基于线粒体途径探讨安寐汤抑制失眠大鼠皮质神经元兴奋机制
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摘要
目的
本研究主要基于中医藏象学说心脑相关理论,从最高级中枢大脑皮层水平首先探讨失眠的阳盛阴衰、阴阳失交与大脑皮层兴奋抑制失衡之间的关系,主要观察大鼠经对氯苯丙氨酸(PCPA)腹腔注射进行失眠造模的不同时间点线粒体的变化和失眠造模对不同月龄大鼠皮层线粒体的影响；其次在上一阶段实验的基础上,从线粒体途径探讨安寐汤治疗失眠的机制,为进一步研究中医药改善失眠的作用机制和临床治疗提供理论参考。
方法
第一部分：首先采用3月龄SPF级SD大鼠,雌雄不分,3-21月龄,按文献对氯苯丙氨酸(PCPA)腹腔注射的方法进行失眠模型制作,并分别于造模后第1、2、3和4天观察额叶皮质神经元线粒体形态功能的改变；采用超活染色和电镜分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量。其次,基于上述最佳变化时间点采用3、6、9、12月龄大鼠进行造模3天后分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量。
第二部分：基于第一部分最佳时间点和最佳月龄,采用PCPA法对9月龄大鼠进行造模,随机分为正常对照组、模型组、安定治疗组、褪黑素治疗组和安寐汤低、中、高剂量治疗组；采用超活染色和电镜分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量,以及黄嗓吟氧化法、硫代巴比妥酸法检测皮层线粒体SOD和MDA的变化。
两部分方法具体如下：
1.模型制作
失眠模型制作采用对氯苯丙氨酸(PCPA)腹腔注射的方法。除正常组注射生理盐水外,其余组按0.3ml/100g腹腔注射PCPA(?)昆悬液(浓度为0.1g/m1)。
2.线粒体制备
实验结束后各组大鼠经10%水合氯醛(350mg/kg)腹腔注射麻醉,迅速断头取脑并立即置入冰冷的生理盐水中,洗净血污,剥离皮层并于2m1的分离介质中剪碎,迅速转移至玻璃匀浆管中(1min之内完成),电动匀浆,分离介质稀释,离心,所得沉淀用分离介质悬浮,制成lml混悬液。装在离心管中于碎冰中保存。全程冰上操作,提取完成后立即测定。线粒体蛋白含量以考马斯亮兰(Bradford)法进行测定。
3.线粒体超活染色观察
实验结束后用脊椎脱臼法处死大鼠,在枕骨大孔处迅速剪断延髓,去颅骨,完全暴露脑组织,取出脑组织,分离出额叶皮质后,在冰冷生理盐水中洗净后,在冰浴条件下充分匀浆,离心,取上清液待用。詹纳斯绿B (JGB)可特异性地使具有活性功能的线粒体呈现蓝绿色,从而判断线粒体功能的完整性。具体按照线粒体功能詹纳斯绿B (JGB)染色试剂盒说明书操作。
4.线粒体的超微结构观察
动物实验结束后,1%戊巴比妥钠(0.2ml／l0g)腹腔注射麻醉动物,灌注后取脑,留取大脑额叶皮质直径3mm的组织块,放入混合固定液中固定。修1mm×1mm×1mm大小组织块,2.5%戊二醛和1%四氧化锇酸双重固定,漂洗,丙酮逐级脱水,Epon812包埋,半薄切片光学定位,超薄切片厚度为50nm,醋酸铀及枸橼酸铅双重染色后于透射电镜下观察。每只大鼠观察1张铜网,每张铜网由左上角至右下角斜线上下移动,随机拍摄5张照片,底片放大1.5万倍。
5.线粒体Na+-K+-ATP, Ca2+-Mg2+-ATPase测定
各组大鼠实验结束后断头处死,迅速在冰盘上取大鼠脑部皮质,暂时保存待测。用滤纸吸干大脑皮质外部残血,精确称重,加入冰生理盐水,制备成组织匀浆。取组织匀浆用普通离心机离心,弃沉淀取上清,再低温离心,沉淀物为线粒体(线粒体的提取按试剂盒要求操作,中性红-詹纳斯绿B染色鉴定线粒体),线粒体中Na+-K+-ATP, Ca2+-Mg2+-ATP酶严格按照南京建成生物的试剂盒操作步骤进行测定。
6.线粒体膜电位测定
取出新鲜切除的大鼠额叶皮质样本0.5cm并压平,加入37℃预热的清理液2ml清洗皮质组织并小心抽去清理液,再用900u1染色工作液浸没整个组织,放进37℃培养箱孵育20min,抽去染色液,再用清理液一遍后开始切片约100um厚,然后置于载玻片上,盖上盖玻片,用手指轻轻压平,即刻在荧光显微镜下观察并拍照。
结果
第一部分：
1.动物失眠模型的建立
模型组大鼠在注射后的第一天昼夜节律正常,未见躁动不安；连续注射2d后,大鼠白天活动增多,对外界声、光刺激敏感;造模后第三天出现昼夜节律消失,白天夜晚活动不停；连续注射4d后,大鼠躁动不安非常明显。
2.线粒体超活染色观察
高倍光镜下可见3月龄各组线粒体形态有的呈短棒状,有的呈线状和近椭圆状,并且部分有聚集现象,其中造模各组的染色线粒体数目较正常对照组明显减少,尤其是以造模后第3天和第4天减少最为明显。而不同月龄大鼠造模3天后,和各正常对照组相比,以9月龄模型组的额叶皮质神经元线粒体的数目减少最为显著；而在各模型组中,又以12月龄大鼠模型组的线粒体最少。
3.线粒体超微结构观察
3月龄大鼠造模后第一天额叶皮层线粒体有轻微的肿胀,嵴有少许的断裂；造模后第二天线粒体肿胀开始明显,嵴断裂较多且出现边集化；造模后第三天线粒体进一步肿胀,嵴大部分断裂且开始出现空泡化；造模后第四天线粒体空泡化进一步加重。
3、6月龄正常大鼠的额叶皮质神经元线粒体的体积不大,而9月龄的则明显变大；12月龄的线粒体体积不仅变大,同时内部的嵴也现了少许的断裂,说明12月龄大鼠脑内的线粒体出现凋亡,提示线粒体的功能出现下降。
各正常对照组的线粒体体积不大,单位面积内嵴的数量也比较多,而相应的各模型组的线粒体体积较大,单位面积内嵴多有断裂,数量较少,有的甚至出现空泡化。其中,9月龄正常组大鼠线粒体略有肿胀,相应的模型组大鼠线粒体肿胀非常明显,嵴基本断裂,空泡化较为严重。而12月龄正常组大鼠线粒体不仅略有肿胀,而且嵴开始有部分断裂,相应的模型组大鼠线粒体肿胀也非常明显,嵴几乎全部断裂边集化且明显空泡化。
4.线粒体膜电位测定
模型组大鼠的膜电位与3月龄正常大鼠相比都下降,可见红色荧光逐渐减少,其中以造模后第4天下降最明显。3月龄正常大鼠造模开始后,绿色荧光也开始增加,并呈现由低到高再到低的趋势；说明造模后第4天大鼠额叶皮质神经元线粒体出现量的变化。9月龄正常大鼠的额叶皮质红色荧光最强,说明线粒体膜电位正常且线粒体功能最为旺盛；而12月龄正常大鼠随着月龄的增长,红色荧光减少且出现了棕黄色荧光,提示线粒体的膜电位出现了下降,线粒体功能开始减弱。与9月龄正常大鼠相比,各模型组大鼠额叶皮质红色荧光明显减弱,而绿色荧光逐渐增强。随着月龄增大,黄绿色荧光开始变成深绿色,说明部分线粒体的膜电位开始下降。
5.线粒体Na+-K+-ATP, Ca2+-Mg2+-ATPase测定
