【作者】 刘志元；

【导师】 张林；

【作者基本信息】 苏州大学 ， 体育教育训练学， 2010， 博士

【摘要】 研究目的:运动应激是运动训练与运动健身产生效应的生物学基础,但是运动应激时的下丘脑-垂体-肾上腺轴(HPA)轴中枢反应机制研究较少,尤其对于不同运动应激时一氧化氮(NO)通过糖皮质激素受体(GR)与糖皮质激素(GC)结合而影响HPA轴调节、以及GR、NOS的变化对HPA轴中枢调节系统研究未见报道。本研究以不同负荷跑台运动引起的应激状态为研究对象,从HPA轴外周激素水平变化、中枢相关受体基因水平变化和蛋白表达变化等不同层次和角度,分析不同负荷运动应激的HPA轴的应激程度及中枢调节规律,揭示不同运动负荷应激时的中枢调节机制。研究方法:11周龄雄性SD大鼠130只,随机分为长期运动组、一次运动组及对照组。长期运动组大鼠以高、中、低三种不同负荷进行跑台训练,6天/周,1小时/天,实验期8周;一次运动组亦采用三种不同负荷运动,采用长期训练组同等负荷末次运动方案。测试方法与指标:双抗体夹心酶标免疫分析法测定血清CORT、T、ACTH、下丘脑CRH含量;免疫组化法测海马CA1区、下丘脑弓状核、腹内侧核、背内侧核、室旁核、中央杏仁核进行形态观察,并测定GR、NOS等阳性神经元数量变化;RT-PCR法测定海马、下丘脑、杏仁核GR、MR、NOS基因转录水平,Western blotting法测定上述核团GR、NOS蛋白表达量。研究结果:(1)长期大负荷运动组海马MR、GR转录减少、GR表达减少(P<0.05),NOS转录及表达水平无明显变化;下丘脑兴奋提高,GR、MR转录水平降低,NOS表达增多(P<0.05);杏仁核GR基因转录水平无明显变化,杏仁核NOS基因转录增多(P<0.05);ACTH升高,CRH、CORT浓度降低。(2)长期中等负荷运动组海马GR、NOS基因转录及表达增多(P<0.01),海马兴奋性降低;下丘脑兴奋性提高,GR转录减少,蛋白浓度升高,NOS基因转录及表达增多(P<0.05);杏仁核兴奋性提高,GR、NOS基因转录及表达减少,各核团MR基因转录减少;CRH、ACTH、CORT浓度升高(P<0.05)。(3)长期低负荷运动组海马兴奋性无明显变化,MR基因转录增多,GR转录水平降低,NOS基因转录及表达增多(P<0.05);下丘脑兴奋,NOS、GR基因转录及表达增多(P<0.05);杏仁核NOS基因转录及表达减少,GR基因转录及表达无明显变化,杏仁核及下丘脑MR基因转录减少,CRH、CORT浓度升高,ACTH无明显变化。(4)一次大负荷运动组海马NOS基因转录及表达增多,GR基因转录增多, MR基因转录减少,海马兴奋性降低;下丘脑兴奋性降低,NOS基因转录及表达增多,GR、MR基因转录减少,GR蛋白升高(P<0.05);杏仁核兴奋性降低,NOS、GR基因转录及表达减少,MR基因转录增多(P<0.05);CRH、CORT浓度升高,ACTH浓度降低(P<0.05)。(5)一次中等负荷运动组海马NOS基因转录及表达增多,GR基因转录及表达水平降低,海马兴奋性无明显变化;下丘脑室旁核兴奋,NOS基因转录及表达增多,GR基因转录水平无变化,表达增多(P<0.05);杏仁核GR基因转录及表达增多,NOS基因转录减少,但NOS浓度升高,各核团MR转录降低(P<0.05);CRH、CORT浓度升高,ACTH浓度降低。(6)一次低负荷运动海马兴奋性无变化,NOS、GR基因转录及表达减少,MR基因转录无变化;下丘脑兴奋性提高NOS基因转录无明显变化,NOS表达增多,GR基因转录及表达增多;杏仁核兴奋性提高,NOS基因转录及表达增多,GR基因转录无明显变化,表达增多,杏仁核及下丘脑MR基因转录减少(P<0.05);CRH、CORT浓度升高,ACTH浓度降低(P<0.05)。研究结论:(1)长期大负荷运动应激后海马、下丘脑及杏仁核兴奋性降低,HPA轴抑制;一次大负荷运动应激造成下丘脑及杏仁核兴奋性升高,HPA轴激活。(2)长期大负荷运动后海马抑制是由于海马兴奋性损伤导致;一次大负荷运动后海马抑制是由于外周高水平GC的负反馈调节。(3)运动应激反应中海马的兴奋性与NOS无相关性,即NOS仅参与海马兴奋性损伤,而不参与其兴奋性调节。(4)长期中等负荷运动与一次中等负荷运动应激HPA轴主要不同在于ACTH变化,长期中等负荷ACTH升高由于杏仁核NOS参与对垂体的调节,一次中等负荷后ACTH下降主要受外周GC、下丘脑与杏仁核的GR负反馈调节所致。(5)小负荷运动后海马兴奋性无明显变化,杏仁核与下丘脑兴奋性提高,长期小负荷运动后杏仁核NOS参与垂体ACTH调节,而一次负荷ACTH下降则受GC与海马GR的反馈调节。(6)长期运动HPA轴的应激反应中枢调节多于外周调节;一次运动主要以HPA轴各环节的反馈调节为主。更多还原

【Abstract】 Objective: Exercise Stress is the biological basis of sports training and sports fitness that can produce effects. However, there was less study of the central response mechanism of HPA axis in exercise stress. In particular, there was no report on the study of HPA axis regulation affected by NO through the combination of glucocorticoid receptor (GR) and glucocorticoid (GC) as well as the changes of GR NOS in HPA Axis central regulatory systems. The research took different load treadmill exercise- induced stress state as the research objective. It analysed different load of exercise stress level of HPA axis and central regulatory rule from the change of the levels of peripheral hormones HPA axis, central-related receptor gene and protein expression, and so on. It revealed the central regulatory mechanisms in the stress of different exercise load.Methods: 11-week 130 male SD rats were randomly divided into three groups: long-term exercise group, one exercise group and control group. The rats of that long-term exercise group were trained to run on the treadmill with three