【作者】 黄攀攀；

【导师】 王平；

【作者基本信息】 湖北中医药大学 ， 中医基础理论， 2010， 博士

【摘要】 目的：本研究通过对《内经》睡眠理论进行总结归纳,认为睡眠是以神的活动为纲纪,营卫二气的运行出入为枢机,阴阳跷脉的气血流注为兆始,五脏生理功能(藏精化气生神)为根柢的重要生命活动。心藏神,指心有统帅全身脏腑、经络、形体、官窍的生理活动和主司精神、意识、思维、情志等心理活动的功能。《素问·灵兰秘典论》：“心者,君主之官,神明出焉”。《内经》认为睡眠由心神主导和控制,老年失眠病位在心,心神失养或心神不宁是其主要病机,养心安神是其主要治法。本实验在D-半乳糖制作亚急性衰老模型基础上,采用咖啡因腹腔注射叠加多平台水环境持续睡眠剥夺法制作老年失眠大鼠模型,通过观察老年失眠大鼠发病过程中呼吸频率、心率等指标变化、学习记忆能力、内分泌激素的昼夜节律、心肌超微结构,肽能神经元的表达(VIP、AVP)以及VIPmRNA、AVPmRNA表达等实验研究,探讨VIP、AVP在调节老年失眠睡眠-觉醒机制中的作用,以及天王补心丹调节睡眠-觉醒机制的可能作用途径。方法：将实验动物随机分成4组：空白对照组、模型组、天王补心丹组、艾司唑仑组。120mg.kg-1.d-1标准颈背部皮下注射D-半乳糖28d制作衰老模型,10mg.kg-1.d-1标准颈背部皮下注射D-半乳糖叠加腹腔注射30mg.kg-1.d-1咖啡因7d,运用自制造模装置进行多平台水环境法进行睡眠剥夺7d,建立老年失眠大鼠模型。具体检测方法如下：1、观察动物睡眠情况、活动量、精神状况、双目、口唇、爪甲、皮毛等一般情况及测量呼吸频率、心率、血压、力竭性游泳时间、血液血红蛋白总数、血红蛋白浓度、摄食量、体重、耳温等一般情况；2、运用Morris水迷宫检测大鼠学习记忆能力；3、放射免疫法测定大鼠血浆ACTH、Cort、TSH、GH等4种内分泌激素,采用群体余弦法分析昼夜节律变化；4、透射电镜检测大鼠心肌超微结构；5、免疫组织化学方法检测心脏和SCN中VIP、AVP表达；6、RT-PCR检测心脏和SCN中VIPmRNA、AVPmRNA表达。结果：1、模型组动物时有呵欠,打瞌睡,精神萎靡,活动量少,多数动物目光呆滞,双目、口唇呈苍白色,少数呈淡粉红色,皮毛重度枯槁蓬乱呈淡象牙色,湿透,爪甲粗糙,抓取时无力反抗；天王补心丹组动物白天时有呵欠,精神稍萎靡,双目、口唇呈白色,爪甲粗糙,皮毛略枯槁蓬乱,抓取时反抗较剧。模型组与空白对照组比较,呼吸频率显著加快(P<0.01),心率明显加快(P<0.01),收缩压显著升高(P<0.01),力竭性游泳时间显著缩短(P<0.01),外周血红细胞总数显著减少(P<0.01),外周血红蛋白浓度显著降低(P<0.01),摄食量显著减少(P<0.01),体重显著减轻(P<0.01),耳温显著升高(P<0.01)；天王补心丹组与模型组比较,呼吸频率显著降低(P<0.01),心率显著减慢(P<0.01),收缩压降低(P<0.05),力竭性游泳时间显著延长(P<0.01),外周血红细胞总数显著增加(P<0.01),外周血红蛋白浓度显著升高(P<0.01),摄食量显著增加(P<0.01),体重增加(P<0.05),耳温显著降低(P<0.01)；艾司唑仑组与模型组比较,呼吸频率显著降低(P<0.01),心率减慢但无统计学意义(P>0.05),收缩压降低(P<0.05),力竭性游泳时间显著延长(P<0.01),外周血红细胞总数和血红蛋白浓度增加,但均无统计学意义(P>0.05),摄食量增加(P<0.05),体重增加但无统计学意义(P>0.05),耳温降低(P<0.05)。天王补心丹总体效果优于艾司唑仑。2、模型组与空白对照组比较,潜伏期时间显著延长(P<0.01),平台象限的游泳距离占总距离百分比显著降低(P<0.01),池壁20%和40%区域游泳距离百分比显著升高(P<0.01),跨过原平台位置次数显著下降(P<0.01)；天王补心丹组、艾司唑仑组与模型组比较,潜伏期时间显著缩短(P<0.01),平台象限的游泳距离占总距离百分比显著升高(P<0.01),池壁20%和40%区域游泳距离百分比显著降低(P<0.01),跨过原平台位置次数显著增加(P<0.01)。天王补心丹总体效果优于艾司唑仑。3、模型组与空白对照组比较,ACTH、Cort活性存在昼夜节律波动(P<0.05,P<0.001),但ACTH峰值相位提前,振幅缩小,Cort峰值相位推后,振幅增加,TSH、GH活性昼夜节律均消失(P>0.05,P>0.05)；天王补心丹组与空白对照组比较,ACTH、Cort、GH活性亦存在明显地昼夜节律(P<0.001, P<0.05, P<0.001), ACTH峰值相位推后,振幅减小,Cort峰值相位提前,振幅减小,GH峰值相位提前,振幅减小,但TSH昼夜节律消失(P>0.05)；艾司唑仑组与空白对照组比较,ACTH、Cort活性昼夜节律消失(P>0.05), TSH、GH活性存在昼夜波动(P<0.05,P<0.001),TSH活性峰值相位提前,振幅增加,GH活性峰值相位提前,振幅减小。天王补心丹总体效果优于艾司唑仑。4、模型组与空白对照组比较,心肌肌丝排列紊乱、断裂,肌小节排列紊乱、断裂,少数细胞核可见浓缩变小,染色质轻度溶解及凝聚,核膜皱缩尚完整。线粒体嵴结构模糊。肌浆网囊性扩张,成大囊泡状；天王补心丹组少数细胞核有轻度损伤,染色质轻度溶解及凝聚,核膜尚完整。肌浆网囊性扩张,成大囊泡状。可见少数线粒体嵴结构模糊；艾司唑仑组大部分心肌细胞核结构正常,少数细胞核有轻度损伤,染色质溶解、凝聚。肌浆网囊性扩张。线粒体内、外膜及嵴结构模糊,不清晰,闰盘结构损害较重,可见局部囊性扩张,部分闰盘崩解。