对于3月龄大鼠,经PCPA腹腔注射造模,随着天数的增加,造模的各模型组Na+-K+-ATP和Ca2+-Mg2+-ATP酶活性逐渐下降,与正常对照组相比均具有统计学意义(p<0.05)；其中造模后第三天和第四天下降尤为明显,且两组之间无统计学意义。同时,随着月龄的增加,正常大鼠大脑额叶皮质线粒体的Na+-K+-ATP和Ca2+-Mg2+-ATP酶有先升后降的趋势。6月龄正常大鼠的酶活性最强,12月龄正常大鼠的酶活性最低,而9月龄和3月龄正常大鼠的酶活性相比较则无统计学意义。各模型组与正常对照组相比都具有统计学意义；但9月龄和12月龄大鼠造模后酶活性却不具有统计学意义,说明造模选择9月龄的大鼠为佳。
第二部分：
1.线粒体超活染色观察
高倍光镜下可见造模后大鼠额叶皮质线粒体数量较正常大鼠明显减少,安定和褪黑素给药后线粒体的数量又显著增加；而低剂量安寐汤两个疗程灌胃后线粒体有所增加,中剂量安寐汤灌胃后较为明显,高剂量安寐汤灌胃后线粒体数目增加明显。
2.线粒体超微结构观察
9月龄正常大鼠造模后额叶皮层线粒体明显肿胀,嵴大部分断裂且出现边集化甚至空泡化,说明线粒体功能下降明显；经安定和褪黑素治疗后线粒体肿胀明显减轻,线粒体比较完整；而安寐汤低剂量治疗后线粒体肿胀有所减轻,但体积仍较正常组增大,线粒体嵴仍有部分断裂,线粒体不完整；中剂量和高剂量安寐汤灌胃治疗后线粒体较为完整,体积不大,嵴几乎无断裂。
3.线粒体膜电位测定
模型组大鼠的膜电位与9月龄正常大鼠相比明显下降,可见红色荧光明显减少,而绿色荧光明显增加,提示线粒体膜电位下降。安定和褪黑素治疗组也见红色荧光较模型组明显增加；而低剂量和中、高剂量安寐汤治疗组红色荧光则逐渐增多,说明线粒体膜电位逐渐好转。
4.线粒体Na+-K+-ATP,Ca2+-Mg2+-ATPase测定
各组与模型组相比大鼠额叶皮质神经元线粒体Na+-K+-ATPase都具有统计学意义(p<0.01),说明造模后各种药物治疗是有效果的；但与正常对照组相比只有模型组和安寐汤低、中剂量治疗组具有统计学意义(p<0.05),这提示尽管安寐汤低、中剂量有疗效但仍不足以恢复到正常水平,同时也说明安定、褪黑素和安寐汤高剂量的疗效和正常组大鼠接近而差不多；安定治疗组与安寐汤低剂量治疗组和模型组相比具有统计学意义(p<0.05),说明低剂量的安寐汤疗效不显著,同时尽管随着安寐汤剂量的增加Na+-K+-ATPase的含量也增加,但低中高三种剂量的疗效之间却无统计学意义。另一方面,无论是模型组还是各治疗组与正常对照组相比,大鼠额叶皮质神经元线粒体Ca2+-Mg2+-ATPase都有所下降,但都具有统计学意义(p<0.05)；同时各治疗组与模型组相比也都具有统计学意义(p<0.01)；安寐汤低、中剂量的疗效与安定和正常大鼠相比还是有统计学意义的(p<0.01),但低剂量与褪黑素、中与高剂量的疗效之间却无统计学意义,这说明尽管随着安寐汤剂量的增加Ca2+-Mg2+-ATPase的含量也增加,以及低与高剂量疗效之间有统计学意义(p<0.05),但中高三种剂量的疗效之间却无统计学意义。
综上所述
1、3月龄大鼠造模后线粒体数量明显减少,超微结构以第3天变化最为显著;额叶皮层线粒体膜电位和ATP酶活性第2天开始明显下降,第3天下降最明显(p<0.05),第4天略有回升。
2、9月龄大鼠造模后线粒体超微结构空泡化、膜电位和酶活性与12月龄模型组均显著,且无统计学意义。
3、安寐汤治疗后大鼠额叶皮质神经元线粒体数目明显增加,超微结构趋于好转,线粒体膜电位增加,同时随着安寐汤剂量的增加线粒体Na+-K+-ATPase、 Ca2+-Mg2+-ATPase的含量也增加,但不具有统计学意义。
结论
1、以3月龄大鼠进行PCPA制作失眠模型过程中额叶皮层线粒体超微结构和功能变化以连续注射3天最明显。
2、PCPA对失眠大鼠模型制作以9月龄为佳且可通过影响额叶皮层线粒体的形态和功能来导致失眠发生。
3、安寐汤可以增加失眠大鼠额叶皮质神经元线粒体的数目和改善线粒体的超微结构变性；安寐汤可改善额叶皮层神经元线粒体膜电位和增加Na+-K+-ATPase、 Ca2+-Mg2+-ATPase的含量,这可能是其改善失眠的作用机制之一；安寐汤可增加Na+-K+-ATPase, Ca2+-Mg2+-ATPase的含量,但剂量依赖性不明显。
4、失眠大鼠皮层线粒体的形态功能的减退与改善与皮层的兴奋抑制平衡失调和失眠的阳盛阴衰、阴阳失交可能存在密切的联系。
创新点
1、中医药治疗失眠的临床报道较多,基础研究也多是基于西医神经递质理论从低位脑干和丘脑等水平探讨,本研究首次从高位的最高级中枢大脑皮层水平来研究失眠的发病机理和安寐汤的改善作用。
2、本研究基于中医藏象学说的心脑相关理论,在以往中医药失眠研究没有形态学和分子水平上的证实的前提下,从线粒体角度来探讨失眠阳盛阴衰、阴阳失交与皮层兴奋与抑制失衡之间的联系以及安寐汤改善失眠的机理。
存在问题
1、限于大鼠饲养周期和经费问题,本研究没能在大鼠的年龄跨度上开展多个月龄的实验,也没能在造模后更多的时间点上开展探讨；
2、本研究未能从分子生物学的角度在基因、mRNA和蛋白表达等不同的水平展开进一步的检测,后期可做连续深入的探索,也可为失眠的大脑皮层角度的基础外围研究提供参考。
Objective
In this study, based on the correlation theory between heart and brain from theory of visceral manifestations, we explored the relationship between yinyang imbalance of insomnia and the imbalance of excitation and suppression of the cerebral cortex, and mainly observed the changes of the cortical mitochondria of rats at different time points which were made from insomnic models by PCPA peritoneal injection, and also observed the effect of PCPA on the structure and function of the cortical mitochondria of different month rat. Followed by an experimental basis in the previous phase, the next experiment will go on in order to study the effect mechanism of Anmeitang from mitochondrial pathway, and urther develop medicine to improve the symptom of insomnia and provide the some evidence for clinic treatment.
