after than immediately after exercise,and the levels of corticosterone in the hypothalamus were higher in the endurance training rats.Serum ACTH levels were not significantly different in the conditions.Serum corticosterone levels were significantly lower immediately after than 15min after the water environment or exercise condition,and significantly higher immediately after than lhr after the water or exercise condition.Correlation analysis of hypothalamus corticosterone and serum corticosterone showed that the two displayed a mildly negative correlation,R=-0.228,p＜0.05.Estrogen in the hypothalamus were higher in the endurance training and water environment rats than in the single water environment or single exercise rats,but only the ratio of the long-term water environment to single was significantly different;significantly higher in the single water environment,long-term acute water environment after long-term water environment,single exercise and endurance training immediately,lhr and 24hr after acute exercise than 15min,and up to 24hr after exercise,serum testosterone was significantly higher in a single water environment significantly higher than that in the long-term,acute exercise and endurance training acute exercise rats.serum testosterone reached its maximum levels 24hr after exercise in the various groups with significant difference.Hippocampal signal protein content.Hippocampal estrogen receptor levels in the single water environment,long-term water environment and single exercise conditions were significantly higher immediately after the factors than 15min,1hr and 24hr after the factors,whereas a decreasing trend emerged immediately after exercise and an increase occurred 24hr after exercise.Hippocampal glucocorticoid receptors saw the same trend in the endurance training rats as the other rats,at the same points of sampling but with insignificant difference.As a whole,Hippocampus NF-κB was not significantly different when the factors and points of sampling were considered, but was significantly lower immediately after than lhr and 24hr after the single water environment,and significantly lower than in the long-term water environment,single exercise and endurance training conditions immediately after exercise.24hr after exercise,hippocampal NF-κB was significantly higher in the endurance training rats than in the untrained rats.In addition,NF-κB was the highest in the untrained rats immediately after exercise,and in the endurance training rats 24hr after exercise.In terms of hippocampus NMDAR2A,the overall significance was higher in the endurance training rats than in the single exercise rats,in the endurance training rats than in the long-term water environment rats immediately after exhaustive exercise, and in the endurance training and untrained rats immediately after than 1hr and 24hr after exercise.A significant difference in CaMKⅡαlevels occurred between the single acute exercise rats and the single water environment rats,between the long-term water environment rats and the single water environment rats,between 24hr after the factors and immediately and 1hr after the factors;the levels were significantly higher in the endurance training rats than in the long-term water environment rats and the untrained rats 24hr after exercise.Correlation analysis.The time for exhaustive swimming in the rats correlated with hypothalamus CRH and hippoeampus ERα,the partial correlation coefficient being 0.573,p＜0.001 and-0.584,p＜0.001,respectively.Other significant bivariate Pearson correlations were:NF-κB and CaMKⅡα(r= 0.253,p＜0.05),ERα,and GR, estrogen in the hypothalamus,and hypothalamus corticosterone(r= 0.428,p＜0.001; r=-0.250,p＜0.05;r=-0.219,p＜0.05),hydroxyl radical hippocampal signal intensity and serum testosterone and corticosterone(r= 0.232,p＜0.05;r=0.443,p＜0.001), serum eorticosterone and eorticosterone in the hypothalamus(r=-0.220,p＜0.05), corticosterone in the hypothalamus and CRH(r=0.214,p＜0.05).Conclusion:CRH and hypothalamus corticosterone,rather than serum corticosterone,play an important role in the regulation of HPA axis hormones;increased hypothalamic stress (increased corticosterone) levels accompanied with increased estradiol levels.The ultrastructures of the hippocampus may change due to environment and swimming excitation,which may return to normal within a short period of time.It may not be damage change,but more likely to be stage adaptation in the process of learning and memory.The negative feedback regulation of the hippocampus on the HPA axis is related to the overall hippocampal functions,and hippocampus ERαand hydroxyl free radicals are related to the negative feedback regulation of HPA.NF-κB may more important in delayed repair that may affect adaption progress.ERαin hippocampal is closely related to exercise ability(probably the first piece of empirical evidence).The above conclusion is generalizable to male SD rats.更多还原