5、模型组与空白对照组比较,心脏中VIP和AVP阳性细胞计数均显著增加(P<0.01,P<0.01),天王补心丹组与模型组比较,VIP、AVP阳性细胞计数均减少(P<0.05,P<0.01)；艾司唑仑组与模型组比较,VIP阳性细胞计数减少但无统计学意义(P>0.05), AVP阳性细胞计数显著减少(P<0.05)；模型组与空白对照组比较,SCN中VIP和AVP阳性细胞计数均显著减少(P<0.01,P<0.01),天王补心丹组与模型组比较,VIP和AVP阳性细胞计数均显著增加(P<0.01,P<0.01),艾司唑仑组与模型组比较,VIP阳性细胞计数减少(P<0.05),而AVP阳性细胞计数增加(P<0.01)。天王补心丹效果优于艾司唑仑。6、模型组与空白对照组比较,心脏中VIPmRNA和AVPmRNA均显著增加(P<0.01,P<0.01),天王补心丹组与模型组比较,VIPmRNA、AVPmRNA表达均显著减少(P<0.01,P<0.01),艾司唑仑组与模型组比较,VIPmRNA表达显著减少(P<0.01),而AVPmRNA表达亦减少但无统计学意义(P>0.05)；模型组与空白对照组比较,SCN中VIPmRNA、AVPmRNA表达均显著减少(P<0.05、P<0.01),天王补心丹组与模型组比较,VIPmRNA、AVPmRNA表达均增加(P<0.01,P<0.01)；艾司唑仑组与模型组比较,VIPmRNA、AVPmRNA表达均增加(P<0.01,P<0.01)。天王补心丹效果优于艾司唑仑。结论：1、通过对《内经》睡眠理论进行总结归纳,认为睡眠是以神的活动为纲纪,营卫二气的运行出入为枢机,阴阳跷脉的气血流注为兆始,五脏生理功能(藏精化气生神)为根柢的重要生命活动。睡眠由心神主导和控制,老年失眠病位在心,心神失养或心神不宁是其主要病机,养心安神是其主要治法,天王补心丹是其代表方剂。2、本实验在D-半乳糖制作亚急性衰老模型基础上,采用咖啡因腹腔注射叠加多平台水环境持续睡眠剥夺法制作老年失眠大鼠模型,在发病过程、证候表现方面与阴虚血少证有很强的相关性,基本符合临床失眠特点,是一种新的证候模型制作方法。3、根据《内经》“心藏神”理论,本实验在D-半乳糖制作亚急性衰老模型基础上,采用咖啡因腹腔注射叠加多平台水环境持续睡眠剥夺法制作老年失眠大鼠模型,通过观察心率、呼吸频率、血压、力竭性游泳时间等与心脏功能相关证候指标、心肌超微结构、心脏中参与心衰发生发展的VIP、AVP及VIPmRNA、AVPmRNA表达等指标,因此,本模型三种造模因素在一定程度上导致实验动物的心血管功能失常,影响到中医理论中“心主血脉”功能,在一定程度上模拟了“心藏神”失司致失眠的病理机制,阐释了“心藏神”主睡眠的科学内涵。4、天王补心丹能使失眠大鼠模型呼吸频率显著降低,心率显著减慢,动脉血压下降,力竭性游泳时间显著增长,摄食量增加,体重增加,血红蛋白总数和浓度均提高,耳温降低；提高学习记忆能力；恢复ACTH、Cort.GH等3种内分泌激素昼夜节律；降低心肌超微结构损伤；上调SCN中相关睡眠物质VIP、AVP及VIPmRNA、AVPmRNA表达,从而达到调整昼夜节律的作用；下调心脏中相关体液因子VIP、AVP及VIPmRNA、AVPmRNA表达,达到降低心血管系统损伤作用,提示其治疗失眠的作用机制可能与其滋阴养血、养心安神的功能有关。更多还原

【Abstract】 Objective:The study have established a senile insomnia model through D-galactose, caffeine and sleep deprivation. According to the theory of "heart is in charge of the spirit" in NeUing, through the observation of behavioral changes in rats, learning and memory, the circadian rhythm of hormone, myocardial ultrastructure, expression of peptide neurons (VIP, AVP),the study have discussed the mechanism that VIP, AVP regulated sleep-wake in rats, and Tianwang Buxin Pills (TWBXP) regulated sleep-wake’s mechanisms.Methods:Male healthy SD rats were randomly allocated into four groups: normal control group,model group, TWBXP group, Estazolam group.Animals were administered with D-gal (120 mg/kg for 28 days), sleep deprivation (multi-platform water environment methods for 7days), caffeine(30 mg/kg for 7 days) to establish the senile insomnia model in rats.1. To observe the sleep, activity amount, mental state, the eyes,lips,onyx,the skin and hair.Breath frequency,heart rate,blood pressure, swimming time, the total number and