【作者】 郭时印；

【导师】 孙振球； 黄忆明；

【作者基本信息】 中南大学 ， 流行病与卫生统计学， 2009， 博士

【摘要】 第一章前言疲劳是人脑力或体力到达一定阶段时必然出现的一种正常的生理现象,是机体复杂的生理生化变化过程。随着社会节奏的加快,社会竞争日益激烈,疲劳已成为威胁人类健康的重要因素。因而延缓疲劳的发生和促进疲劳的恢复成为营养卫生学、航天医学、军事医学、运动医学、保健医学、康复医学等学科的研究重点。目前,消除疲劳的主要措施有休息、睡眠、按摩及某些物理方法等,这些虽然能消除疲劳,但在效果上仍有不如人意的地方。在寻找更安全、更有效的抗疲劳方法中,从天然产物中提取的活性物质由于其副作用少,不含对人体有害的兴奋剂成分而成为近年来抗疲劳研究的热点。辣椒素（capsaicin）是辣椒中的主要辛辣成分,可特异地作用于辣椒素敏感C类纤维（CSPNs）的辣椒素受体（VR）,继而操纵非选择性阳离子通道,引起一些活性物质释放,进而发挥作用。研究表明,辣椒素具有提高运动能力的功能,说明辣椒素可能具有抗疲劳的作用,但目前关于辣椒素抗疲劳的作用机理及其量效研究还不深入和系统,辣椒素对海马神经元的保护作用及相关机制的研究尚未见报道。第二章辣椒素抗疲劳作用的动物实验研究目的:观察辣椒素对小鼠的抗疲劳效果。方法:将168只雄性昆明种小鼠按体重随机分为7组:（1）正常对照组:灌胃等体积蒸馏水。（2）溶媒对照组:灌胃等体积的溶媒（10%乙醇+10%吐温80液+80%的生理盐水）。（3）低剂量辣椒素组:按3mg/kg·wet·d的剂量灌胃辣椒素。（4）中剂量辣椒素组:按6mg/kg·wet·d的剂量灌胃辣椒素。（5）中高剂量辣椒素组:按10mg/kg·wet·d的剂量灌胃辣椒素。（6）高剂量辣椒素组:按15mg/kg·wet·d的剂量灌胃辣椒素。（7）红景天（Rhodiola Rosea）阳性对照组:按干品1g/kg·wet·d的剂量灌胃。所有动物于每日晨起8时定时灌胃1次。灌胃28d后检测小鼠的体重,并检测小鼠的爬杆时间、负重游泳时间以及小鼠运动后血乳酸水平、肝糖原含量、血尿素氮含量及血乳酸脱氢酶的活力,作为抗疲劳实验的检测指标;以常压耐缺氧时间和急性脑缺血性耐缺氧时间,亚硝酸钠中毒时间作为抗缺氧实验的检测指标。结果:（1）辣椒素对小鼠体重增长无明显影响;（2）辣椒素可以明显延长小鼠的爬杆时间、负重游泳时间,低剂量处理组（3mg/kg）与正常对照组相比差异无统计学意义;中剂量、中高剂量、高剂量组与正常对照组相比,差异有统计学意义,且以高剂量（15mg/kg）时作用最明显;（3）辣椒素低剂量组（3mg/kg）小鼠常压耐缺氧时间和急性脑缺血性耐缺氧时间与正常对照组差异均无统计学意义（P＞0.05）;而辣椒素中、中高及高剂量组小鼠耐缺氧时间均显著延长,与正常对照组相比差异有统计学意义（P＜0.05）,其中以高剂量组（15mg/kg）作用最明显,其常压耐缺氧时间和耐急性脑缺血性缺氧时间分别提高了约64%和107%,且高剂量组与红景天阳性对照组差异无统计学意义。（4）辣椒素灌胃组小鼠运动后肝糖原、肌糖原含量及血乳酸脱氢酶活力均显著高于正常对照组,而运动后的血乳酸水平、血尿素氮水平均显著低于正常对照组,其中均以高剂量组（15mg/kg）作用最明显。（5）辣椒素灌胃组小鼠亚硝酸钠中毒存活时间与正常对照组相比,显著延长,差异有统计学意义（P＜0.05）。结论:辣椒素在6～15mg/kg范围内,可以显著延长小鼠的爬杆和力竭游泳时间,提高其抗缺氧的能力,并可以抑制运动后血乳酸和血尿素氮的生成,维持运动后小鼠肝脏和肌肉组织内糖原的含量,具有明显的抗运动疲劳的作用。第三章辣椒素抗疲劳作用的生化机制研究目的:在第二章的研究基础上,选择合适剂量的辣椒素,进一步探讨辣椒素抗疲劳的作用机制。方法:分别从以下4方面探讨辣椒素抗疲劳的作用机制:（1）抗氧化系统:①检测各组小鼠运动前后肝组织超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）及脂质过氧化产物丙二醛（MDA）含量;②检测血浆中肌细胞损伤的标志性酶肌酸激酶（CK）及肌酸激酶MB亚型（CK-MB）含量。（2）能量代谢系统:①检测各组小鼠运动前后血糖的水平,肌、肝糖原含量;②检测各组小鼠运动前后血浆游离脂肪酸（FFA）浓度:③检测血浆总蛋白（TP）、白蛋白（Al）、球蛋白（Glob）含量;（3）中枢系统:①各组大鼠进行睡眠剥夺72h后,进行旷场实验,计算各组睡眠剥夺后的旷场实验评分;②将各组大鼠处死后,迅速取大鼠下丘脑、海马组织,检测5-羟色胺（5-HT）,去甲肾上腺素,肾上腺素,多巴胺,5-羟基吲哚乙酸（5-HIAA）的含量,并检测血氨含量。结果:（1）抗氧化系统:①运动前,各组肝脏SOD、GSH-Px活性和肝脏MDA的水平差异无统计学意义（P＞0.05或P＞0.01）;运动后,不同浓度辣椒素灌胃组肝脏SOD和GSH-Px活性与正常对照组相比,显著升高,而肝脏MDA的水平显著降低,差异均有统计学意义（P＜0.05或P＜0.01）,其中以高剂量组（15mg/kg）作用最明显。②运动前辣椒素灌胃组与正常对照组相比,血浆CK、CK-MB含量差异无统计学意义（P＞0.05）。长时间运动后,不同浓度辣椒素处理组的血浆CK及CK-MB的含量显著降低,差异有统计学意义（P＜0.05或P＜0.01）,其中以高剂量辣椒素处理组（15mg/kg）作用最明显。（2）能量代谢系统:①静止状态下,各组小鼠的血糖水平差异无统计学意义（P＞0.05）:运动后,各组小鼠的血糖均有所降低,但运动后不同浓度辣椒素处理组的血糖水平均较正常运动对照组的血糖水平显著增高,差异有统计学意义（P＜0.05或P＜0.01）;运动后不同浓度辣椒素处理组肝糖原和肌糖原含量均较正常运动对照组显著升高,差异有统计学意义（P＜0.05或P＜0.01）。②运动后,分别检测各组血浆FFA的含量,结果表明红景天能显著降低运动后血FFA;而不同浓度的辣椒素处理组的血浆FFA的含量与空白运动组相比均显著增高,且与空白运动组相比差异有统计学意义（P＜0.01）。③各组小鼠运动前后的血浆TP、AL和Glob水平差异均无统计学意义。（3）中枢系统:①睡眠剥夺72h后,与空白睡眠组相比,空白睡眠剥夺组的OFT评分显著降低;咖啡因阳性对照组及不同浓度的辣椒素处理组与空白睡眠剥夺组相比,OFT得分显著升高,差异有统计学意义（P＜0.05或P＜0.01）,且以辣椒素高剂量组（15mg/kg）作用最明显,差异有统计学意义（74±9 vs 49±7,P＜0.01）。