【作者】 雷龙鸣；

【导师】 孙国杰；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2011， 博士

【摘要】 目的观察艾灸“印堂”与“神阙”穴对亚健康脑力疲劳模型大鼠学习记忆能力的影响及其可能机制,为临床应用艾灸印堂与神阙调治亚健康脑力疲劳提供现代医学理论依据。本课题的研究目的具体包括以下4个方面。(1)建立并评价部分睡眠剥夺所致亚健康脑力疲劳大鼠动物模型,观察艾灸“印堂”与“神阙”穴对模型大鼠学习记忆能力的影响。(2)探讨部分睡眠剥夺对模型大鼠下丘脑-垂体-肾上腺皮质轴(hypothalamus-pituitary-adrenocortex axis, HPA)相关激素水平的影响,观察艾灸“印堂”与“神阙”穴对HPA轴激素水平的调节作用。(3)观测部分睡眠剥夺所致亚健康脑力疲劳模型大鼠海马神经元形态学变化,探讨艾灸“印堂”与“神阙”穴对海马神经元形态学的影响。(4)检测部分睡眠剥夺所致亚健康脑力疲劳模型大鼠海马细胞凋亡相关蛋白表达变化,探讨艾灸“印堂”与“神阙”穴对模型大鼠海马细胞凋亡相关蛋白表达的影响。方法采用随机对照的动物实验研究方法进行本课题研究。实验分为以下4个部分。(1)将健康成年SD大鼠30只(雌雄各半)按体重大小及雌雄随机分成3组：正常组、模型组和艾灸组,每组10只。模型组和艾灸组采用睡眠剥夺水箱进行每天不足5-6小时睡眠的睡眠剥夺,正常组在同一时间段内放入模拟水环境水箱内,连续14天。造模期间,艾灸组另给予艾灸“印堂”与“神阙”穴,每穴每次灸15分钟,每天1次。观察3组大鼠的一般状况,并每周称重一次。在实验进行第2周,采用八臂迷宫对3组大鼠进行学习训练,并检测其学习记忆能力。(2)实验结束当天,处死大鼠并采血。采用双抗体两步夹心酶联免疫吸附法(ELISA法)检测各组大鼠血清促肾上腺皮质激素(adrenocorticotropic hormone,简写为ACTH)和内源性糖皮质激素(glucocorticoid,简写为GC)浓度。(3)实验结束当天处死大鼠后取材并制作标本。组织病理切片观察3组大鼠海马CA3区神经元形态学,原位末端标记法(TUNEL法)观测海马细胞凋亡情况。(4)采用免疫组织化学法检测3组大鼠海马CA3区Bcl-2、Bax与Caspase-3等凋亡相关基因蛋白的表达情况。结果上述实验完成后,得到如下观测结果。(1)一般状况：实验前,3组大鼠一般状况好。实验开始造模初期,模型组与艾灸组大鼠外观无明显改变,但其行为的兴奋性与警觉性提高,对周围刺激反应敏捷。随着造模时间的延长,模型组与艾灸组大鼠披毛毛色逐渐发黄而少光泽,且开始出现掉毛,表现出易“激惹”现象,对抓取、抚摸等刺激表现出逃避行为。造模1周后,模型组大鼠明显消瘦,披毛蓬乱,毛色枯黄无光泽且容易掉落,出现疲劳与虚弱行为。正常对照组从实验开始到结束,外观表现大致如实验前。艾灸组自造模1周起外观与行为表现介于正常组和模型组之间。(2)体重变化：正常组实验第7天与实验前相比体重增加,但差异无显著性(P>0.05)；模型组、艾灸组实验第7天与实验前相比,体重均减轻,差异有显著性(P<0.05)；实验第7天,模型组与艾灸组大鼠体重比较,差异无显著性(P>0.05)。实验第7天及第14天,模型组、艾灸组体重与正常组相比为轻,差异有显著性(P<0.05)。(3)八臂迷宫训练成绩：3组大鼠觅食时间比较,模型组最长,艾灸组次之,正常组最短,3组之间两两比较,差异均有显著性(P<0.01)。自训练测试第3天起,正常组及艾灸组错误臂数与模型组比较均为少(P<0.05),艾灸组与正常组错误臂数比较,差异无显著性(P>0.05)。自训练测试第5天起,正常组及艾灸组正确臂数与模型组比较均为多(P<0.05)。(4)血清ACTH与GC浓度：模型组及艾灸组血清ACTH浓度明显高于正常组(P<0.05),艾灸组血清ACTH浓度明显低于模型组(P<0.05)。模型组血清GC浓度明显高于正常组(P<0.05)；艾灸组GC浓度明显低于模型组(P<0.05),与正常组比较,差异无显著性(P>0.05)。(5)海马切片神经元形态学观察结果海马切片HE染色后光镜可见：正常组海马CA3区有大量排列规则整齐的锥体细胞,模型组锥体细胞形态不规则,细胞数量减少,艾灸组锥体细胞排列比较规则、整齐。海马切片细胞凋亡观察可见：正常组海马组织可见少量TUNEL阳性细胞,模型组及艾灸组阳性细胞散布于整个海马组织；艾灸组阳性细胞数明显少于模型组(P<0.05),艾灸组阳性细胞数与正常组比较,差异无显著性(P>0.05)。(6)海马切片免疫组化染色后光镜观察结果Bcl-2蛋白检测结果：与正常组比较,模型组、艾灸组Bcl-2蛋白阳性细胞数目均为多(P<0.05)；艾灸组Bcl-2蛋白阳性细胞数目较模型组多,差异有显著性(P<0.05)。Bax蛋白检测结果：与正常组比较,模型组、艾灸组Bax蛋白阳性细胞数目均为多(P<0.05)；艾灸组Bax蛋白阳性细胞数目较模型组少,差异有显著性(P<0.05)。Caspase-3蛋白检测结果：与正常组比较,模型组与艾灸组Caspase-3蛋白阳性细胞数目均为多(P<0.05)；艾灸组Caspase-3蛋白阳性细胞数目较模型组比较少,差异有显著性(P<0.05)。结论通过本课题研究,可以得出以下结论。(1)采用每天不足5-6小时睡眠、连续14天的部分剥夺睡眠方法建立的亚健康脑力疲劳大鼠模型,其行为学与人类亚健康脑力疲劳临床表现相类似,是研究亚健康脑力疲劳较为理想的动物模型。(2)艾灸“印堂”与“神阙”穴具有改善亚健康脑力疲劳模型大鼠学习记忆能力等亚健康样症状的作用。(3)HPA轴功能亢进,高GC水平下进而引起的海马损伤,是亚健康脑力疲劳发生的机制之一。艾灸“印堂”与“神阙”穴具有调节亚健康脑力疲劳模型大鼠HPA轴的功能,这是艾灸能够改善亚健康脑力疲劳模型大鼠亚健康样症状的机制之一。(4)艾灸“印堂”与“神阙”穴可以阻止脑力疲劳模型大鼠海马锥体细胞带神经元丢失,减轻海马神经元损伤,维持海马神经元的正常结构；艾灸还可以抑制海马神经细胞的凋亡,保护海马神经元。(5)睡眠剥夺导致模型大鼠海马神经元Bax与Casepase-3基因蛋白表达增加,促使海马神经细胞凋亡,是亚健康脑力疲劳的又一发生机制。艾灸“印堂”与“神阙”穴可通过下调海马促凋亡蛋白Bax及凋亡相关酶Casepase-3的表达,上调抗凋亡蛋白Bcl-2的表达,从而抑制大鼠海马神经细胞凋亡,保护海马神经元,此为艾灸改善脑力疲劳模型大鼠学习记忆能力的另一主要机制。更多还原

