调肝方药加味四逆散分时给药对CMUS大鼠昼夜节律的影响
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摘要
一、研究目的
抑郁症是一类严重危害人类身心健康的常见疾病。其终生患病率高达15%-20%,对人类危害极大。目前临床使用的抗抑郁剂大多是基于抑郁症发病机制的单胺递质假说而研发的,存在着一些明显的缺陷。鉴于此,寻求新的抗抑郁剂研发策略十分必要。
大量研究表明,抑郁症患者存在着明显的生物节律尤其是昼夜节律紊乱,如睡眠障碍,以及部分激素、神经递质及细胞因子等合成、分泌、代谢的昼夜节律失调。由此可见,生物节律的紊乱可能是抑郁症发病机制的一个重要组成部分
本课题组早期实验证实,调肝方药加味四逆散具有确切的抗抑郁效应。在此基础上,结合中医时间医学分时给药的理论基础,在宏观上将抑郁症昼夜节律失调和中医肝主疏泄、调畅情志理论以及天人相应、阴阳平衡理论结合起来,采用调肝方药加味四逆散卯时、酉时分时给药的方法,从调节机体昼夜节律角度,探讨其抗抑郁的作用机理。这种研究可作为中药复方抗抑郁研究的一个探索,并为临床用药提供可靠的药理学依据,从而减少传统中医用药中存在的局限性和盲目性,使中药治疗抑郁症病理改变的针对性更强。同时,本研究也将在一定程度上揭示中医时间医学中分时给药理论的科学内涵。
二、研究方法
1.CMUS动物模型的建立
SPF级SD大鼠80只,雄性,体重150g-180g,自由摄食饮水,在光暗周期为12h,温度为23±2℃的安静环境适应两周后,根据糖水消耗实验结果和体重情况分为8组,每组10只。造模采用Willner P慢性轻度不可预计应激动物模型,并加以修改。接受应激处理的组别大鼠与空白组大鼠分房饲养,在21天接受不同应激源的刺激,应激源包含有狭小空间(束缚)、45。斜笼、湿笼、新入侵者(并笼)、持续光照、禁食、禁水、空瓶。于造模周期结束后,对所有实验动物进行糖水消耗实验,并称量体重,通过前后对比分析判断模型建立情况以及各给药组别的抗抑郁效应。
2.药物处理
在造模的同时,即开始对各组别大鼠进行不同的药物处理,其中,空白组(不灌胃、不施加任何刺激)、模型组(每天7：30灌胃双蒸水,3ml／只、施加刺激)、JWSNS卯时组(每天7：30灌胃中药JWSNS,3ml/只、施加刺激)、JWSNS酉时组(每天17：30灌胃中药JWSNS,3ml/只、施加刺激)、JWSNS卯酉分时组(每天7：30、17：30分别灌胃中药JWSNS,1.5ml/只、施加刺激)、双蒸水卯酉分时组(每天7：30、17：30分别灌胃双蒸水,1.5ml/只、施加刺激)、MT清晨组(造模前期每天7：30灌胃双蒸水,后期每天7：30灌胃MT溶液,3ml/只、施加刺激)、MT傍晚组(造模前期每天17：30灌胃双蒸水,后期每天17：30灌胃MT溶液,3ml/只、施加刺激)。其中空白组与其他各组分房饲养。
3.指标的检测
体温：在末次糖水消耗实验结束后三天内,对所有大鼠进行昼夜24小时的体温检测,每四小时一次,检测时间点分别为10：00、14：00、18：00、22：00、2：00和6：00。将所得数据进行拟合,得出各组大鼠体温变化的余弦方程,并对其体温中值、峰值、谷值、振幅以及峰值时间、谷值时间进行比较,观察不同处理对大鼠24小时体温节律的影响。
激素神经递质：在体温检测结束后三天内,对所有大鼠进行昼夜24小时的眶下静脉丛乙醚麻醉下采血,每四小时一次,采集时间点分别为10：00、14：00、18：00、22：00、2：00和6：00。血液采集结束立即离心,取血清部分-20℃冻存备用。采血结束后七天内,对所得样本进行酶联免疫吸附试验(ELISA),检测其血清中5-羟色胺、褪黑素、皮质酮含量,并将所得数据进行拟合,得出此三种物质昼夜变化的余弦方程,并对其血清浓度的中值、峰值、谷值、振幅以及峰值时间、谷值时间进行比较,观察不同处理对大鼠24小时5-羟色胺、褪黑素、皮质酮节律的影响。
三、实验结果
1.成功建立大鼠CMUS实验动物模型
实验结果显示,模型组大鼠表现为体重增长缓慢和糖水偏爱度的显著下降(P<0.01)。且在整个实验周期的第22天左右,模型组动物开始出现典型的毛无光泽、竖毛现象,其余除正常组外各组毛色也较之前晦暗。据此认定CMUS动物模型复制成功。
2.调肝方药加味四逆散及阳性药物褪黑素抗抑郁行为效应
在体重变化方面,阳性药物褪黑素清晨、傍晚给药对CMUS造成的大鼠体重下降有抑制趋势,且此种作用优于加味四逆散各给药组别；且加味四逆散卯酉分时给药优于卯时给药优于酉时给药。考虑为药物本身的质地、口感及单次灌胃量等因素对动物的食欲的影响以及灌胃时的机械刺激对大鼠的影响,进而影响其体重的增加。
在糖水偏爱度的变化方面,方药加味四逆散卯时、酉时、卯酉分时给药以及褪黑素清晨、傍晚给药均可以抑制由对慢性应激带来的糖水偏爱度下降。
因此得出结论,调肝方药加味四逆散卯时、酉时、卯酉分时给药以及阳性药物褪黑素清晨、傍晚给药均具有抗抑郁效应。
3.慢性应激带来的体温及激素神经递质的改变
对于本实验检测的四个指标——体温、血清5-羟色胺含量、血清褪黑素含量、血清皮质酮含量。研究结果显示,在正常情况下,大鼠体温呈现昼夜节律变化,其中光时相(白昼)体温较低,暗时相(夜间)体温较高,体温谷值出现在9：00左右,峰值出现在与之相对应的21：00左右,昼夜体温波动范围在(0.2-0.6)℃之间。CMUS可导致实验大鼠昼夜24小时平均体温下降,峰值抬高,谷值降低,体温波动幅度变大,时相推后。
血清5-羟色胺、褪黑素、皮质酮含量同样存在着昼夜节律的变化,三者表现为大致相同的波动规律,都是光时相(白昼)含量较低,暗时相(夜间)含量较高。其中,5-羟色胺和皮质酮的血清浓度峰值大致出现在21：00左右,谷值出现在9：00左右,褪黑素的时相向后延迟约一个小时,峰值时间出现在22：00左右,谷值时间出现在10：00左右。在慢性应激状态下,实验大鼠表现出血清5-羟色胺和褪黑素整体水平的下降,波动幅度减小,节律性减弱以及时相的大幅度提前,甚至出现昼夜节律的颠倒；而血清皮质酮则表现为整体水平的升高,波动幅度减小,节律性减弱以及时相的大幅度提前。