different loads: high-load, middle-load, and low-load, 6 days/week, one hour/day, and the experimental period was 8 weeks. One exercise group was also divided into three different load movements, adopting the last sports program of the same load of the long-term group. Testing methods and indices: It determined the content of serum CORT, T, ACTH, hypothalamic CRH levels with the method of double-antibody sandwich enzyme linked immunosorbent. With the method of Immunohistochemistry, it tested hippocampal CA1, hypothalamic arcuate nucleus, ventromedial nucleus, dorsomedial nucleus, paraventricular nucleus, central amygdaloid nucleus, and observed the morphological changes, determining the number of changes of GR, NOS-positive neurons, and so on. With the method of PT-PCR, it determined the gene transcription level of hippocampus, hypothalamus, and amygdala GR, MR, NOS. With the method of Western blotting, it determined the protein expression of GR and NOS of the above-mentioned nuclei.Results:(1) Hippocampus MR, GR transcription in the long-term heavy load exercise group decreased, and GR expression also decreased(P<0.05). Transcription and expression of NOS did not change significantly. The excitability in hypothalamus was elevated. GR, MR transcription decreased. NOS expression increased (P<0.05). GR gene transcription in the amygdale had no significant change, and NOS gene transcription in the amygdale increased (P<0.05). ACTH increased, but CRH, CORT concentration decreased.(2) Hippocampus GR, NOS gene transcription and expression increased in the long-term moderate load exercise group (P<0.01). The excitability in Hippocampus decreased, whereas the excitability in hypothalamus was elevated. GR transcription decreased, but protein concentration increased. NOS gene transcription and expression increased (P<0.05). Amygdala excitability increased; GR, NOS gene transcription and expression decreased, and the nuclei of MR decreased; CRH, ACTH, CORT concentration increased (P<0.05).(3) Hippocampal excitability in the long-term low-load exercise group had no significant change. MR gene transcription increased, whereas GR transcription level decreased. NOS gene transcription and expression increased (P<0.05); hypothalamus was excited. NOS, GR gene transcription and expression increased (P <0.05); NOS gene transcription and expression in amygdala decreased. GR gene transcription and expression showed no significant changes. MR gene transcription in the amygdala and hypothalamus decreased, CRH, CORT concentration increased. ACTH had no significant changes.(4) Hippocampus NOS gene transcription and expression increased in heavy load exercise group. GR gene transcription increased, but MR gene transcription decreased. Hippocampal excitability decreased. The excitability of hypothalamus decreased. NOS gene transcription and expression increased. GR, MR gene transcription decreased, whereas GR protein increased ( P<0.05 ) . Amygdala excitability decreased. NOS, GR gene transcription and expression decreased. MR gene transcription increased (P<0.05); CRH, CORT concentration increased, whereas ACTH concentration decreased (P <0.05).