【Abstract】 Objective:The experiment aims to investigate the effect and the possible mechanisms of moxibustion on Yintang(GV29) and Shenque(RN08) for learning and memorizing capacity in sub-health mental fatigue model rats, providing the modern medical theoretical basis for the clinical application of moxibustion on Yintang(GV29) and Shenque(RN08) for the treatment of sub-health mental fatigue. The detailed purpose of this project includes the following four aspects:(1)To establish and appraise the sub-health mental fatigue model in rats induced by partial sleep deprivation, and to observe the effect of the moxibustion on Yintang(GV29) and Shenque(RN08) on learning and memorizing capacity in rats.(2)To investigate the effects of partial sleep deprivation on hypothalamus-pituitary-adrenocortex axis (HPA) related hormone levels in rats, and to investigate the regulatory role of the moxibustion in HPA axis related hormone levels.(3) To observe morphological changes of hippocampal neurons in sub-health mental fatigue model rats caused by partial sleep deprivation, and to investigate the effects of the moxibustion on Yintang (GV29) and Shenque (RN08) on the morphology of hippocampal neurons.(4)To detect the expressionnal changes of apoptosis relatedproteins in hippocampal neurons in model rats caused by partial sleep deprivation, and to investigate the effect of the moxibustion on Yintang(GV29) and Shenque(RN08) on the expression of apoptosis related proteins in hippocampal neurons in model rats.Methods:To apply the researching method of randomized controlled animal trials on this subject. The experiment was divided into the following four parts:(1) 30 healthy adult Sprague-Dawley (SD) rats (in half male and female) were randomly divided into 3 groups:Normal group, model group and moxibustion group (10 rats per group). Rats in model group and moxibustion group were placed in sleep deprivation tank with no more than 5-6 hours of sleep a day for partial sleep deprivation, while the rats in normal group were put into a tank of water environment under simulated conditions. The whole process lasted for 14 days. During the period of modeling, another moxibustion of Yintang (GV29) and Shenque (RN08) were given to the rats in moxibustion group,15 minutes for each point at a time per day. The general condition of the rats in 3 groups was observed everyday and the body weights were recorded weekly throughout the experiment. In the second week of the experiment, eight-arm maze was used for the the learning training in all the rats. Learning and memorizing capacity were then tested.(2) On the day when the experiment finished,all rats were put to death. At the time of sacrifice, blood samples of each group were collected. Two steps by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of serum adrenocorticotropic hormone (ACTH) and endogenous glucocorticoid(GC) in each rat.(3) At the time of sacrifice, brain samples of each group were collected. Histological and pathological changes of hippocampal neurons in CA3 area were observed for the 3 groups of rats. TdT-mediated dUTP nick end labeling (TUNEL) assay was used to evaluate the apoptosis of hippocampal neurons.(4) The immunohistochemistry staining was used to test the expression of Bcl-2, Bax and Caspase-3 in hippocampal neurons of CA3 area.Results:After the above experiments, the following results were obtained.