Methods
Part I:First,3-month-old SPF SD rats, male and female accounted for half, were dealed with chlorobenzene alanine (PCPA) by intraperitoneal injection for insomnic model according to the literature, and we observed the changes of the mitochondrial morphology and function of the frontal cortical neurons on1st,2nd,3rd and4th days after intraperitoneal injection by the super live staining and electron microscopy, which can measure the number and ultrastructure of mitochondria, meanwhile we also used fluorescence microscopy to detect mitochondrial membrane potential and WST-1to determine the ATP enzyme content of mitochondria. Secondly, based on the best time of change point, different aged rats,3,6,9, and12-month-old rats, will be produced for insomnic models by the intraperitoneal injection for3days, then we will observe the changes of the number and ultrastructure of mitochondria using the the super live staining and electron microscopy, while we will use the fluorescence microscopy to detect mitochondrial membrane potential and the WST-1to determine the ATP enzyme content of the mitochondria.
Part Ⅱ:Based on the best time point and the best month age of the first part experiment, the9-month-old rats produced by PCPA for insomnic models were randomly divided into normal control group, model group, antipsychotic treatment group, melatonin treatment group and Anmeitang low, medium and high dose treatment group; and we observed the changes of the number and ultrastructure of mitochondria by super live staining and electron microscopy, and we also measured the membrane potential and ATP enzyme content of mitochondria by the fluorescence microscopy and WST-1method.
As follows are the methods of two parts.
1, preparation of animal model
All rats were made for the insomnic model with p-chlorophenyl alanine (PCPA) by intraperitoneal injection. In addition to the normal saline group, the other groups received intraperitoneal injection of PCPA0.3ml/100g suspension (concentration0.1g/ml).
2, preparation of mitochondria
The rats were anaesthetized with10%chloral hydrate (350mg/kg) by intraperitoneal injection at the end of the experiment, and the brains of rats were removed quickly and immediately placed in ice-cold saline, then blood was washed. We peeled the cortex and placed them into the separation medium (2ml), and cut the cortex into pieces, then quickly transferred to a glass homogenizer tube (completed within lmin), electric homogenate diluted separation medium, centrifuged, and the resulting precipitate was suspended with a separation medium into lml suspension. The samples were placed into the centrifuge tube, which were stored in the trash ice. The target would immediately complete after the extraction. Mitochondrial protein content was measured with the method of Coomassie brilliant blue (Bradford).
3, mitochondrial super live staining observation
The rats were sacrificed by spinal dislocation in the foramen magnum at the end of the experiment, then we cut the medulla oblongata quickly, peeled the skull, and the brain tissue was completely exposed, and then picked up the brain tissue, isolated frontal cortex after washing the cortex with the cold saline bath conditions, fully homogenized, centrifuged, and the supernatant set aside. Janus Green B (JGB) could specifically stain the activated mitochondria into blue-green, which could help to determine the integrity of mitochondrial function. The specific steps of super-live staining we can refer to the kit instructions of Janus Green B (JGB).
4, the observation of mitochondrial ultrastructure
Animals were anesthetized with1%sodium pentobarbital (0.2ml/10g) by intraperitoneal injection at the end of the experiment, then perfused, and the brain would taken out;3mm diameter specimens from frontal cortex were made, then placed into the mixed stationary liquid. Next, the specimens would be trimed into1mm×1mm×1mm size tissue blocks, and placed into the mixed stationary liquid of2.5%glutaraldehyde and1%osmium tetroxide acid, rinsed, dehydrated in acetone, Epon812embedded; Next we made the semi-thin section,which would be positioned by optical microscope, and ultrathin sections(50nm) were made for observation after the stain of uranyl acetate and lead citrate by electron microscopy. A copper mesh for each rat was observed, and each copper mesh was moved from top left to bottom right diagonal moves up and down, and five photographs randomly photographed, whose negatives would magnified to15000times.
5, the measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase
The rats were decapitated rapidly and their brains were taken out in the ice tray at the end of the experiment, and the frontal cortexs were cut out for the test, then the residual blood outside the cerebral cortex, accurately weighed, was dried with filter paper, and the icy physiological saline was added to prepare a tissue homogenate, which would be centrifuged with the centrifugal machine. Next, the supernatant was reserved and the pellet was discarded, and then centrifuged, and the precipitate was mitochondria. All the procedure of kit was followed.
6, determination of mitochondrial membrane potential
the frontal cortex samples excised freshly0.5cm were flatten, and the cleaning solution2ml preheated to37℃was used to clean the cortical tissue and then carefully removed. The entire samples were placed into the900ul working fluid, then put into37℃incubator for20min, and then the staining fluid should be removed, and the slices were made about100um thick after the samples were cleaned once again, and then placed on a slide, cover glass, flatten gently with your fingers, and we should immediately observe the slices under a fluorescence microscope and photograph.
Results
Part I:
1, Animal model of insomnia
The circadian rhythm of the insomnic model was normal on the first day after the injection, and no restlessness occurred;but the activity of the insomnic model increased, and the models were very sensitive to the sound and light outside on the second day after the injection; the circadian rhythm disappeared on the third day after the injection, and all models walked non-stop day and night; on the fourth day after the injection, the restlessness of all models were very obvious.
2, observation of super live staining of mitochondria
The mitochondrial morphology of the three-month rats in each group appeared different shaps, some looked like short stick, and some were nearly linear and oval-shaped, and some appeared aggregated state in high-powered optical microscope. The number of mitochondria of each model group was significantly reduced compared with normal control group, especially on the third、fourth day after the injection. The number of mitochondria of the different model group reduced comparing with the normal control group, and the mitochondrial number of In-9m-3d group was the minimum, but the mitochondrial number of In-12m-3d group rised a little. The decrease of the number of mitochondria of the different month group gradually accompanied with the the rising age, and the number of12-month group was minimum.
3, observation of mitochondrial ultrastructure
The morphology of motochondria of the frontal cortex appeared slight swollen, and some cristae of mitochondrial inner membrane broke on the first day after the injection; the mitochondria swollen obviously, and many cristae broke and assembled at the edge of the inner membrane on the second day after the injection; the mitochondria swollen further, and most of the cristae broke and the whole mitochondria became a vacuole on the third day after the injection; the mitochondria nearly turned into vacuolation on the fourth day after the injection. The mitochondrial size of the frontal cortex of the normal rats,3,6month old, was small, but the size of9month old rats became significantly larger; not only the size of12month old rats was larger, but also a little cristae inside broke, which indicating that the mitochondrial apoptosis of the frontal cortex of12-month-old rat happened, and mitochondrial function started to decline. The mitochondrial size of normal control group was small, and the number of cristae per unit area was large, while the mitochondria of the corresponding model groups larger and the number of cristae per unit area was small, many cristae fractured, even some mitochondria became vacuoles. The mitochondria of nine-month-old normal rats slightly swollen, and the mitochondria of the corresponding model rats swollen seriously, and most of the cristae fractured, then serious vacuolization occured. The mitochondria of12-month-old normal rats not only swollen slightly, but some cristae broke; but the mitochondria of the corresponding model rats swollen very clearly, and almost all cristae broken and gathered to the edge of the membrane, so the clear vacuoles appeared.