②空白睡眠剥夺组与空白睡眠组相比下丘脑和海马的5-HT、5-HIAA水平显著升高,差异有统计学意义（14.38±1.37vs 12.76±1.25,P＜0.01）:咖啡因阳性对照组及不同浓度的辣椒素处理组与空白睡眠剥夺组相比下丘脑和海马的5-HT、5-HIAA水平显著降低,差异有统计学意义（P＜0.05或P＜0.01）,且以辣椒素高剂量组（15mg/kg）作用最明显（11.19±1.26vs 14.38±1.37,P＜0.01）,与咖啡因阳性对照组相比差异无统计学意义（P＞0.05）。③与空白正常睡眠组相比,空白睡眠剥夺组下丘脑组织的肾上腺素、去甲肾上腺素及多巴胺的含量均显著降低,差异有统计学意义（P＜0.01）;与空白睡眠剥夺组相比,咖啡因阳性对照组,不同浓度的辣椒素处理组下丘脑的肾上腺素、去甲肾上腺素及多巴胺的含量均显著升高,差异有统计学意义（P＜0.05或P＜0.01）,且以高剂量（15mg/kg）作用最明显,与咖啡因阳性对照组相比差异无统计学意义（P＞0.05）。④睡眠剥夺前,各组大鼠的血NH₃水平差异无统计学意义。空白睡眠剥夺组与空白睡眠组相比血NH₃水平显著升高,差异有统计学意义（318.6±34.8 vs 166.7±20.8,P＜0.01）;咖啡因阳性对照组及不同浓度的辣椒素处理组与空白睡眠剥夺组相比血NH₃水平显著降低,差异有统计学意义（P＜0.05或P＜0.01）,且辣椒素的作用为高剂量组（15mg/kg）作用最明显,（158.5±27.9vs 318.6±34.8,P＜0.01）,与咖啡因阳性对照组相比差异无统计学意义（P＞0.05）。结论:辣椒素具有显著的抗疲劳作用,其机制可能为①增强小鼠抗氧化酶的活性,清除氧自由基,减少长时间运动引起的心肌的损伤;②增强脂肪的代谢,从而减少血糖的消耗,维持运动后的血糖水平;③减少中枢的5-羟色胺和血NH₃的生成,并可升高肾上腺素、去甲肾上腺素及多巴胺的含量,从而使多巴胺/5-羟色胺的比值维持在较高水平,调节中枢活动。第四章辣椒素对缺氧/复氧诱导的海马神经元凋亡的影响目的:辣椒素对H/R诱导神经元凋亡的保护作用并进一步探讨了该作用可能涉及的细胞分子机制。方法:选用妊娠18d的SD大鼠的胎鼠,分离和培养海马神经元组织,微管结合蛋白2（MAP2）抗体免疫荧光鉴定;取培养8-10 d生长良好的海马神经元用于实验。3h缺氧（1%O₂）和随后24h复氧诱导海马神经元细胞损伤。原位TUNEL法检测细胞凋亡程度;比色法试剂盒检测caspase-3活性;Western blot检测Akt蛋白磷酸化;荧光法检测细胞内活性氧（ROS）水平。结果:①海马神经元的培养及鉴定:取培养8 d的细胞,进行MAP2抗体免疫荧光鉴定,结果显示神经元纯度为94.5±2.1%。②TUNEL染色结果显示,H/R能诱导海马神经元凋亡,与对照组比较差异有统计学意义（P＜0.05或P＜0.01）。同时,H/R也能显著增加神经元caspase-3的活性。而处理辣椒素（3-30μM）和caspase-3特异性抑制剂DEVD-CHO（100M）能显著抑制H/R诱导的海马神经元凋亡和caspase-3活性的增加。③H/R能显著增加细胞内ROS生成,而预先给予细胞内抗氧化剂PDTC（50M）或辣椒素（3-30M）能显著抑制H/R诱导的ROS生成增加。预先给予PDTC也能显著抑制H/R诱导的caspase-3活性增加。④辣椒素能显著诱导Akt磷酸化;PI3K抑制剂LY294002（10M）能完全取消辣椒素诱导的Akt磷酸化。而且,LY294002也能显著抑制辣椒素降低细胞内ROS生成和抗凋亡作用。⑤VR1受体阻断剂辣椒辣素（1M）能完全取消辣椒素诱导的Akt磷酸化作用,同时也能阻断辣椒素抗凋亡作用。预处理[Ca²⁺]_i的鳌合剂BAPTA/AM（5M）也能取消辣椒素诱导的Akt磷酸化和抗凋亡作用。结论:辣椒素能抑制H/R诱导的海马神经元凋亡,其机制与激活VR1—Ca²⁺通路,经PI3K/Akt信号转导途径抑制氧化应激和caspase-3激活有关。辣椒素的神经元保护作用可能是其抗脑I/R损伤的细胞分子机制之一。更多还原

【Abstract】 PartⅠBackgroundFatigue is a normal physiological phenomenon and it occurs when mental or physical strength reseaches a certain level.Fatigue is a complicated physiological change.With the speeding up of the social rhythm and increasing of social competition,people always stay in tension when working and studying and their physical strength and energy are always in the long-term overdrawn.According to the investigation result of WHO,over 35%of people are in fatigue state,the rate of fatigue among middle aged men reached to 60.70%,nearly 70% of the middle-aged are in fatigue state in China.Fatigue can lead to the decline of people’s performance.If the fatigue can’t recover in time, "Overtraining syndrome,OTS" and "Chronic fatigue syndrome,CFS" would occur and lead to endocrine disturbance,immunity decrease and threaten people’s health.At present,various measures to eliminate fatigue include rest,sleep, massage and other physical methods;People also depend on nutritive fatigue supplements