(1) General condition:During the initial period of.the modeling experiment, the appearance of the model group and the moxibustion group had no obvious change, but the excitement and alertness of their behaviour improved. And they responded quickly to the stimulus of the surroundings. With the extension of the modeling time, the outer coat color of the model and moxibustion group gradually turned yellow and few gloss. The rats started to get hair loss, displayed the "irritability" phenomenon and escaped to the stimulation such as capture and touch. One week after modeling, model group became emaciated obviously with unkempt coat, which was of dull brown and easy to drop. The fatigue and weakness behaviour appeared. From the beginning to the end of the experiment, the appearance of the normal group performed approximately like it did before the experiment. The appearance and behaviour of the moxibustion group was situated between the normal group and the model group since one week after modeling.(2) The change of body weight:There was no significant difference between normal group weighed on the day 7 and before the experiment, (P>0.05); Significant reductions in body weights in model group and moxibustion group on day 7 than initial weight were observed (P<0.05); There was no significant difference in body weight changes between the model group and the moxibustion group on the 7th day of the experiment(P>0.05). The model and moxibustion groups had distinctly lower body weights than normal group on the 7th and 14th day of the experiment (P<0.05)(3) Eight-arm maze training results:There were significantly differences in foraging time among each group (P<0.05), while the model group was the longest one, followed by the moxibustion group, and the normal group was the shortest. Pairwise comparison among the 3 groups had significant difference (P<0.05). Since the 3rd day of the training test, the error number of the arms for the normal and moxibustion group was fewer compared with the model group. The comparison between the moxibustion group and the model group had no significant difference, (P>0.05). Since the 5th day of the training test, the correct number of the arms for normal and moxibustion group was more than the model group (P<0.05)(4) The concentration of ACTH and GC in serum:the ACTH concentration in serum of model and moxibustion group was significantly higher than the normal group (P<0.05). While the moxibustion group was significantly lower than model group (P<0.05). The GC concentration in serum of model group was significantly higher than the normal group (P<0.05),While the moxibustion group was significantly lower than the model group (P<0.05). The comparison between the moxibustion and normal group hasdno significant difference (P>0.05)(5) Morphological observations on hippocampal neurons sectionThere were large amount of well-defined pyramidal cells in the CA3 area of the hippocampus in normal group under microscope. The irregular pyramidal cells with decreased numbers of model group could be observed as well. And the pyramidal cells of the moxibustion group were well-defined.Apoptosis of hippocampus:Few TUNEL-positive cells in the hippocampus tissue of the normal group could be observed under microscope. And