4.阳性药物褪黑素抗抑郁机理的推断
实验结果显示,在调节由慢性应激带来的体温及激素神经递质改变方面,褪黑素清晨给药组表现为对体温振幅的调节和对5-羟色胺、皮质酮时相的调节,而对含量改变不明显。具体表现为通过调节夜间体温(峰值)使体温波动幅度减小,更接近正常值；使5-羟色胺分泌时相、皮质酮分泌时相更接近空白组。褪黑素傍晚组表现为对体温振幅、时相的调节,对5-羟色胺含量、振幅的调节和对皮质酮含量的调节。具体表现为使体温振幅减小,时相提前；使5-羟色胺含量增加,节律性更加明显；使皮质酮含量减少。纵观褪黑素作用的大体趋势,以对生物节律(振幅、时相)的调节为主。
因此可以做出推断,阳性药物褪黑素主要是通过调节时间节律来达到抗抑郁效果的。
5.方药加味四逆散各给药组抗抑郁机理的推断
对于由慢性应激带来的体温及激素神经递质改变的调节,方药加味四逆散三个给药组别中：
卯时给药主要通过改变时间节律来达到抗抑郁的作用。表现为对体温时相、血清5-羟色胺和血清褪黑素分泌时相的调节,而对其含量几乎没有影响；具体表现为对于CMUS作用下实验大鼠24小时体温峰值时间、谷值时间的延迟性改变起到抑制作用,从而其时相更加接近于空白组；并对慢性应激带来的血清5-羟色胺、褪黑素时相的大幅度提前有调节作用,使其时相更加接近于空白组。
酉时给药主要通过改变激素分泌量来达到抗抑郁的作用。首先在体温方面,酉时给药主要通过对昼夜体温(峰值、谷值)的双向调节达到使体温波动幅度减小的结果,从而使其体温变化更接近正常值。在对激素神经递质的调节方面,酉时给药均能对慢性应激带来的大鼠5-羟色胺、褪黑素、皮质酮含量波动不明显,节律性减弱这一现象起到一定的抑制作用。并可调节血清中5-羟色胺、褪黑素的量,抑制其含量的降低,调节血清中皮质酮水平,抑制其升高。
卯酉分时给药的抗抑郁效应则是通过改变激素分泌量和调整其节律性两方面起作用的。体温方面,卯酉分时给药主要通过对日间体温(谷值)使体温波动幅度减小,更接近正常值。在对激素神经递质的调节方面,卯酉分时给药能抑制血清5-HT含量的降低,抑制血清皮质酮水平的升高,并对5-羟色胺、皮质酮的振幅起到调节作用,使其波动更接近空白组。此外,卯酉分时给药还起到调整褪黑素、皮质酮时相变化的作用。
因此可以认为,对于方药加味四逆散来讲,卯时给药主要是通过调整时间节律来达到抗抑郁效果的,而酉时给药主要是通过调整激素神经递质的分泌量来达到抗抑郁效果的,卯酉分时给药则同时从调整激素神经递质分泌量和调整时间节律两个方面来发挥其抗抑郁的效应。
四、研究结论
本研究所得结论如下：
1.慢性应激性抑郁症可以带来生物体昼夜节律的紊乱,包括体温整体水平的降低、波动幅度的加大、时相的延迟；血清5-羟色胺、褪黑素水平的降低,节律性的减弱,时相的提前；血清皮质酮水平的升高,节律性的减弱,时相的提前。
2.方药加味四逆散卯时、酉时、卯酉分时给药以及褪黑素清晨、傍晚给药等各治疗组都有明显的抗抑郁效应。
3.阳性对照药物褪黑素主要是通过调节时间节律来达到抗抑郁效果
4.调肝方药加味四逆散卯时给药主要通过调整时间节律来达到抗抑郁效果
5.调肝方药加味四逆散酉时给药主要通过改变激素分泌量来达到抗抑郁效果
6.调肝方药加味四逆散卯酉分时给药主要通过改变激素分泌量和调整其节律性两方面达到抗抑郁效果
综上所述,可认为调肝方药加味四逆散对慢性应激性抑郁症带来的体温及血清激素水平昼夜节律的紊乱具有调节作用。其不同时间点给药作用机制有所不同,卯时给药主要通过改变时间节律来达到抗抑郁效果,酉时给药主要通过改变激素分泌量来达到抗抑郁效果,卯酉分时给药主要通过改变激素分泌量和调整其节律性两方面达到抗抑郁效果。而目前应用于调节抑郁症生物节律紊乱的药物褪黑素,其作用机理与加味四逆散卯时给药相类似,主要通过改变时间节律来达到抗抑郁效果。
Objective
Depression is one kind of mental disorder who is extremely harmful to mankind. Its lifetime prevalence high up to 15%-20%. Rightnow, antidepressant in use are mostly based on the pathogenesis of depression monoamine hypothesis, which often show some obvious flaws. In view of this, the search for a new antidepressant strategy is necessary.
Numerous studies show that patients with depression often shows a clear circadian rhythm disorder, particularly the day/night circadian rhythm disorder, such as sleep disturbance, and hormones, neurotransmitters also cytokines circadian rhythm disorders in its synthesis, secretion and metabolism. Thus, biological rhythm disorders may be one important component of depression.