and traditional medical therapy aiming at relieving. However,these effects also have something unsatisfactory.Consequently, active substances extracted from the natural products have become the focus of the research for a safer,more effective methords of relieving fatigue since they have less side effects and not contain harmful components of doping for people.Capsaicin,the major pungent ingredient in red pepper,exerts extensive biological and pharmacological activities via activating vanilloid receptor-1（VR1）,which may be fully released in the cerebral and periphery nervous system.Recently,it is reported that capsaicin could improve the capacity of exercise,which means that capsaicin may have the effect of antifatigue.However,so far,the study of the effect of capsaicin on antifatigue and its dose-effect relation is still not so profound and systematic now and there are no reports about the related signal molecular and the signal translation of capsaicin in the antifatigue effect study.PartⅡ.The Antifatigue effects of Capsaicin on miceAIM:To explore the antifatigue effects of capsaicin on mice.METHODS:168 normal male KunMing mice were randomly divided into 7 groups based on body weight:low,medium,medium-high, high dose capsaicin intervention groups,control group and positive control group.The control group was given distilled water,the four intervention groups were given different doses of capsaicin（3、6、10、15mg/kg·bw） respectively and the positive control group were treated with Rhodiola Rosea（1g/kg·bw/d）.After 4 weeks,the weight of each mouse was measured.In addition,the pole-climbing time,the loading swimming time,serum urea nitrogrn,the hepatic glycogen,the muscle glycogen,the content of blood lactic acid and the activity of lactate dehydrogenase are all taken as the index of the antifatigue effects;and the toxicosis experiment of the second sodium,the survival time of mice under anoxia are taken as the index of anti-anoxic.RESULTS:（1） There is no significant difference between the capsaicin treated group and the control group.（2） Capsaicin significantly increased the time of climbing pole and loading swimming time of the mice,the maximum effect of capsaicin was observed at 15mg/kg·bw.（3） There was no significant difference of the survival time of mice under normal pressure anoxia and acute ischemic hypoxia between the low dose capsaicin treated group（3mg/kg） and the control group （P＞0.05）;different concentration of capsaicin（6、10、15mg/kg）can significantly extend the survival time of mice under the normal pressure anoxia and acute ischemic hypoxia compared with the control group（P＜0.05）;the maximal effect was observed at 15mg/kg·bw,the survival time under normal pressure anoxia and acute ischemic hypoxia were extended 64%and 107%respectively,there was no significant difference between the capsaicin 15mg/kg·bw treated group and the Rhodiola Rosea（1g/kg·bw/d） treated group.