the concentrstion of hemoglobin,body weight, food intake and temperature of ears was measured repectively.2. The learning and memory was measured by Morris water maze. 3. The concentration of ACTH, Cort, TSH, GH was detected through radio-immunity method, then the circadian rhythms changes were analysed by Cosinor Method.4. The myocardial ultrastructure was detected by transmission electron microscope.5.The expression of VIP,AVP both in heart and SCN was measured by immunohistochemical method and the image was analysed.6. The expression of VIPmRNA, AVPmRNA both in heart and SCN was measured by semi-quantitative analysis of a retrovirus-polymerase chain reaction (RT-PCR).Results:1. The model animals appeared insomnia symptoms, severe fatigue performance; Compared to normal control group, model group looked gray-faced and sleeping, lethargy, appetite decreased, weakness, breath frequency and heart rate quickened, blood pressure heightened,body weight and food intake decreased, temperature of ears raised, the total number and the concentration of hemoglobin decreased. TWBXP groups improved most of the symptoms, while Estazolam just improved some of the symptoms.2. In the positioning navigation experiments, compared to normal control group, model group rats’ latent period of four days and two days were significantly reduced (P<0.01). In the search space experiments, the platform quadrant swimming distance percentage of the total distance was significantly falled (P<0.01), the wall 20% and 40% from the percentage of the regional swimming results showed the rising trend(P<0.01),Compared to normal control group, model group, TWBXPgroup, Estazolam group’s number of crossing the location of the original platforms decreased significantly (P<0.01). 3. Compared to normal control group, model group’s circadian rhythm of ACTH, Cort appeared (P<0.05,P< 0.001) while circadian rhythm of TSH、GH had not been appeared (P> 0.05, P> 0.05); Compared to normal control group,TWBXP group’s circadian rhythm of ACTH、Cort、GH appeared (P <0.001, P<0.05, P<0.001) while circadian rhythm of TSH had not been appeared (P>0.05).4. Compared to normal control group, model group’s myocardial ultrastructure had showed that myofilament had arranged disorderly, some of nucleus had been concentrated and diminished, chromatin had been dissolved and condensed,the structure of ridge in chromatin had been vagued, sarcoplasmic reticulum had been