Early experiments confirmed, Liver recipe Jiawei Sini Decoction have exactly antidepressant effect. Basis on this, combin with the theory time-based theory of traditional Chinese medicine. Give the Modified Liver Herbs at Mao Shi, You Shi hourly, inorder to perspective the mechanism of its antidepressant effect from the body regulating circadian rhythm. This research can be used as an explore to compound Chinese medicine in antidepressant, and provide basis for traditional Chinese in clinical pharmacology. Methods
1.CMUS animal model
SPF SD rats 80, male, weighing 150g-180g, free ingestion of water, light/dark period of 12h, temperature 23±2℃in a quiet environment to adapt for two weeks. According to sugar consumption and body weight divided into 8 groups. Use Willner P modeling with an unpredictable chronic mild stress model. Groups of rats received shock treatment and control group rats kept housing allocation. In 21 days, animals receiving the stimulation of different stressors, stress source contains small space (binding),45°oblique cage, wet cage, a new invasion, continuous light, fast, ban water, empty bottles. At the end of the modeling period, all experimental animals on the sugar consumption of the experiment and weighed, to judge by comparative analysis before and after the model as well as the antidepressant effects of the medication group.
2. Treatment
While construction of the model that all groups of rats were started on a different drug treatment, in which the control group (not fed, not to impose any stimulation), model group(7:30 per day administered distilled water,3ml/only, imposition of stimulation), JWSNS Maoshi group(7:30 per day administered medicine JWSNS,3ml/only, exert stimulus), JWSNS Youshi group (fed daily 17:30 medicine JWSNS,3ml/only, imposition of stimulation), JWSNS Maoyou time-group (daily 7:30,17:00 were administered medicine JWSNS, 1.5ml/only, imposition of stimulation), double distilled water Maoyou time-group (daily 7:30,17:00 were administered distilled water, 1.5ml/only, exert stimulus), MT morning group (7:30 per day administered early modeling double-distilled water, post-gavage daily 7:30 MT solution, 3ml/only, exert stimulation), MT evening group(17:30 per day administered early modeling double-distilled water, post-gavage daily 17:30 MT solution, 3ml/only exert stimulus). The control group were feed in another room.
3. Detection
Temperature:In the last three days after the end of the sugar consumption experiment, all rats were 24-hour day and night temperature screening, once every four hours, testing time were 10:00,14:00,18:00,22:00,2:00 and 6:00. The data will fit the changes in body temperature of rats obtained the cosine equation, and its body temperature value, peak, trough, amplitude and peak time, valley time, to observe the change of body temperature rhythm in different treatment groups.
Neurotransmitter hormone:After three days detection in body temperature, 24 hours a day all the rats of the infraorbital venous plexus under ether anesthesia blood was collected once every four hours, collection time points were10:00,14:00,18:00,22:00,2:00 and 6:00. The end of the blood collected immediately centrifuged, and the serum frozen spare parts-20℃. Within seven days of the end of blood collection, the samples from enzyme-linked immunosorbent assay (ELISA), detection of the serum of 5-hydroxytryptamine, melatonin, corticosterone levels, and fitting the data obtained diurnal variation of these three substances the cosine equation, and the median of their serum concentration, peak, valley, amplitude and peak time, valley time, to observe the change of 5-hydroxytryptamine, melatonin, corticosterone rhythm in different treatment groups. Results
1. Successful animal model of CMUS
Experimental results show that model group showed weight gain and a significant sucrose preference decreased (P<0.01). And throughout the experimental period of 22 days, the animals began to model a typical dull hair, hair shaft phenomenon, other than the normal groups, the coat color is also darker than before. Accordingly, animal model identified CMUS successed.
2. Liver Decoction Jiawei Sihi Decoction and positive behavioral effects of antidepressant drugs melatonin
Changes in body weight, the positive drug melatonin morning, evening administration of the CMUS weight loss in rats caused an inhibitory trend, and this effect than the administration of Jiawei Sini Decoction Group; and Sini San Mao You time-dose administration than Maoshi than Youshi administration.
Changes in sucrose preference, the prescription of Jiawei Sini San Mao Shi, You Shi, Mao You times and melatonin morning, evening administration can reduce the escend of sucrose preference caused by chronic stress.
According to the result liver recipe Sini San Mao Shi, You Shi, Mao You timeshare and positive drug melatonin morning, evening administration all take an antidepressant effects.
3. Changes in body temperature and hormonal neurotransmitter by CMUS
For assay of the four indicators-body temperature, serum 5-HT, melatonin and serum corticosterone levels. The results showed that in normal circumstances, the rats showed circadian rhythm of body temperature, in which light phase (daytime) temperature low, dark phase (night) high body temperature, day and night temperature fluctuation range (0.2-0.6)℃. CMUS could lead to an average of 24 hours temperature drops, fluctuations become larger, phase postponed.
Serum of 5-hydroxytryptamine, melatonin, corticosterone levels also exist changes in circadian rhythm, all three showed similar fluctuations, light phase(day)is lower, the dark phase(night)were higher. In chronic stress, the experimental rats showed a decline in 5-HT and melatonin level, and volatility reduced, rhythm also weakened or even reversed. Corticosterone levels were elevated, volatility reduced, rhythm and phase of a significant weakening in advance.
4. Positive inference mechanism of antidepressant drugs melatonin
Experimental results show that in the regulation of body temperature caused by chronic stress and hormonal changes in neurotransmitters, the melatonin morning treatment group showed a regulation of body temperature and the amplitude of 5-HT, corticosterone phase, but no significant change to the content. It also can adjust 5-HT, CORT secretion phase closer to control group. Evening melatonin group showed on the temperature amplitude, phase adjustment, and regulation of 5-HT, corticosterone levels. So we can extrapolate positive drug melatonin rhythm in the regulation of time to achieve antidepressant effects.
5. Prescription anti-depressant mechanism of Jiawei Sini Decoction
For'the changes in body temperature and hormone regulation of neurotransmitters, caused by chronic stress:Mao Shi group, mainly through changes in rhythm to achieve antidepressant effects. Manifested on the body temperature,5-HT and melatonin secretion phase, while almost no effect on its content. You Shi administration, mainly through changes in hormone secretion to achieve antidepressant effects. Mao You antidepressant effect of time-sharing administration is by changing the hormone secretion and adjust its role both in terms of rhythm.