（4） Compared with the control group,the capsaicin has significantly improved the content of hepatic glycogen and muscle glycogen and the activity of the lactate dehydrogenase after exercise,meanwhile,the level of serum urea nitrogrn and the content of blood lactic acid were significantly decreased,the maximal effect were both observed at 15mg/kg.（5） Compared with the control group,capsaicin of different concentration can significantly lengthen the survival time under the toxicosis experiment of the sodium nitrite（P＜0.05）.CONCLUSION:Capsaicin has obvious antifatigue effects.It has significantly lengthened the time of pole climbing and loading swimming of the mice,improved the capacity of the anti-hypoxia,and inhibited the generation of blood lactic acid and serum urea nitrogen after exercise, maintained the content of the hepatic and muscle glycogen.PartⅢ.The antifatigue mechanism of CapsaicinAIM:To investigate the antifatigue mechanism of Capsaicin on mice from several aspects including antioxidant activity,energy metabolism and central system using the appropriate concentration based depending on the results from PartⅠ.METHODS:（1） antioxygen system:①measuring the activity of hepatic superoxide dismutase,glutathion peroxidase and the content of hepatic malonaldehyde before and after exercise;②measuring the plasma content of creatine kinase and MB isoenzyme of creatine kinase of the mice before and after exercise as the indexes of the damage of muscle cells.（2）energy metabolism system:①detecting the blood glucose、muscle and hepatic glycogen of mice before and after exercise; ②detecting the content of plasma free fatty acid before and after exercise;③measuring content of total protein,albumin and globulin plasma.（3） central system:①After sleep deprivation for 48h and 72h,all the rats were put to an openfield test and then the calculation of the score of openfield test follows;②Killed the rats and took the organs of hypothalamus,brain stem immediately,then measured the 5-hydroxytryptamine, NE,Dopamine,Ad,the content of 5-HIAA of the hypothalamus and brain stem,and detecting the content of plasma NH₃.RESULTS:（1） antioxygen system:①There is no significant differentce in the activity of hepatic SOD,GSH-Px and the level of MDA between the control and the capsaicin treated group before exercise(P＞0.05;after exercise,the activity of hepatic SOD,GSH-Px of each group all decreased,however,capsaicin significantly increased the activityies of hepatic SOD,GSH-Px compared with the control group after exercise and reduced the content of MDA（P＜0.05 or P＜0.01 ）,the maximal effect was observed at 15mg/kg·bw.