(5) Hippocampal NOS gene transcription and expression in moderate load exercise group increased. The level of GR gene transcription and expression decreased. There was no significant change in hippocampal excitability. Hypothalamic paraventricular nucleus was excited, and NOS gene transcription and expression increased. There was no change in the level of GR gene transcription, and the expression increased(P<0.05); GR gene transcription and expression in amygdala increased, whereas NOS gene transcription decreased. NOS concentration increased, but MR transcription of the nuclei decreased (P<0.05). CRH, CORT concentration increased, but ACTH concentration decreased.(6) There was no change in hippocampus excitability in a low-load exercise group. NOS, GR gene transcription and expression decreased. There was no change in MR gene transcription; Excitability of hypothalamic increased. NOS gene transcription showed no significant changes. NOS expression increased, GR gene transcription and expression increased, amygdala excitability increased, and NOS gene transcription and expression increased. GR gene transcription showed no significant changes, and expression increased. MR gene transcription of amygdala and hypothalamus decreased (P<0.05); CRH, CORT concentration increased, but ACTH concentration decreased (P<0.05).Conclusions(1) The stress of long-term heavy load exercise led to the decrease of the excitability in hypothalamus and amygdala, and the suppression of HPA Axis. The stress of one heavy load exercise led to the increase of the excitability in hypothalamus and amygdala. HPA axis was activated.(2) The suppression of hippocampal of long-term heavy load post-exercise was led by the injury of the hippocampus excitability. The suppression of hippocampus of one heavy load exercise was due to peripheral glucocorticoid feedback regulation.(3) In the response of the exercise stress, the excitability of the hippocampus had no correlation with NOS. NOS had participated only in the hippocampus excitatory damage, not participated in the regulation of their excitability.(4) The main difference of HPA axis of the long-term moderate load exercise and one moderate load exercise lied in the change of ACTH. The rise of ACTH of long-term moderate load exercise was due to the participation of the regulation to pituitary between the amygdale and NOS. The decrease of ACTH one moderate load exercise was caused by negative feedback regulation of peripheral GC, and GR of hypothalamus and amygdala.(5) There was no significant change for hippocampal excitability of low-load post-exercise. However, the excitability in amygdala and hypothalamus was elevated. After a long-term low-loaded exercise, amygdala NO participated in the regulation of pituitary ACTH. ACTH of one loaded exercise decreased, which was affected by the feedback regulation of GC and hippocampus GR.(6) The stress response of central regulation of HPA axis of a long-term exercise was more than peripheral regulation. One exercise gave priority to HPA axis feedback regulation of various sectors.更多还原