4, observation of mitochondrial membrane potential
Compared with membrane potentials (MP) of3-month-old normal rats, MPs of the rats in model group all declined, and the red fluorescence intensity in model group declined gradually, and the largest decrease occurred on the fourth day after the injection. The green fluorescence intensity began to increase with the day going on after the injection, and presented the tendency of shifting from low to high, which suggested that the mitochondrial amount of the frontal cortex began to change on the fourth day after the injection. The red fluorescence intensity in frontal cortex of9-month-group normal group was the strongest, which suggested that the mitochondrial membrane potential was normal and mitochondrial function was the most exuberant, with the month increased, the red fluorescence intensity of12-month-old normal rats became to decline and tan fluorescence appeared, which illustrated that mitochondrial membrane potential began to descend and mitochondrial function was subsiding. Compared to9-month-old normal rats, the red fluorescence intensity of frontal cortex of all the other model groups declined more obviously, but the green fluorescence intensity enhanced. As the rats grow older, yellow-green fluorescence turned to dark green, which showed that the membrane potential of some mitochondria initiated to go down.
5, measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase
To3-month-old rats, the activity of Na+-K+-ATP, Ca2+-Mg2+-ATPase of each model group decreased gradually with the day going on after the injection, which was statistically significant comparing to the normal control group (p<0.05); moreover, the decrease was the most significant on the3rd,4th day after the intraperitoneal injection, but no significance exsisted between the two model groups. Meanwhile, with the increase of month age, the mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase activity of the frontal cortex of the normal group rose first and then decreased. The enzyme activity of6-month-rat was the strongest in the normal groups, while the enzyme activity of12-month-rat was the lowest, but there was no significance between9-month-old and3-month-old normal rats. The table shows that ATP enzyme activity of the model group and normal control group was statistically significant, but there was no statistical significance between the enzyme activity 9-month-old and12-month-old rats after the injection, which indicated that the9-month-old rats were better for the insomnic model.
Part II:
1, observation of super live staining of mitochondria
The mitochondrial number of the frontal cortex of the model rats decreased obviously compared with the normal rats in high-powered optical microscope, while the mitochondrial number of after administration of diazepam and melatonin increased significantly; in addition, the mitochondrial number of the frontal cortex also increased a little after two courses of low-dose Anmeitang treatment, and the mitochondrial number of the frontal cortex increased more obviously after two courses of middle-dose Anmeitang treatment, while the mitochondrial number of the frontal cortex increased the mo.st obviously after two courses of high-dose Anmeitang treatment,
2, observation of mitochondrial ultrastructure
The size of the frontal cortical mitochondria of9-month-old rats swollen obviously after the injection of PCPA, and most of the cristae broke and gathered at the edge of the mitochondrial membrane even vacuolation occurred, which indicated that mitochondrial function decreased significantly; the swelling mitochondria of the frontal cortex got better after two courses of diazepam and melatonin treatment, and the mitochondrial size were relatively integrated; while the swelling reduced to a certain extent, yet larger than the normal after low-dose treatment of Anmeitang, and some cristae broke; but the mitochondrial size was next door to that of the normal mitochondria after two courses of middle-dose and high-dose treatment of Anmeitang, and the mitochondrial integrality was betrer than that of loe-dose treatment of Anmeitang, and there was almost no breakage in the cristae.
3, Determination of mitochondrial membrane potential
The mitochondrial membrane potential of the model group significantly decreased compared with the9-month-old normal rats. It could be seen that the red fluorescence obviously reduced, while the green fluorescence significantly increased.
the red fluorescence of diazepam and melatonin treatment increased compared with the model group, while the red fluorescence of low, middle, high-dose treatment of Anmeitang increased gradually, indicating that the mitochondrial membrane potential began to improve little by little.
4, measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase
as for Na+-K+-ATP, Ca2+-Mg2+-ATPase of the frontal cortex, there was statistical significance (p<0.01) between the model group and all the treatment groups, which hinted that a variety of drug was effective after two courses of treatment. However, compared with the control group, only the model group and Anmeitang low, middle-dose group were statistically significant (p<0.05), suggesting that Anmei tang low and middle dose were effective, but still not enough to return to normal level; moreover, showing the curative effect of high-dose Anmeitang treatment was close to diazepam and melatonin treatment group and the normal group; there was statistical significance (p<0.05) between diazepam treatment group and low-dose treatment group of Anmeitang, tne model group, indicating that low doses of Anmeitang effect was not significant. With the increasing doses of Anmeitang and Na+-K+-ATP content also increasing, but there was no statistical significance among the low, middle, high-dose treatment groups of Anmeitang.
On the other hand, Ca2+-Mg2+-ATPase of the frontal cortex of the model group and all the treatment groups declined compared with the control group, but they were statistically significant (p<0.05); while there was statistical significance between each treatment group and the model group (p<0.01); while there was also statistical significance between the normal, diazepam treatmeng group and low, middle-dose treatment group of Anmeitang (p <0.01), but there was no statistical significance between the low-dose Anmeitang treatment and the melatonin treatment, middle, high-dose Ameitang treatment groups, which indicated that although Ca2+-Mg2+-ATPase content also increased with increasing doses of Anmeitang, as well as the statistical significance between low-dose Anmeitang treatment and high-dose Anmeitang treatment (p<0.05), there was no statistical significance between middle-dose Anmeitang treatment and high-dose Anmeitang treatment, which suggested that the dose-dependent of Anmeitang was not obvious.
In summary
1, the mitochondrial number and ultrastructure of the cortex of3-month-old model rats reduced significantly on the3rd day after intraperitoneal injection; and the mitochondrial membrane potential and ATP activity started to decline obviously on the2n day, and the most significantly(p<0.05) on the3rd day, but rise slightly on the fourth day.
2, the most obviously changes of the mitochondrial ultrastructure, membrane potential and ATP activity of the cortex existed in the9-month-old model group, but these changes were not statistically significant compare with12-month-old model group.
3, the mitochondrial number began to increase, and the mitochondrial ultrastructure and the mitochondrial membrane potential of the cortex also began to get better after the continuous treatment of Anmeitang; and the content of Na+-K+-ATPase Ca2+-Mg2+-ATPase also began to increase along with the increase of Anmeitang dose, but no significance existed in the both.
Conclusion
1, To3-month-old rats, the most obvious changes of the mitochondrial ultrastructure and function of the frontal cortex of the model rats after a continuous injection of3days were on3rd day.
2,9-month-old rats were the best choice for the insomnic model by PCPA, and the possible insomnic mechanism is the damage of the mitochondrial morphology and function of the prefrontal cortex by the intraperitoneal injection of PCPA.
3, Anmeitang can increase the number of mitochondria of the prefrontal cortex of the model rats, and improve the degeneration of the mitochondrial ultrastructure; while Anmeitang can also increase the mitochondrial membrane potential and the content of Na+-K+-ATPase、Ca2+-Mg2+-ATPase; the dose-dependent of effectivenessin is not obvious although the content of Na+-K+-ATPaseN Ca2+-Mg2+-ATPase increase along with the dose increase of Anmeitang.
4, The decrease and the improvement of the morphology and function of the cortical mitochondria is possibly related with the cortical excitation and inhibition imbalance and excess of Yin and shortage of Yang.
Innovation
1, it is frequent for us to see the clinical reports of Chinese medicine treatment about the insomnia, but the basic research are less, and these basic researches are performed based on the theory of Western neurotransmitter of the lower brain stem and hypothalamus levels. But we firstly investigated the etiopathogenesis of the insomnic models by PCPA and the effect mechanism of Anmeitang from the highest level of the neurous system.
2, we fistly discussed the relationship between the structure and function of mitochondria and the cortical excitation and inhibition imbalance, excess of Yin and shortage of Yang of insomnia.
Inadequacy
1, we don't carry out more experiments about month-ages and time points of model rats because of the difficulty of the rat breeding cycle and funding issues.
2, our study does not carry out the further examination at different levels of the gene, mRNA and protein expression, but we will perform the next continuous, exhaustive exploration in order to provide some references for the basis of external research.