It follows that, for Jiawei Sini Decoction speaking, Mao Shi administered mainly by adjusting the rhythm of time to achieve antidepressant effects, and Youshi administration mainly by adjusting the amount of hormone secretion of neurotransmitters to achieve antidepressant effects, Mao You are also from the adjustment of time-dose hormone secretion of neurotransmitters and adjust the time to play both rhythm antidepressant effect. Conclusions
1. CMUS can bring the disorder of circadian rhythm, including body temper ature level reduce, band increase and phase delay; serum 5-hydroxytryptam ine, melatonin levels decrease, rhythm weakened, phase advance; serum corti costerone levels increased, rhythm weakened and phase advance.
2. Recipe Jiawei Sini San and melatonin treatment groups had significant antidepressant effect
3. Positive control drug melatonin mainly in regulation of time to achieve antidepressant effects
4. Liver Decoction Jiawei Sini Decoction Mao Shi administration, mainly through changes in rhythm to achieve antidepressant effects
5. Liver Decoction Jiawei Sini Decoction Youshi administration, mainly through changes in hormone secretion to achieve antidepressant effects
6. Liver Decoction Jiawei Sini Decoction Maoyou administration by both changing the hormone secretion and to adjust its rhythm to achieve antidepressant effects
抑郁症是一类严重危害人类身心健康的常见疾病。其终生患病率高达15%-20%,对人类危害极大。目前临床使用的抗抑郁剂大多是基于抑郁症发病机制的单胺递质假说而研发的,存在着一些明显的缺陷。鉴于此,寻求新的抗抑郁剂研发策略十分必要。
大量研究表明,抑郁症患者存在着明显的生物节律尤其是昼夜节律紊乱,如睡眠障碍,以及部分激素、神经递质及细胞因子等合成、分泌、代谢的昼夜节律失调。由此可见,生物节律的紊乱可能是抑郁症发病机制的一个重要组成部分
本课题组早期实验证实,调肝方药加味四逆散具有确切的抗抑郁效应。在此基础上,结合中医时间医学分时给药的理论基础,在宏观上将抑郁症昼夜节律失调和中医肝主疏泄、调畅情志理论以及天人相应、阴阳平衡理论结合起来,采用调肝方药加味四逆散卯时、酉时分时给药的方法,从调节机体昼夜节律角度,探讨其抗抑郁的作用机理。这种研究可作为中药复方抗抑郁研究的一个探索,并为临床用药提供可靠的药理学依据,从而减少传统中医用药中存在的局限性和盲目性,使中药治疗抑郁症病理改变的针对性更强。同时,本研究也将在一定程度上揭示中医时间医学中分时给药理论的科学内涵。
二、研究方法
1.CMUS动物模型的建立
SPF级SD大鼠80只,雄性,体重150g-180g,自由摄食饮水,在光暗周期为12h,温度为23±2℃的安静环境适应两周后,根据糖水消耗实验结果和体重情况分为8组,每组10只。造模采用Willner P慢性轻度不可预计应激动物模型,并加以修改。接受应激处理的组别大鼠与空白组大鼠分房饲养,在21天接受不同应激源的刺激,应激源包含有狭小空间(束缚)、45。斜笼、湿笼、新入侵者(并笼)、持续光照、禁食、禁水、空瓶。于造模周期结束后,对所有实验动物进行糖水消耗实验,并称量体重,通过前后对比分析判断模型建立情况以及各给药组别的抗抑郁效应。
2.药物处理
在造模的同时,即开始对各组别大鼠进行不同的药物处理,其中,空白组(不灌胃、不施加任何刺激)、模型组(每天7：30灌胃双蒸水,3ml／只、施加刺激)、JWSNS卯时组(每天7：30灌胃中药JWSNS,3ml/只、施加刺激)、JWSNS酉时组(每天17：30灌胃中药JWSNS,3ml/只、施加刺激)、JWSNS卯酉分时组(每天7：30、17：30分别灌胃中药JWSNS,1.5ml/只、施加刺激)、双蒸水卯酉分时组(每天7：30、17：30分别灌胃双蒸水,1.5ml/只、施加刺激)、MT清晨组(造模前期每天7：30灌胃双蒸水,后期每天7：30灌胃MT溶液,3ml/只、施加刺激)、MT傍晚组(造模前期每天17：30灌胃双蒸水,后期每天17：30灌胃MT溶液,3ml/只、施加刺激)。其中空白组与其他各组分房饲养。
3.指标的检测
体温：在末次糖水消耗实验结束后三天内,对所有大鼠进行昼夜24小时的体温检测,每四小时一次,检测时间点分别为10：00、14：00、18：00、22：00、2：00和6：00。将所得数据进行拟合,得出各组大鼠体温变化的余弦方程,并对其体温中值、峰值、谷值、振幅以及峰值时间、谷值时间进行比较,观察不同处理对大鼠24小时体温节律的影响。
激素神经递质：在体温检测结束后三天内,对所有大鼠进行昼夜24小时的眶下静脉丛乙醚麻醉下采血,每四小时一次,采集时间点分别为10：00、14：00、18：00、22：00、2：00和6：00。血液采集结束立即离心,取血清部分-20℃冻存备用。采血结束后七天内,对所得样本进行酶联免疫吸附试验(ELISA),检测其血清中5-羟色胺、褪黑素、皮质酮含量,并将所得数据进行拟合,得出此三种物质昼夜变化的余弦方程,并对其血清浓度的中值、峰值、谷值、振幅以及峰值时间、谷值时间进行比较,观察不同处理对大鼠24小时5-羟色胺、褪黑素、皮质酮节律的影响。