swelled; TWBXP group’s myocardial ultrastructure had showed that myofilament had arranged disorderly, chromatin had been dissolved, the structure of ridge in chromatin had been vagued, sarcoplasmic reticulum had been swelled slightly.5. Compared to normal control group, the VTP,AVP positive cells in model group’s heart had been increased significantly (P<0.01), and that in TWBXP group had been decreased significantly (P<0.01); Compared to normal control group, the VIP,AVP positive cells in model group’s SCN had been decreased significantly (P<0.01), and that in TWBXP group had been increased significantly (P<0.01).6. Compared to normal control group, the expression of VIPmRNA,AVPmRNA in model group’s heart had been increased significantly (P<0.01), and that in TWBXP group had been decreased significantly (P<0.01); Compared to normal control group, the expression of VIPmRNA,AVPmRNA in model group’s SCN had been decreased significantly (P<0.01), and that in TWBXP group had been increased significantly (P<0.01). Conclusion:1. The multiple model therefore provides a promising model in which we can study treatments for insomnia,which has significant correlation with Yin-weakness and Blood-deficiency syndrome.2.D-gal,caffeine and SD had lead to some important changes of the cardiovascular function in animals,such as breath frequency,heart rate,blood pressure, swimming time et al,which also had resulted in the changes of myocardial ultrastructure and the expression of VIP、AVP、VTPmRNA. AVPmRNA. The multiple model had damaged "heart governed blood and vessels" function in traditional Chinese medicine theory,that had simulated the pathomechanism of "heart is in charge of spirit " disfunction in some degree, illuminated the scitific intension of "heart is in charge of spirit" which governs the sleep.3. TianWang BuXin Pills can significantly reduce the respiratory rate, depress heart rate and arterial blood pressure, extend exhaustive swimming time, increase food intake and weight gain, increase the total number and concentration of hemoglobin, decrease ear temperature, improve learning and memory, restore the circadian rhythm of ACTH, Cort and GH hormone, reduce the damage of myocardial ultrastructure,increase the expression of VIP、AVP and VIPmRNA、AVPmRNA in SCN while decrease the expression of VIP、AVP and VIPmRNA、AVPmRNA in heart.It may be illustrated by the function of TWBXP’s ziyinyangxue,buxinanshen.更多还原