本研究主要基于中医藏象学说心脑相关理论,从最高级中枢大脑皮层水平首先探讨失眠的阳盛阴衰、阴阳失交与大脑皮层兴奋抑制失衡之间的关系,主要观察大鼠经对氯苯丙氨酸(PCPA)腹腔注射进行失眠造模的不同时间点线粒体的变化和失眠造模对不同月龄大鼠皮层线粒体的影响；其次在上一阶段实验的基础上,从线粒体途径探讨安寐汤治疗失眠的机制,为进一步研究中医药改善失眠的作用机制和临床治疗提供理论参考。
方法
第一部分：首先采用3月龄SPF级SD大鼠,雌雄不分,3-21月龄,按文献对氯苯丙氨酸(PCPA)腹腔注射的方法进行失眠模型制作,并分别于造模后第1、2、3和4天观察额叶皮质神经元线粒体形态功能的改变；采用超活染色和电镜分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量。其次,基于上述最佳变化时间点采用3、6、9、12月龄大鼠进行造模3天后分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量。
第二部分：基于第一部分最佳时间点和最佳月龄,采用PCPA法对9月龄大鼠进行造模,随机分为正常对照组、模型组、安定治疗组、褪黑素治疗组和安寐汤低、中、高剂量治疗组；采用超活染色和电镜分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量,以及黄嗓吟氧化法、硫代巴比妥酸法检测皮层线粒体SOD和MDA的变化。
两部分方法具体如下：
1.模型制作
失眠模型制作采用对氯苯丙氨酸(PCPA)腹腔注射的方法。除正常组注射生理盐水外,其余组按0.3ml/100g腹腔注射PCPA(?)昆悬液(浓度为0.1g/m1)。
2.线粒体制备
实验结束后各组大鼠经10%水合氯醛(350mg/kg)腹腔注射麻醉,迅速断头取脑并立即置入冰冷的生理盐水中,洗净血污,剥离皮层并于2m1的分离介质中剪碎,迅速转移至玻璃匀浆管中(1min之内完成),电动匀浆,分离介质稀释,离心,所得沉淀用分离介质悬浮,制成lml混悬液。装在离心管中于碎冰中保存。全程冰上操作,提取完成后立即测定。线粒体蛋白含量以考马斯亮兰(Bradford)法进行测定。
3.线粒体超活染色观察
实验结束后用脊椎脱臼法处死大鼠,在枕骨大孔处迅速剪断延髓,去颅骨,完全暴露脑组织,取出脑组织,分离出额叶皮质后,在冰冷生理盐水中洗净后,在冰浴条件下充分匀浆,离心,取上清液待用。詹纳斯绿B (JGB)可特异性地使具有活性功能的线粒体呈现蓝绿色,从而判断线粒体功能的完整性。具体按照线粒体功能詹纳斯绿B (JGB)染色试剂盒说明书操作。
4.线粒体的超微结构观察
动物实验结束后,1%戊巴比妥钠(0.2ml／l0g)腹腔注射麻醉动物,灌注后取脑,留取大脑额叶皮质直径3mm的组织块,放入混合固定液中固定。修1mm×1mm×1mm大小组织块,2.5%戊二醛和1%四氧化锇酸双重固定,漂洗,丙酮逐级脱水,Epon812包埋,半薄切片光学定位,超薄切片厚度为50nm,醋酸铀及枸橼酸铅双重染色后于透射电镜下观察。每只大鼠观察1张铜网,每张铜网由左上角至右下角斜线上下移动,随机拍摄5张照片,底片放大1.5万倍。
5.线粒体Na+-K+-ATP, Ca2+-Mg2+-ATPase测定
各组大鼠实验结束后断头处死,迅速在冰盘上取大鼠脑部皮质,暂时保存待测。用滤纸吸干大脑皮质外部残血,精确称重,加入冰生理盐水,制备成组织匀浆。取组织匀浆用普通离心机离心,弃沉淀取上清,再低温离心,沉淀物为线粒体(线粒体的提取按试剂盒要求操作,中性红-詹纳斯绿B染色鉴定线粒体),线粒体中Na+-K+-ATP, Ca2+-Mg2+-ATP酶严格按照南京建成生物的试剂盒操作步骤进行测定。
6.线粒体膜电位测定
取出新鲜切除的大鼠额叶皮质样本0.5cm并压平,加入37℃预热的清理液2ml清洗皮质组织并小心抽去清理液,再用900u1染色工作液浸没整个组织,放进37℃培养箱孵育20min,抽去染色液,再用清理液一遍后开始切片约100um厚,然后置于载玻片上,盖上盖玻片,用手指轻轻压平,即刻在荧光显微镜下观察并拍照。
结果
第一部分：
1.动物失眠模型的建立
模型组大鼠在注射后的第一天昼夜节律正常,未见躁动不安；连续注射2d后,大鼠白天活动增多,对外界声、光刺激敏感;造模后第三天出现昼夜节律消失,白天夜晚活动不停；连续注射4d后,大鼠躁动不安非常明显。
2.线粒体超活染色观察
高倍光镜下可见3月龄各组线粒体形态有的呈短棒状,有的呈线状和近椭圆状,并且部分有聚集现象,其中造模各组的染色线粒体数目较正常对照组明显减少,尤其是以造模后第3天和第4天减少最为明显。而不同月龄大鼠造模3天后,和各正常对照组相比,以9月龄模型组的额叶皮质神经元线粒体的数目减少最为显著；而在各模型组中,又以12月龄大鼠模型组的线粒体最少。
3.线粒体超微结构观察
3月龄大鼠造模后第一天额叶皮层线粒体有轻微的肿胀,嵴有少许的断裂；造模后第二天线粒体肿胀开始明显,嵴断裂较多且出现边集化；造模后第三天线粒体进一步肿胀,嵴大部分断裂且开始出现空泡化；造模后第四天线粒体空泡化进一步加重。
3、6月龄正常大鼠的额叶皮质神经元线粒体的体积不大,而9月龄的则明显变大；12月龄的线粒体体积不仅变大,同时内部的嵴也现了少许的断裂,说明12月龄大鼠脑内的线粒体出现凋亡,提示线粒体的功能出现下降。
各正常对照组的线粒体体积不大,单位面积内嵴的数量也比较多,而相应的各模型组的线粒体体积较大,单位面积内嵴多有断裂,数量较少,有的甚至出现空泡化。其中,9月龄正常组大鼠线粒体略有肿胀,相应的模型组大鼠线粒体肿胀非常明显,嵴基本断裂,空泡化较为严重。而12月龄正常组大鼠线粒体不仅略有肿胀,而且嵴开始有部分断裂,相应的模型组大鼠线粒体肿胀也非常明显,嵴几乎全部断裂边集化且明显空泡化。
4.线粒体膜电位测定
模型组大鼠的膜电位与3月龄正常大鼠相比都下降,可见红色荧光逐渐减少,其中以造模后第4天下降最明显。3月龄正常大鼠造模开始后,绿色荧光也开始增加,并呈现由低到高再到低的趋势；说明造模后第4天大鼠额叶皮质神经元线粒体出现量的变化。9月龄正常大鼠的额叶皮质红色荧光最强,说明线粒体膜电位正常且线粒体功能最为旺盛；而12月龄正常大鼠随着月龄的增长,红色荧光减少且出现了棕黄色荧光,提示线粒体的膜电位出现了下降,线粒体功能开始减弱。与9月龄正常大鼠相比,各模型组大鼠额叶皮质红色荧光明显减弱,而绿色荧光逐渐增强。随着月龄增大,黄绿色荧光开始变成深绿色,说明部分线粒体的膜电位开始下降。
5.线粒体Na+-K+-ATP, Ca2+-Mg2+-ATPase测定