三、实验结果
1.成功建立大鼠CMUS实验动物模型
实验结果显示,模型组大鼠表现为体重增长缓慢和糖水偏爱度的显著下降(P<0.01)。且在整个实验周期的第22天左右,模型组动物开始出现典型的毛无光泽、竖毛现象,其余除正常组外各组毛色也较之前晦暗。据此认定CMUS动物模型复制成功。
2.调肝方药加味四逆散及阳性药物褪黑素抗抑郁行为效应
在体重变化方面,阳性药物褪黑素清晨、傍晚给药对CMUS造成的大鼠体重下降有抑制趋势,且此种作用优于加味四逆散各给药组别；且加味四逆散卯酉分时给药优于卯时给药优于酉时给药。考虑为药物本身的质地、口感及单次灌胃量等因素对动物的食欲的影响以及灌胃时的机械刺激对大鼠的影响,进而影响其体重的增加。
在糖水偏爱度的变化方面,方药加味四逆散卯时、酉时、卯酉分时给药以及褪黑素清晨、傍晚给药均可以抑制由对慢性应激带来的糖水偏爱度下降。
因此得出结论,调肝方药加味四逆散卯时、酉时、卯酉分时给药以及阳性药物褪黑素清晨、傍晚给药均具有抗抑郁效应。
3.慢性应激带来的体温及激素神经递质的改变
对于本实验检测的四个指标——体温、血清5-羟色胺含量、血清褪黑素含量、血清皮质酮含量。研究结果显示,在正常情况下,大鼠体温呈现昼夜节律变化,其中光时相(白昼)体温较低,暗时相(夜间)体温较高,体温谷值出现在9：00左右,峰值出现在与之相对应的21：00左右,昼夜体温波动范围在(0.2-0.6)℃之间。CMUS可导致实验大鼠昼夜24小时平均体温下降,峰值抬高,谷值降低,体温波动幅度变大,时相推后。
血清5-羟色胺、褪黑素、皮质酮含量同样存在着昼夜节律的变化,三者表现为大致相同的波动规律,都是光时相(白昼)含量较低,暗时相(夜间)含量较高。其中,5-羟色胺和皮质酮的血清浓度峰值大致出现在21：00左右,谷值出现在9：00左右,褪黑素的时相向后延迟约一个小时,峰值时间出现在22：00左右,谷值时间出现在10：00左右。在慢性应激状态下,实验大鼠表现出血清5-羟色胺和褪黑素整体水平的下降,波动幅度减小,节律性减弱以及时相的大幅度提前,甚至出现昼夜节律的颠倒；而血清皮质酮则表现为整体水平的升高,波动幅度减小,节律性减弱以及时相的大幅度提前。
4.阳性药物褪黑素抗抑郁机理的推断
实验结果显示,在调节由慢性应激带来的体温及激素神经递质改变方面,褪黑素清晨给药组表现为对体温振幅的调节和对5-羟色胺、皮质酮时相的调节,而对含量改变不明显。具体表现为通过调节夜间体温(峰值)使体温波动幅度减小,更接近正常值；使5-羟色胺分泌时相、皮质酮分泌时相更接近空白组。褪黑素傍晚组表现为对体温振幅、时相的调节,对5-羟色胺含量、振幅的调节和对皮质酮含量的调节。具体表现为使体温振幅减小,时相提前；使5-羟色胺含量增加,节律性更加明显；使皮质酮含量减少。纵观褪黑素作用的大体趋势,以对生物节律(振幅、时相)的调节为主。
因此可以做出推断,阳性药物褪黑素主要是通过调节时间节律来达到抗抑郁效果的。
5.方药加味四逆散各给药组抗抑郁机理的推断
对于由慢性应激带来的体温及激素神经递质改变的调节,方药加味四逆散三个给药组别中：
卯时给药主要通过改变时间节律来达到抗抑郁的作用。表现为对体温时相、血清5-羟色胺和血清褪黑素分泌时相的调节,而对其含量几乎没有影响；具体表现为对于CMUS作用下实验大鼠24小时体温峰值时间、谷值时间的延迟性改变起到抑制作用,从而其时相更加接近于空白组；并对慢性应激带来的血清5-羟色胺、褪黑素时相的大幅度提前有调节作用,使其时相更加接近于空白组。
酉时给药主要通过改变激素分泌量来达到抗抑郁的作用。首先在体温方面,酉时给药主要通过对昼夜体温(峰值、谷值)的双向调节达到使体温波动幅度减小的结果,从而使其体温变化更接近正常值。在对激素神经递质的调节方面,酉时给药均能对慢性应激带来的大鼠5-羟色胺、褪黑素、皮质酮含量波动不明显,节律性减弱这一现象起到一定的抑制作用。并可调节血清中5-羟色胺、褪黑素的量,抑制其含量的降低,调节血清中皮质酮水平,抑制其升高。
卯酉分时给药的抗抑郁效应则是通过改变激素分泌量和调整其节律性两方面起作用的。体温方面,卯酉分时给药主要通过对日间体温(谷值)使体温波动幅度减小,更接近正常值。在对激素神经递质的调节方面,卯酉分时给药能抑制血清5-HT含量的降低,抑制血清皮质酮水平的升高,并对5-羟色胺、皮质酮的振幅起到调节作用,使其波动更接近空白组。此外,卯酉分时给药还起到调整褪黑素、皮质酮时相变化的作用。
因此可以认为,对于方药加味四逆散来讲,卯时给药主要是通过调整时间节律来达到抗抑郁效果的,而酉时给药主要是通过调整激素神经递质的分泌量来达到抗抑郁效果的,卯酉分时给药则同时从调整激素神经递质分泌量和调整时间节律两个方面来发挥其抗抑郁的效应。
四、研究结论
本研究所得结论如下：
1.慢性应激性抑郁症可以带来生物体昼夜节律的紊乱,包括体温整体水平的降低、波动幅度的加大、时相的延迟；血清5-羟色胺、褪黑素水平的降低,节律性的减弱,时相的提前；血清皮质酮水平的升高,节律性的减弱,时相的提前。
2.方药加味四逆散卯时、酉时、卯酉分时给药以及褪黑素清晨、傍晚给药等各治疗组都有明显的抗抑郁效应。
3.阳性对照药物褪黑素主要是通过调节时间节律来达到抗抑郁效果
4.调肝方药加味四逆散卯时给药主要通过调整时间节律来达到抗抑郁效果
5.调肝方药加味四逆散酉时给药主要通过改变激素分泌量来达到抗抑郁效果
6.调肝方药加味四逆散卯酉分时给药主要通过改变激素分泌量和调整其节律性两方面达到抗抑郁效果
综上所述,可认为调肝方药加味四逆散对慢性应激性抑郁症带来的体温及血清激素水平昼夜节律的紊乱具有调节作用。其不同时间点给药作用机制有所不同,卯时给药主要通过改变时间节律来达到抗抑郁效果,酉时给药主要通过改变激素分泌量来达到抗抑郁效果,卯酉分时给药主要通过改变激素分泌量和调整其节律性两方面达到抗抑郁效果。而目前应用于调节抑郁症生物节律紊乱的药物褪黑素,其作用机理与加味四逆散卯时给药相类似,主要通过改变时间节律来达到抗抑郁效果。
Objective
Depression is one kind of mental disorder who is extremely harmful to mankind. Its lifetime prevalence high up to 15%-20%. Rightnow, antidepressant in use are mostly based on the pathogenesis of depression monoamine hypothesis, which often show some obvious flaws. In view of this, the search for a new antidepressant strategy is necessary.