the positive cells of the model group spread widely throughout the entire hippocampal tissue. The positive cells of the moxibustion group were significantly fewer than the model group (P<0.05). The comparison between the moxibustion and normal group had no significant difference (P>0.05)(6) Results of the immunohistochemical staining in hippocampal sectionThe results of Bcl-2 expression test:Bcl-2 positive cells in model and moxibustion group were increased significantly compared to those in normal group (P<0.05); Bcl-2 positive cells in moxibustion group were also significantly increased compared to those in model group (P<0.05).The results of Bax expression test:Bax positive cells in model and moxibustion group were increased compared to those in normal group (P<0.05); Bax positive cells in moxibustion group were significantly decreased compared to those in model group (P<0.05).The results of Caspase-3 expression test:Caspase-3 positive cells in model and moxibustion group were increased compared to those in normal group (P<0.05); Caspase-3 positive cells in moxibustion group were decreased compared to those in model group, the difference was significant (P<0.05).Conclusion:(1) Sub-health mental fatigue rat model, induced by partial sleep deprivation methods with less than 5-6 hours of sleep a day for 14 days, was similar to the clinical manifestations of human sub-health mental fatigue. It is identified to be an ideal animal model for studying sub-health mental fatigue.(2) The moxibustion on Yintang(GV29) and Shenque (RN08) acupoint has the function of improving subhealth-like symptoms (such as decreased learning and memorizing capacity) in sub-health mental fatigue rat.(3) The hippocampus damage caused by HPA axis hyperactivity and high GC level is one of the mechanisms for the onset of sub-health mental fatigue. The moxibustion on Yintang(GV29) and Shenque(RN08) acupoint has the function of regulating HPA axis in sub-health mental fatigue rat. This is also one of the mechanisms for improving the subhealth-like symptoms of the mental fatigue rat by moxibustion.(4) The moxibustion on Yintang(GV29) and Shenque(RN08) acupoint can prevent the neurons loss in hippocampus pyramidal cell layer in the mental fatigue rat and reduce the damage of hippocampal neurons, which is beneficial to maintain the normal structure of hippocampal neurons; Moxibustion can also inhibit the apoptosis in hippocampal neurons and finally protect hippocampal neurons.(5) The upregulation of Bax and Casepase-3 proteins expression in hippocampal neurons of the model rat induced by sleep deprivation promote the incidence of apoptosis in the hippocampus. It is another possible mechanism of the sub-health mental fatigue. The moxibustion on Yintang(GV29) and Shenque (RN08) acupoint can reduce the expression of pro-apoptotic proteins Bax and apoptosis-related enzymes Casepase-3, increase the expression of Bcl-2 to inhibit the hippocampus neuronal apoptosis of the rats and finally protect hippocampus neurons. It is another main mechanism for improvement of learning and memorizing capacity in mental fatigue rat by moxibustion.更多还原