②Before exercise,there is no significant difference of the levels of CK and CK-MB in plasma between the control and the capsaicin treated group.After exercise,capsaicin significantly reduced the levels of CK and CK-MB in plasma compared with the control group（P＜0.05 or P＜0.01 ）,the maximal effect was observed at 15mg/kg·bw.（2） energy metabolism system:①There is no significant differentce in the blood glucose between the control and the capsaicin treated group before exercise（P＞0.05）;After exercise,the blood glucose all decreased,however,the capsaicin significantly increased the blood glucose（P＜0.05 or P＜0.01 ）,the maximal effect was observed at 15mg/kg·bw.Capsaicin significantly increased the content of hepatic and muscle glycogen after exercise.②Capsaicin increased the serum free fatty acid content after exercise,the maximal effect was observed at 15mg/kg·bw the high-dose Capsaicin treated group and the control （P＜0.01）③There is no significant difference among each group before and after exercise. （3） central system:①The open field test scores of the mice after sleep deprivation for 72h was significantly reduced compared with normal sleep group;caffeine and capsaicin significantly improved the open field test scores after sleep deprivation for 72h compared with the rats with normal sleep（P＜0.05 or P＜0.01 ）;the the maximal effect was observed at 15mg/kg·bw（74±9 vs 49±7,P＜0.01 ）.②The content of hypothalamus and brain stem 5-HTand 5-HIAA after sleep deprivation for 72h was significantly increased compared with normal sleep group （14.38±1.37vs 12.76±1.25,P＜0.01）;caffeine and capsaicin significantly reduced the content of hypothalamus and brain stem 5-HTand 5-HIAA after sleep deprivation for 72h compared with the normal sleep group（P＜0.05 or P＜0.01 ）;and the the maximal effect was observed at 15mg/kg·bw（11.19±1.26vs 14.38±1.37,P＜0.01 ） and there is no significant difference compared with caffeine treated group（P＞0.05）.③The content of Ad,NE and DA in the brain stem was significantly reduced after sleep deprivation for 72h compared with normal sleep group（P＜0.01）;Caffeine and Capsaicin significantly decreased the the content of Ad,NE and DA in the brain stem（P＜0.05 or P＜0.01 ）;and the the maximal effect was observed at 15mg/kg·bw and there is no significant difference compared with caffeine treated group（P＞0.05）.