Numerous studies show that patients with depression often shows a clear circadian rhythm disorder, particularly the day/night circadian rhythm disorder, such as sleep disturbance, and hormones, neurotransmitters also cytokines circadian rhythm disorders in its synthesis, secretion and metabolism. Thus, biological rhythm disorders may be one important component of depression.
Early experiments confirmed, Liver recipe Jiawei Sini Decoction have exactly antidepressant effect. Basis on this, combin with the theory time-based theory of traditional Chinese medicine. Give the Modified Liver Herbs at Mao Shi, You Shi hourly, inorder to perspective the mechanism of its antidepressant effect from the body regulating circadian rhythm. This research can be used as an explore to compound Chinese medicine in antidepressant, and provide basis for traditional Chinese in clinical pharmacology. Methods
1.CMUS animal model
SPF SD rats 80, male, weighing 150g-180g, free ingestion of water, light/dark period of 12h, temperature 23±2℃in a quiet environment to adapt for two weeks. According to sugar consumption and body weight divided into 8 groups. Use Willner P modeling with an unpredictable chronic mild stress model. Groups of rats received shock treatment and control group rats kept housing allocation. In 21 days, animals receiving the stimulation of different stressors, stress source contains small space (binding),45°oblique cage, wet cage, a new invasion, continuous light, fast, ban water, empty bottles. At the end of the modeling period, all experimental animals on the sugar consumption of the experiment and weighed, to judge by comparative analysis before and after the model as well as the antidepressant effects of the medication group.
2. Treatment
While construction of the model that all groups of rats were started on a different drug treatment, in which the control group (not fed, not to impose any stimulation), model group(7:30 per day administered distilled water,3ml/only, imposition of stimulation), JWSNS Maoshi group(7:30 per day administered medicine JWSNS,3ml/only, exert stimulus), JWSNS Youshi group (fed daily 17:30 medicine JWSNS,3ml/only, imposition of stimulation), JWSNS Maoyou time-group (daily 7:30,17:00 were administered medicine JWSNS, 1.5ml/only, imposition of stimulation), double distilled water Maoyou time-group (daily 7:30,17:00 were administered distilled water, 1.5ml/only, exert stimulus), MT morning group (7:30 per day administered early modeling double-distilled water, post-gavage daily 7:30 MT solution, 3ml/only, exert stimulation), MT evening group(17:30 per day administered early modeling double-distilled water, post-gavage daily 17:30 MT solution, 3ml/only exert stimulus). The control group were feed in another room.
3. Detection
Temperature:In the last three days after the end of the sugar consumption experiment, all rats were 24-hour day and night temperature screening, once every four hours, testing time were 10:00,14:00,18:00,22:00,2:00 and 6:00. The data will fit the changes in body temperature of rats obtained the cosine equation, and its body temperature value, peak, trough, amplitude and peak time, valley time, to observe the change of body temperature rhythm in different treatment groups.
Neurotransmitter hormone:After three days detection in body temperature, 24 hours a day all the rats of the infraorbital venous plexus under ether anesthesia blood was collected once every four hours, collection time points were10:00,14:00,18:00,22:00,2:00 and 6:00. The end of the blood collected immediately centrifuged, and the serum frozen spare parts-20℃. Within seven days of the end of blood collection, the samples from enzyme-linked immunosorbent assay (ELISA), detection of the serum of 5-hydroxytryptamine, melatonin, corticosterone levels, and fitting the data obtained diurnal variation of these three substances the cosine equation, and the median of their serum concentration, peak, valley, amplitude and peak time, valley time, to observe the change of 5-hydroxytryptamine, melatonin, corticosterone rhythm in different treatment groups. Results
1. Successful animal model of CMUS
Experimental results show that model group showed weight gain and a significant sucrose preference decreased (P<0.01). And throughout the experimental period of 22 days, the animals began to model a typical dull hair, hair shaft phenomenon, other than the normal groups, the coat color is also darker than before. Accordingly, animal model identified CMUS successed.
2. Liver Decoction Jiawei Sihi Decoction and positive behavioral effects of antidepressant drugs melatonin
Changes in body weight, the positive drug melatonin morning, evening administration of the CMUS weight loss in rats caused an inhibitory trend, and this effect than the administration of Jiawei Sini Decoction Group; and Sini San Mao You time-dose administration than Maoshi than Youshi administration.
Changes in sucrose preference, the prescription of Jiawei Sini San Mao Shi, You Shi, Mao You times and melatonin morning, evening administration can reduce the escend of sucrose preference caused by chronic stress.
According to the result liver recipe Sini San Mao Shi, You Shi, Mao You timeshare and positive drug melatonin morning, evening administration all take an antidepressant effects.
3. Changes in body temperature and hormonal neurotransmitter by CMUS
For assay of the four indicators-body temperature, serum 5-HT, melatonin and serum corticosterone levels. The results showed that in normal circumstances, the rats showed circadian rhythm of body temperature, in which light phase (daytime) temperature low, dark phase (night) high body temperature, day and night temperature fluctuation range (0.2-0.6)℃. CMUS could lead to an average of 24 hours temperature drops, fluctuations become larger, phase postponed.
Serum of 5-hydroxytryptamine, melatonin, corticosterone levels also exist changes in circadian rhythm, all three showed similar fluctuations, light phase(day)is lower, the dark phase(night)were higher. In chronic stress, the experimental rats showed a decline in 5-HT and melatonin level, and volatility reduced, rhythm also weakened or even reversed. Corticosterone levels were elevated, volatility reduced, rhythm and phase of a significant weakening in advance.