对于3月龄大鼠,经PCPA腹腔注射造模,随着天数的增加,造模的各模型组Na+-K+-ATP和Ca2+-Mg2+-ATP酶活性逐渐下降,与正常对照组相比均具有统计学意义(p<0.05)；其中造模后第三天和第四天下降尤为明显,且两组之间无统计学意义。同时,随着月龄的增加,正常大鼠大脑额叶皮质线粒体的Na+-K+-ATP和Ca2+-Mg2+-ATP酶有先升后降的趋势。6月龄正常大鼠的酶活性最强,12月龄正常大鼠的酶活性最低,而9月龄和3月龄正常大鼠的酶活性相比较则无统计学意义。各模型组与正常对照组相比都具有统计学意义；但9月龄和12月龄大鼠造模后酶活性却不具有统计学意义,说明造模选择9月龄的大鼠为佳。
第二部分：
1.线粒体超活染色观察
高倍光镜下可见造模后大鼠额叶皮质线粒体数量较正常大鼠明显减少,安定和褪黑素给药后线粒体的数量又显著增加；而低剂量安寐汤两个疗程灌胃后线粒体有所增加,中剂量安寐汤灌胃后较为明显,高剂量安寐汤灌胃后线粒体数目增加明显。
2.线粒体超微结构观察
9月龄正常大鼠造模后额叶皮层线粒体明显肿胀,嵴大部分断裂且出现边集化甚至空泡化,说明线粒体功能下降明显；经安定和褪黑素治疗后线粒体肿胀明显减轻,线粒体比较完整；而安寐汤低剂量治疗后线粒体肿胀有所减轻,但体积仍较正常组增大,线粒体嵴仍有部分断裂,线粒体不完整；中剂量和高剂量安寐汤灌胃治疗后线粒体较为完整,体积不大,嵴几乎无断裂。
3.线粒体膜电位测定
模型组大鼠的膜电位与9月龄正常大鼠相比明显下降,可见红色荧光明显减少,而绿色荧光明显增加,提示线粒体膜电位下降。安定和褪黑素治疗组也见红色荧光较模型组明显增加；而低剂量和中、高剂量安寐汤治疗组红色荧光则逐渐增多,说明线粒体膜电位逐渐好转。
4.线粒体Na+-K+-ATP,Ca2+-Mg2+-ATPase测定
各组与模型组相比大鼠额叶皮质神经元线粒体Na+-K+-ATPase都具有统计学意义(p<0.01),说明造模后各种药物治疗是有效果的；但与正常对照组相比只有模型组和安寐汤低、中剂量治疗组具有统计学意义(p<0.05),这提示尽管安寐汤低、中剂量有疗效但仍不足以恢复到正常水平,同时也说明安定、褪黑素和安寐汤高剂量的疗效和正常组大鼠接近而差不多；安定治疗组与安寐汤低剂量治疗组和模型组相比具有统计学意义(p<0.05),说明低剂量的安寐汤疗效不显著,同时尽管随着安寐汤剂量的增加Na+-K+-ATPase的含量也增加,但低中高三种剂量的疗效之间却无统计学意义。另一方面,无论是模型组还是各治疗组与正常对照组相比,大鼠额叶皮质神经元线粒体Ca2+-Mg2+-ATPase都有所下降,但都具有统计学意义(p<0.05)；同时各治疗组与模型组相比也都具有统计学意义(p<0.01)；安寐汤低、中剂量的疗效与安定和正常大鼠相比还是有统计学意义的(p<0.01),但低剂量与褪黑素、中与高剂量的疗效之间却无统计学意义,这说明尽管随着安寐汤剂量的增加Ca2+-Mg2+-ATPase的含量也增加,以及低与高剂量疗效之间有统计学意义(p<0.05),但中高三种剂量的疗效之间却无统计学意义。
综上所述
1、3月龄大鼠造模后线粒体数量明显减少,超微结构以第3天变化最为显著;额叶皮层线粒体膜电位和ATP酶活性第2天开始明显下降,第3天下降最明显(p<0.05),第4天略有回升。
2、9月龄大鼠造模后线粒体超微结构空泡化、膜电位和酶活性与12月龄模型组均显著,且无统计学意义。
3、安寐汤治疗后大鼠额叶皮质神经元线粒体数目明显增加,超微结构趋于好转,线粒体膜电位增加,同时随着安寐汤剂量的增加线粒体Na+-K+-ATPase、 Ca2+-Mg2+-ATPase的含量也增加,但不具有统计学意义。
结论
1、以3月龄大鼠进行PCPA制作失眠模型过程中额叶皮层线粒体超微结构和功能变化以连续注射3天最明显。
2、PCPA对失眠大鼠模型制作以9月龄为佳且可通过影响额叶皮层线粒体的形态和功能来导致失眠发生。
3、安寐汤可以增加失眠大鼠额叶皮质神经元线粒体的数目和改善线粒体的超微结构变性；安寐汤可改善额叶皮层神经元线粒体膜电位和增加Na+-K+-ATPase、 Ca2+-Mg2+-ATPase的含量,这可能是其改善失眠的作用机制之一；安寐汤可增加Na+-K+-ATPase, Ca2+-Mg2+-ATPase的含量,但剂量依赖性不明显。
4、失眠大鼠皮层线粒体的形态功能的减退与改善与皮层的兴奋抑制平衡失调和失眠的阳盛阴衰、阴阳失交可能存在密切的联系。
创新点
1、中医药治疗失眠的临床报道较多,基础研究也多是基于西医神经递质理论从低位脑干和丘脑等水平探讨,本研究首次从高位的最高级中枢大脑皮层水平来研究失眠的发病机理和安寐汤的改善作用。
2、本研究基于中医藏象学说的心脑相关理论,在以往中医药失眠研究没有形态学和分子水平上的证实的前提下,从线粒体角度来探讨失眠阳盛阴衰、阴阳失交与皮层兴奋与抑制失衡之间的联系以及安寐汤改善失眠的机理。
存在问题
1、限于大鼠饲养周期和经费问题,本研究没能在大鼠的年龄跨度上开展多个月龄的实验,也没能在造模后更多的时间点上开展探讨；
2、本研究未能从分子生物学的角度在基因、mRNA和蛋白表达等不同的水平展开进一步的检测,后期可做连续深入的探索,也可为失眠的大脑皮层角度的基础外围研究提供参考。
Objective
In this study, based on the correlation theory between heart and brain from theory of visceral manifestations, we explored the relationship between yinyang imbalance of insomnia and the imbalance of excitation and suppression of the cerebral cortex, and mainly observed the changes of the cortical mitochondria of rats at different time points which were made from insomnic models by PCPA peritoneal injection, and also observed the effect of PCPA on the structure and function of the cortical mitochondria of different month rat. Followed by an experimental basis in the previous phase, the next experiment will go on in order to study the effect mechanism of Anmeitang from mitochondrial pathway, and urther develop medicine to improve the symptom of insomnia and provide the some evidence for clinic treatment.