④There is no significant difference in the content of blood NH₃ among each group before sleep deprivation.The content of blood NH₃ after sleep deprivation for 72h was significantly increased compared with normal sleep group;Caffeine and Capsaicin significantly decreased the content of blood NH₃ after sleep deprivation for 72h compared with the rats with normal sleep（P＜0.05 or P＜0.01 ）;and the the maximal effect was observed at 15mg/kg·bw（158.5±27.9vs 318.6±34.8,P＜0.01 ） and there is no significant difference compared with caffeine treated group（P＞0.05）.CONCLUSIONS:Capsaicin has significant antifatigue effect and the mechanism may be related to①increase the antioxidase activity, eliminate free radicals,and reduce the damage of cardic muscle;②promote the metabolism of fatty acids,reduce glycogen consumption and maintain the level of blood glucose after exercise;③decrease the generation of 5-HT and blood NH₃,and simultaneously increase the content of NA,Ad and DA,thus maintain the ratio of DA/5-HT at a high level to adjust the central activities.PartⅣ.Effect of capsaicin on I/R-induced apoptotic death of primary rat hippocampal neurons and the mechanismsAIM:To observe the effect of capsaicin on I/R-induced apoptotic death of primary rat hippocampal neurons and to explore its underlying mechanisms.METHODS:Primary cultures of rat hippocampal neurons were prepared from day 18 Sprague-Dawley rat embryos,and all experiments were performed on cells cultured for 8-10 days.Neuronal damage was induced by three-hour hypoxia（1%O₂） and consequent 24-hour reoxygenation.Apoptosis was evaluated by in situ terminal uridine nick end labeling（TUNEL） analysis and caspase-3 activity.Akt activation was characterized by western bloting with antibodies to phosphorylated Akt.Intracellular oxidant productions were measured using H₂DCF,a fluorescent indicator.RESULTS:（1）At 8th day of in vitro culture,embryonic rat hippocampus grown were identified as 94.5±2.1%microtubule-associated protein 2-positive cells,suggesting these cells contained mainly neurons.（2）H/R significantly increased the apoptotic death of hippocampal neurons as shown by increases in both TUNEL-positive cell number and caspase-3 activity.Pretreatment with capsaicin（3-30μM） or caspase-3 specific inhibitor DEVD-CHO（100μM） could markedly attenuate H/R-induced apoptosis in hippocampal neurons.（3）Capsaicin markedly induced the phosphorylation of Akt and PI3K inhibitor LY294002（10μM） prevented the capsaicin-induced survival effect in hippocampal neurons.（4）Intracellular reactive oxygen species（ROS） levels were significantly increased after H/R,which was concentration-dependent inhibited by capsaicin.The effects of capsaicin on intracellular ROS levels and apoptosis were blocked by LY294002（10μM）.（5）VR1 antagonist capsazepine（1μM） or intracellular Ca²⁺ chelator BAPTA/AM（5μM） could eliminate these effects of capsaicin mentioned above.CONCLUSION:The present data suggest that capsaicin protects against H/R-induced apoptosis of hippocampal neurons via VR1-[Ca²⁺]_i-dependent and PI3K/Akt-mediated signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.更多还原