4. Positive inference mechanism of antidepressant drugs melatonin
Experimental results show that in the regulation of body temperature caused by chronic stress and hormonal changes in neurotransmitters, the melatonin morning treatment group showed a regulation of body temperature and the amplitude of 5-HT, corticosterone phase, but no significant change to the content. It also can adjust 5-HT, CORT secretion phase closer to control group. Evening melatonin group showed on the temperature amplitude, phase adjustment, and regulation of 5-HT, corticosterone levels. So we can extrapolate positive drug melatonin rhythm in the regulation of time to achieve antidepressant effects.
5. Prescription anti-depressant mechanism of Jiawei Sini Decoction
For'the changes in body temperature and hormone regulation of neurotransmitters, caused by chronic stress:Mao Shi group, mainly through changes in rhythm to achieve antidepressant effects. Manifested on the body temperature,5-HT and melatonin secretion phase, while almost no effect on its content. You Shi administration, mainly through changes in hormone secretion to achieve antidepressant effects. Mao You antidepressant effect of time-sharing administration is by changing the hormone secretion and adjust its role both in terms of rhythm.
It follows that, for Jiawei Sini Decoction speaking, Mao Shi administered mainly by adjusting the rhythm of time to achieve antidepressant effects, and Youshi administration mainly by adjusting the amount of hormone secretion of neurotransmitters to achieve antidepressant effects, Mao You are also from the adjustment of time-dose hormone secretion of neurotransmitters and adjust the time to play both rhythm antidepressant effect. Conclusions
1. CMUS can bring the disorder of circadian rhythm, including body temper ature level reduce, band increase and phase delay; serum 5-hydroxytryptam ine, melatonin levels decrease, rhythm weakened, phase advance; serum corti costerone levels increased, rhythm weakened and phase advance.
2. Recipe Jiawei Sini San and melatonin treatment groups had significant antidepressant effect
3. Positive control drug melatonin mainly in regulation of time to achieve antidepressant effects
4. Liver Decoction Jiawei Sini Decoction Mao Shi administration, mainly through changes in rhythm to achieve antidepressant effects
5. Liver Decoction Jiawei Sini Decoction Youshi administration, mainly through changes in hormone secretion to achieve antidepressant effects
6. Liver Decoction Jiawei Sini Decoction Maoyou administration by both changing the hormone secretion and to adjust its rhythm to achieve antidepressant effects
引文
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[8]Yasukazu Nakahata, Benedetto Grimaldi, Saurabh Sahar, Jun Hirayama and Pa olo Sassone-Corsi. Current Opinion in Cell Biology,2007,19:230-237
[9]钟历勇.松果体腺、褪黑素与抗应激作用[J].国外医学·生理、病理科学与临床分册,2003；23(5)：522-524
[10]简坤林,陈槐卿,宋开源等.电刺激对军人血浆褪黑激素昼夜节律的影响[J].生物医学工程学杂志,2003；20(3)：494-496
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[13]朱列和,许崇涛.睡眠剥夺抑郁模型大鼠血清皮质酮和行为学的改变[J].中国临床康复,2005；9(32)：157
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[16]王艳红,文勇立,齐莎日娜等.哺乳动物生物钟模型及研究进展[J].四川生理科学杂志,2006；28(1)：33-35
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[21]Henry Olders. Average sunrise time predicts depression prevalence. Jou rnal of Psychosomatic Research.,2003,55:99-105
[22]Bhattacharjee Y. Psychiatric research. Is internal timing key to mental health?Science,2007,317 (5844):1488-1490
[23]Remy Chenevard, Yves Suter, Paul Erne. Effects of the heart-lung machine on melatonin metabolism and mood disturbances. European Journal of Cardio-thoracic Surgery,2008,34:338-343
[24]Katarina Dodig-Curkovic, Sven Kurbel, Vesna Cacinovic Matic. Can inci dence of depression in women be linked to estrogen dependent secretion of various hormone binding proteins?. Medical Hypotheses,2009,72:211-212
[25]严灿,徐志伟.肝主疏泄条畅情志功能的中枢神经生物学机制探讨[J].中国中西医结合杂志,2005；25(5)：459-462
[26]Sergio Damian Paredes, Soledad Sanchez, Ruben Victor Rial, Ana Beatriz R odriguez, Carmen Barriga. Changes in behaviour and in the circadian rhythm s of melatonin and corticosterone in rats subjected to a forced-swimming test. Journal of APPLIED BIOMEDICINE,2005,3:47-57
[27]Kentarou Ushijima, Takuya Morikawa, Hideto To, Shun Higuchi, Shigehiro Oh do. Chronobiological disturbances with hyperthermia and hypercortisolism induced by chronic mild stress in rats. Behavioural Brain Research,2006,17 3:326-330
[28]吴丽丽.运用肝主疏泄理论防治应激性抑郁症的基础研究.广州中医药大学博士论文.中国期刊网博士论文全文数据库.14-20
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[1]Paul Willner, David Benton, Emma Brown, et. al. Depression. increases. cravin g. for sweet rewards in animal and human models of depression and craving. Psychopharmacology,1998;136:272-283
[2]刘芳娥,刘利兵,陈健康等.褪黑素对急性睡眠剥夺大鼠行为的影响及机制[J].第四军医大学学报,2006;27(5)：434
[3]马东,董文心.应激与抑郁症发病的相关性及新药研究进展[J].世界临床药物,2009; 30(9)： 557
[4]Garcia R. Stress, meta plasticity, and anti ressants. Curr MolMed[J],2002, 2:629-638
[5]Naida F. FORBES, Caroline A. STEWART. Chronic Mild Stress snd Sucrose Con sumption:Validity as a Model of Depression. Physiology&Behavior[J], Vol.60, No.6, pp.1481-1484,1996
[1]杨波,刘延友,李颖.生物节律测控和分析系统的研制及应用[J].四川大学学报,2006；37(3)：447
[2]王凌.国外时间生物学进展[J].生物医学工程学杂志,2005；22(11)：185-188
[3]孟小明,王惠玉.可见光疗法对24h生物节律周期的影响及临床应用[J].中国康复,2003；18(6)：383
[4]祝恒琛.中医时辰治疗学[M],北京：华夏出版社,1998,第一版；129-132
[1]World bank. Global economic prospects and the developing countries[M]. Washington, DC:world bank,1993.