Methods
Part I:First,3-month-old SPF SD rats, male and female accounted for half, were dealed with chlorobenzene alanine (PCPA) by intraperitoneal injection for insomnic model according to the literature, and we observed the changes of the mitochondrial morphology and function of the frontal cortical neurons on1st,2nd,3rd and4th days after intraperitoneal injection by the super live staining and electron microscopy, which can measure the number and ultrastructure of mitochondria, meanwhile we also used fluorescence microscopy to detect mitochondrial membrane potential and WST-1to determine the ATP enzyme content of mitochondria. Secondly, based on the best time of change point, different aged rats,3,6,9, and12-month-old rats, will be produced for insomnic models by the intraperitoneal injection for3days, then we will observe the changes of the number and ultrastructure of mitochondria using the the super live staining and electron microscopy, while we will use the fluorescence microscopy to detect mitochondrial membrane potential and the WST-1to determine the ATP enzyme content of the mitochondria.
Part Ⅱ:Based on the best time point and the best month age of the first part experiment, the9-month-old rats produced by PCPA for insomnic models were randomly divided into normal control group, model group, antipsychotic treatment group, melatonin treatment group and Anmeitang low, medium and high dose treatment group; and we observed the changes of the number and ultrastructure of mitochondria by super live staining and electron microscopy, and we also measured the membrane potential and ATP enzyme content of mitochondria by the fluorescence microscopy and WST-1method.
As follows are the methods of two parts.
1, preparation of animal model
All rats were made for the insomnic model with p-chlorophenyl alanine (PCPA) by intraperitoneal injection. In addition to the normal saline group, the other groups received intraperitoneal injection of PCPA0.3ml/100g suspension (concentration0.1g/ml).
2, preparation of mitochondria
The rats were anaesthetized with10%chloral hydrate (350mg/kg) by intraperitoneal injection at the end of the experiment, and the brains of rats were removed quickly and immediately placed in ice-cold saline, then blood was washed. We peeled the cortex and placed them into the separation medium (2ml), and cut the cortex into pieces, then quickly transferred to a glass homogenizer tube (completed within lmin), electric homogenate diluted separation medium, centrifuged, and the resulting precipitate was suspended with a separation medium into lml suspension. The samples were placed into the centrifuge tube, which were stored in the trash ice. The target would immediately complete after the extraction. Mitochondrial protein content was measured with the method of Coomassie brilliant blue (Bradford).
3, mitochondrial super live staining observation
The rats were sacrificed by spinal dislocation in the foramen magnum at the end of the experiment, then we cut the medulla oblongata quickly, peeled the skull, and the brain tissue was completely exposed, and then picked up the brain tissue, isolated frontal cortex after washing the cortex with the cold saline bath conditions, fully homogenized, centrifuged, and the supernatant set aside. Janus Green B (JGB) could specifically stain the activated mitochondria into blue-green, which could help to determine the integrity of mitochondrial function. The specific steps of super-live staining we can refer to the kit instructions of Janus Green B (JGB).
4, the observation of mitochondrial ultrastructure
Animals were anesthetized with1%sodium pentobarbital (0.2ml/10g) by intraperitoneal injection at the end of the experiment, then perfused, and the brain would taken out;3mm diameter specimens from frontal cortex were made, then placed into the mixed stationary liquid. Next, the specimens would be trimed into1mm×1mm×1mm size tissue blocks, and placed into the mixed stationary liquid of2.5%glutaraldehyde and1%osmium tetroxide acid, rinsed, dehydrated in acetone, Epon812embedded; Next we made the semi-thin section,which would be positioned by optical microscope, and ultrathin sections(50nm) were made for observation after the stain of uranyl acetate and lead citrate by electron microscopy. A copper mesh for each rat was observed, and each copper mesh was moved from top left to bottom right diagonal moves up and down, and five photographs randomly photographed, whose negatives would magnified to15000times.
5, the measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase
The rats were decapitated rapidly and their brains were taken out in the ice tray at the end of the experiment, and the frontal cortexs were cut out for the test, then the residual blood outside the cerebral cortex, accurately weighed, was dried with filter paper, and the icy physiological saline was added to prepare a tissue homogenate, which would be centrifuged with the centrifugal machine. Next, the supernatant was reserved and the pellet was discarded, and then centrifuged, and the precipitate was mitochondria. All the procedure of kit was followed.
6, determination of mitochondrial membrane potential
the frontal cortex samples excised freshly0.5cm were flatten, and the cleaning solution2ml preheated to37℃was used to clean the cortical tissue and then carefully removed. The entire samples were placed into the900ul working fluid, then put into37℃incubator for20min, and then the staining fluid should be removed, and the slices were made about100um thick after the samples were cleaned once again, and then placed on a slide, cover glass, flatten gently with your fingers, and we should immediately observe the slices under a fluorescence microscope and photograph.
Results
Part I:
1, Animal model of insomnia
The circadian rhythm of the insomnic model was normal on the first day after the injection, and no restlessness occurred;but the activity of the insomnic model increased, and the models were very sensitive to the sound and light outside on the second day after the injection; the circadian rhythm disappeared on the third day after the injection, and all models walked non-stop day and night; on the fourth day after the injection, the restlessness of all models were very obvious.
2, observation of super live staining of mitochondria
The mitochondrial morphology of the three-month rats in each group appeared different shaps, some looked like short stick, and some were nearly linear and oval-shaped, and some appeared aggregated state in high-powered optical microscope. The number of mitochondria of each model group was significantly reduced compared with normal control group, especially on the third、fourth day after the injection. The number of mitochondria of the different model group reduced comparing with the normal control group, and the mitochondrial number of In-9m-3d group was the minimum, but the mitochondrial number of In-12m-3d group rised a little. The decrease of the number of mitochondria of the different month group gradually accompanied with the the rising age, and the number of12-month group was minimum.
3, observation of mitochondrial ultrastructure
The morphology of motochondria of the frontal cortex appeared slight swollen, and some cristae of mitochondrial inner membrane broke on the first day after the injection; the mitochondria swollen obviously, and many cristae broke and assembled at the edge of the inner membrane on the second day after the injection; the mitochondria swollen further, and most of the cristae broke and the whole mitochondria became a vacuole on the third day after the injection; the mitochondria nearly turned into vacuolation on the fourth day after the injection. The mitochondrial size of the frontal cortex of the normal rats,3,6month old, was small, but the size of9month old rats became significantly larger; not only the size of12month old rats was larger, but also a little cristae inside broke, which indicating that the mitochondrial apoptosis of the frontal cortex of12-month-old rat happened, and mitochondrial function started to decline. The mitochondrial size of normal control group was small, and the number of cristae per unit area was large, while the mitochondria of the corresponding model groups larger and the number of cristae per unit area was small, many cristae fractured, even some mitochondria became vacuoles. The mitochondria of nine-month-old normal rats slightly swollen, and the mitochondria of the corresponding model rats swollen seriously, and most of the cristae fractured, then serious vacuolization occured. The mitochondria of12-month-old normal rats not only swollen slightly, but some cristae broke; but the mitochondria of the corresponding model rats swollen very clearly, and almost all cristae broken and gathered to the edge of the membrane, so the clear vacuoles appeared.
4, observation of mitochondrial membrane potential
Compared with membrane potentials (MP) of3-month-old normal rats, MPs of the rats in model group all declined, and the red fluorescence intensity in model group declined gradually, and the largest decrease occurred on the fourth day after the injection. The green fluorescence intensity began to increase with the day going on after the injection, and presented the tendency of shifting from low to high, which suggested that the mitochondrial amount of the frontal cortex began to change on the fourth day after the injection. The red fluorescence intensity in frontal cortex of9-month-group normal group was the strongest, which suggested that the mitochondrial membrane potential was normal and mitochondrial function was the most exuberant, with the month increased, the red fluorescence intensity of12-month-old normal rats became to decline and tan fluorescence appeared, which illustrated that mitochondrial membrane potential began to descend and mitochondrial function was subsiding. Compared to9-month-old normal rats, the red fluorescence intensity of frontal cortex of all the other model groups declined more obviously, but the green fluorescence intensity enhanced. As the rats grow older, yellow-green fluorescence turned to dark green, which showed that the membrane potential of some mitochondria initiated to go down.
5, measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase
To3-month-old rats, the activity of Na+-K+-ATP, Ca2+-Mg2+-ATPase of each model group decreased gradually with the day going on after the injection, which was statistically significant comparing to the normal control group (p<0.05); moreover, the decrease was the most significant on the3rd,4th day after the intraperitoneal injection, but no significance exsisted between the two model groups. Meanwhile, with the increase of month age, the mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase activity of the frontal cortex of the normal group rose first and then decreased. The enzyme activity of6-month-rat was the strongest in the normal groups, while the enzyme activity of12-month-rat was the lowest, but there was no significance between9-month-old and3-month-old normal rats. The table shows that ATP enzyme activity of the model group and normal control group was statistically significant, but there was no statistical significance between the enzyme activity 9-month-old and12-month-old rats after the injection, which indicated that the9-month-old rats were better for the insomnic model.
Part II:
1, observation of super live staining of mitochondria
The mitochondrial number of the frontal cortex of the model rats decreased obviously compared with the normal rats in high-powered optical microscope, while the mitochondrial number of after administration of diazepam and melatonin increased significantly; in addition, the mitochondrial number of the frontal cortex also increased a little after two courses of low-dose Anmeitang treatment, and the mitochondrial number of the frontal cortex increased more obviously after two courses of middle-dose Anmeitang treatment, while the mitochondrial number of the frontal cortex increased the mo.st obviously after two courses of high-dose Anmeitang treatment,
2, observation of mitochondrial ultrastructure
The size of the frontal cortical mitochondria of9-month-old rats swollen obviously after the injection of PCPA, and most of the cristae broke and gathered at the edge of the mitochondrial membrane even vacuolation occurred, which indicated that mitochondrial function decreased significantly; the swelling mitochondria of the frontal cortex got better after two courses of diazepam and melatonin treatment, and the mitochondrial size were relatively integrated; while the swelling reduced to a certain extent, yet larger than the normal after low-dose treatment of Anmeitang, and some cristae broke; but the mitochondrial size was next door to that of the normal mitochondria after two courses of middle-dose and high-dose treatment of Anmeitang, and the mitochondrial integrality was betrer than that of loe-dose treatment of Anmeitang, and there was almost no breakage in the cristae.
3, Determination of mitochondrial membrane potential
The mitochondrial membrane potential of the model group significantly decreased compared with the9-month-old normal rats. It could be seen that the red fluorescence obviously reduced, while the green fluorescence significantly increased.
the red fluorescence of diazepam and melatonin treatment increased compared with the model group, while the red fluorescence of low, middle, high-dose treatment of Anmeitang increased gradually, indicating that the mitochondrial membrane potential began to improve little by little.
4, measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase
as for Na+-K+-ATP, Ca2+-Mg2+-ATPase of the frontal cortex, there was statistical significance (p<0.01) between the model group and all the treatment groups, which hinted that a variety of drug was effective after two courses of treatment. However, compared with the control group, only the model group and Anmeitang low, middle-dose group were statistically significant (p<0.05), suggesting that Anmei tang low and middle dose were effective, but still not enough to return to normal level; moreover, showing the curative effect of high-dose Anmeitang treatment was close to diazepam and melatonin treatment group and the normal group; there was statistical significance (p<0.05) between diazepam treatment group and low-dose treatment group of Anmeitang, tne model group, indicating that low doses of Anmeitang effect was not significant. With the increasing doses of Anmeitang and Na+-K+-ATP content also increasing, but there was no statistical significance among the low, middle, high-dose treatment groups of Anmeitang.
On the other hand, Ca2+-Mg2+-ATPase of the frontal cortex of the model group and all the treatment groups declined compared with the control group, but they were statistically significant (p<0.05); while there was statistical significance between each treatment group and the model group (p<0.01); while there was also statistical significance between the normal, diazepam treatmeng group and low, middle-dose treatment group of Anmeitang (p <0.01), but there was no statistical significance between the low-dose Anmeitang treatment and the melatonin treatment, middle, high-dose Ameitang treatment groups, which indicated that although Ca2+-Mg2+-ATPase content also increased with increasing doses of Anmeitang, as well as the statistical significance between low-dose Anmeitang treatment and high-dose Anmeitang treatment (p<0.05), there was no statistical significance between middle-dose Anmeitang treatment and high-dose Anmeitang treatment, which suggested that the dose-dependent of Anmeitang was not obvious.
In summary
1, the mitochondrial number and ultrastructure of the cortex of3-month-old model rats reduced significantly on the3rd day after intraperitoneal injection; and the mitochondrial membrane potential and ATP activity started to decline obviously on the2n day, and the most significantly(p<0.05) on the3rd day, but rise slightly on the fourth day.
2, the most obviously changes of the mitochondrial ultrastructure, membrane potential and ATP activity of the cortex existed in the9-month-old model group, but these changes were not statistically significant compare with12-month-old model group.
3, the mitochondrial number began to increase, and the mitochondrial ultrastructure and the mitochondrial membrane potential of the cortex also began to get better after the continuous treatment of Anmeitang; and the content of Na+-K+-ATPase Ca2+-Mg2+-ATPase also began to increase along with the increase of Anmeitang dose, but no significance existed in the both.
Conclusion
1, To3-month-old rats, the most obvious changes of the mitochondrial ultrastructure and function of the frontal cortex of the model rats after a continuous injection of3days were on3rd day.
2,9-month-old rats were the best choice for the insomnic model by PCPA, and the possible insomnic mechanism is the damage of the mitochondrial morphology and function of the prefrontal cortex by the intraperitoneal injection of PCPA.
3, Anmeitang can increase the number of mitochondria of the prefrontal cortex of the model rats, and improve the degeneration of the mitochondrial ultrastructure; while Anmeitang can also increase the mitochondrial membrane potential and the content of Na+-K+-ATPase、Ca2+-Mg2+-ATPase; the dose-dependent of effectivenessin is not obvious although the content of Na+-K+-ATPaseN Ca2+-Mg2+-ATPase increase along with the dose increase of Anmeitang.
4, The decrease and the improvement of the morphology and function of the cortical mitochondria is possibly related with the cortical excitation and inhibition imbalance and excess of Yin and shortage of Yang.
Innovation
1, it is frequent for us to see the clinical reports of Chinese medicine treatment about the insomnia, but the basic research are less, and these basic researches are performed based on the theory of Western neurotransmitter of the lower brain stem and hypothalamus levels. But we firstly investigated the etiopathogenesis of the insomnic models by PCPA and the effect mechanism of Anmeitang from the highest level of the neurous system.
2, we fistly discussed the relationship between the structure and function of mitochondria and the cortical excitation and inhibition imbalance, excess of Yin and shortage of Yang of insomnia.
Inadequacy
1, we don't carry out more experiments about month-ages and time points of model rats because of the difficulty of the rat breeding cycle and funding issues.
2, our study does not carry out the further examination at different levels of the gene, mRNA and protein expression, but we will perform the next continuous, exhaustive exploration in order to provide some references for the basis of external research.
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