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[3]李辉,张建球,赵后峰等.城市人群抑郁症流行病学调查[J].临床心身疾病杂志,2009：15(2)：134
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[6]Johnson MS. Activity and distribution of certain wild mice in relation to biotic communities Journal of Mammalogy,1926;7(4):245-277.
[7]Dunlap JC Loros JJ Merrow M The genetic and molecular dissection of a prototypic circadian system Prog Brain Res 1996;111;11-27.
[8]Yasukazu Nakahata, Benedetto Grimaldi, Saurabh Sahar, Jun Hirayama and Pa olo Sassone-Corsi. Current Opinion in Cell Biology,2007,19:230-237
[9]钟历勇.松果体腺、褪黑素与抗应激作用[J].国外医学·生理、病理科学与临床分册,2003；23(5)：522-524
[10]简坤林,陈槐卿,宋开源等.电刺激对军人血浆褪黑激素昼夜节律的影响[J].生物医学工程学杂志,2003；20(3)：494-496
[11]王正荣,王浴生.时间生物学与时间药理学[J].四川生理科学杂志,1997；19(1)：1-5
[12]李俊旭,郑继旺,梁建辉.脑源性白介素的研究进展[J].中国药理学通报,2003；19(3)：253-256
[13]朱列和,许崇涛.睡眠剥夺抑郁模型大鼠血清皮质酮和行为学的改变[J].中国临床康复,2005；9(32)：157
[14]Luis San, Belen Arranz. Agomelatine:A novel mechanism of antidepressan t action involving the melatonergic and the serotonergic system. European Psychiatry,2008,23:396-402
[15]Stefania Maccari,Sara Morley-Fletcher. Effects of prenatal restraint stress on the hypothalamus-pituitary-adrenal axis and related behavioural and neurobiological alterations. Psychoneuroendocrinology,2007,32:510-515
[16]王艳红,文勇立,齐莎日娜等.哺乳动物生物钟模型及研究进展[J].四川生理科学杂志,2006；28(1)：33-35
[17]Cinnamon Stetler, Sally S. Dickerson, Gregory E. Miller. Uncoupling of soc ial zeitgebers and diurnal cortisol secretion in clinical depression. Psyc honeuroendocrinology,2004,29:1250-1259
[18]David Morawetz. Insomnia and Depression:Which Comes First?. Sleep Resea rch Online,2003,5(2):77-81
[19]祝恒琛.中医时辰治疗学[M],北京：华夏出版社,1998,第一版
[20]David, J. Kupfger. Depression and associated sleep disturbances:patient benefits with agomelatine. European Neuropsychopharmacology,2006,16:5639-5643
[21]Henry Olders. Average sunrise time predicts depression prevalence. Jou rnal of Psychosomatic Research.,2003,55:99-105
[22]Bhattacharjee Y. Psychiatric research. Is internal timing key to mental health?Science,2007,317 (5844):1488-1490
[23]Remy Chenevard, Yves Suter, Paul Erne. Effects of the heart-lung machine on melatonin metabolism and mood disturbances. European Journal of Cardio-thoracic Surgery,2008,34:338-343
[24]Katarina Dodig-Curkovic, Sven Kurbel, Vesna Cacinovic Matic. Can inci dence of depression in women be linked to estrogen dependent secretion of various hormone binding proteins?. Medical Hypotheses,2009,72:211-212
[25]严灿,徐志伟.肝主疏泄条畅情志功能的中枢神经生物学机制探讨[J].中国中西医结合杂志,2005；25(5)：459-462
[26]Sergio Damian Paredes, Soledad Sanchez, Ruben Victor Rial, Ana Beatriz R odriguez, Carmen Barriga. Changes in behaviour and in the circadian rhythm s of melatonin and corticosterone in rats subjected to a forced-swimming test. Journal of APPLIED BIOMEDICINE,2005,3:47-57
[27]Kentarou Ushijima, Takuya Morikawa, Hideto To, Shun Higuchi, Shigehiro Oh do. Chronobiological disturbances with hyperthermia and hypercortisolism induced by chronic mild stress in rats. Behavioural Brain Research,2006,17 3:326-330
[28]吴丽丽.运用肝主疏泄理论防治应激性抑郁症的基础研究.广州中医药大学博士论文.中国期刊网博士论文全文数据库.14-20
[29]张承龙等.中式人体生物钟概论[M],北京：中医古籍出版社,2006,第一版
[30]杨晓林.《内经》中因时针刺初探[J].湖南中医学院学报,1995;15(2)：13-14
[31]姚成增,胡剑北.右归饮择时使用对肾阳虚大鼠睾酮昼夜节律的影响[J].上海中医药杂志,2005：39(2)：53-55
[1]Paul Willner, David Benton, Emma Brown, et. al. Depression. increases. cravin g. for sweet rewards in animal and human models of depression and craving. Psychopharmacology,1998;136:272-283
[2]刘芳娥,刘利兵,陈健康等.褪黑素对急性睡眠剥夺大鼠行为的影响及机制[J].第四军医大学学报,2006;27(5)：434
[3]马东,董文心.应激与抑郁症发病的相关性及新药研究进展[J].世界临床药物,2009; 30(9)： 557
[4]Garcia R. Stress, meta plasticity, and anti ressants. Curr MolMed[J],2002, 2:629-638
[5]Naida F. FORBES, Caroline A. STEWART. Chronic Mild Stress snd Sucrose Con sumption:Validity as a Model of Depression. Physiology&Behavior[J], Vol.60, No.6, pp.1481-1484,1996
[1]杨波,刘延友,李颖.生物节律测控和分析系统的研制及应用[J].四川大学学报,2006；37(3)：447
[2]王凌.国外时间生物学进展[J].生物医学工程学杂志,2005；22(11)：185-188
[3]孟小明,王惠玉.可见光疗法对24h生物节律周期的影响及临床应用[J].中国康复,2003；18(6)：383
[4]祝恒琛.中医时辰治疗学[M],北京：华夏出版社,1998,第一版；129-132