肝应春理论探讨及其调控机制的实验研究
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摘要
“五脏应时”理论是中医“天人相应”理论的重要内容,是中医学从时间医学角度对藏象学说的构建和发展。所以,基于“天人相应”理论,从时间结构认识藏象实质已成为藏象理论研究的重要途径。“肝应春”理论是“五脏应时”理论的重要组成部分,正确认识肝的藏象实质、了解肝“应时而变”的适应性调控机制是“肝应春”理论研究的重要内容,也是深化和创新肝藏象学说的关键环节。
1目的
基于“天人相应”理论,以褪黑素(MT)为切入点,以中医“肝应春”适应性调控机制为研究对象,通过对“肝应春”理论内涵的探讨以及“肝应春”调控机制与MT及其合成限速酶AANAT季节相关性的实验研究,观察MT以及AANAT的季节性变化规律,从时间医学角度,揭示中医“肝应春”的理论内涵及其适应性调控机制的生物学基础,为某些季节性肝系疾病的防治提供理论和实验依据。
2方法
2.1理论研究
利用文献学和逻辑分析的研究方法,对“肝应春”理论源流进行梳理,从肝藏血主疏泄的功能探讨“肝应春”理论的科学内涵,提出“肝应春”适应性调控机制的理论假说。并进一步对“肝应春”调控机制的中医理论内涵以及MT在神经内分泌调控中枢中的作用进行逻辑分析,探讨“肝应春”调控机制的生物学基础。
2.2实验研究
2.2.1实验设计
本实验以肝藏血主疏泄在“肝应春”调控机制中的作用特点与光照在四季变化规律之间的关系为切入点,选择受光照调节具有季节性变化规律的MT为研究对象,观察在生理状态下,仓鼠血清MT含量四季的变化规律,并观察对MT合成与分泌具有限制性作用的AANAT在松果腺、下丘脑和垂体不同层次中的蛋白质水平以及mRNA表达的四季变化规律,分析其相关性。同时,利用人工模拟气候箱反季节模拟四季气候,对模拟气候与自然气候下上述指标进行比较,观察模拟气候与自然气候的差异性。
2.2.2实验动物
雄性仓鼠(叙利亚金黄地鼠),分别在春分、夏至、秋分、冬至前35天购入,适应性饲养1周,随机分为自然季节组和模拟季节组,每组8只。自然季节组采用自然光照、室温,湿度。模拟季节组利用人工气候模拟箱,模拟自然气候,为了排除自然气候变化对模拟气候的影响,采用反季节模拟的方式,即春季时模拟秋季,夏季时模拟冬季,秋季时模拟春季,冬季时模拟夏季。以光照、温度、湿度为主要模拟条件,在光照上,参照各季节天安门升降旗时间人工模拟昼夜变化；在温度上,根据前期实验经验以各季节的平均温度为标准；湿度是以北京气象台各月发布湿度,按季节取平均值。各季节自然气候组和模拟气候组分别至春分、夏至、.秋分和冬至断头处死,取样。
2.2.3检测方法
血清MT含量测定,采用125I标记放射免疫法；AANAT蛋白质水平测定,采用western blot法：AANAT mRNA表达,采用荧光实时定量PCR (Real-time PCR)法。
2.2.4实验结果统计学分析方法
所有数据以x±S表示,组间差异利用SPSS17.0统计软件进行ANOVA检验,两组间比较采用LSD法和t-检验进行统计分析,以P<0.05表示有显著性差异。
3结果
3.1理论研究结果
3.1.1提出“肝应春”理论的内涵是
在当旺的春季,肝的疏泄功能增强,藏血功能减弱发挥着对自身肝系统及其他四脏重要的调控作用。在其他季节肝藏血主疏泄的功能应时而变，促进或抑制其他四脏以维持机体应时而变的调节稳态。
3.1.2提出“肝应春”调控机制与神经内分泌系统密切相关
本研究通过对肝藏血主疏泄功能的现代生物学研究分析,发现“肝应春”调控机制与神经内分泌系统具有相关性,并提出了从与神经内分泌系统的调控中枢松果腺(MT)-下丘脑-垂体的角度来研究“肝应春”的调控机制的研究思路。
3.2实验研究结果
3.2.1四季气候变化对仓鼠血清褪黑素的影响
在自然四季变化下,仓鼠血清MT在各季节之间均有显著性差异,在四季呈现出冬>春>秋>夏的变化规律。
3.2.2四季气候变化对AANAT蛋白质水平的影响
在自然四季变化下,AANAT蛋白质水平在松果腺夏与秋之间无显著性差异(P>0.05),在下丘脑冬与春之间无显著性差异(P>0.05),在垂体秋与春之间无显著性差异(P>O.05),其他各季节之间均有显著性差异(P春>秋>夏的变化趋势,与血清MT的季节变化规律一致,在垂体中呈现冬>秋>春>夏的变化趋势,与血清MT的季节变化在春、秋两季的趋势不一致；AANAT蛋白质水平在松果腺、下丘脑、垂体同一季节中呈现出下丘脑>垂体>松果腺的趋势。
3.2.3四季气候变化对AANAT mRNA表达的影响
在自然四季变化下,AANAT mRNA表达在松果腺春与夏、春与秋、夏与秋无显著性差异(P>0.05),在下丘脑春与夏、冬与秋之间无显著性差异(P>0.05),在垂体春与秋、春与冬之间无显著性差异(P>0.05),其它各季节之间均有显著性差异(P<0.05),AANAT mRNA表达在松果腺、垂体中的四季变化趋势呈现冬>春>秋>夏,与血清MT的季节变化趋势一致,在下丘脑中四季呈冬>秋>春>夏的变化趋势,与血清MT的季节变化在春、秋的变化趋势不一致。
3.2.4模拟四季气候对血清褪黑素的影响
在模拟气候变化下,血清MT春与秋、冬之间无显著性差异(P>0.05),其它各季节之间均有显著性差异(P<0.05),四季变化趋势与自然季节变化冬>春>秋>夏一致。与自然气候同季节相比,各组间无显著性差异。模拟与自然反季节相比,模拟春与自然冬、模拟冬与自然夏之间有显著性差异(P<0.05)。
3.2.5模拟四季气候对AANAT蛋白质水平的影响
在模拟气候变化下,AANAT蛋白质水平在松果腺、下丘脑、垂体各季节之间均有显著性差异(P<0.05)。在松果腺四季呈冬>春>夏>秋的变化趋势,与自然气候在夏、秋两季变化趋势不一致；在下丘脑呈冬>秋>春>夏的变化趋势,与自然气候在春、秋两季变化趋势不一致；在垂体呈冬>秋>春>夏的变化趋势,与自然气候变化趋势一致。模拟与自然同季节相比,AANAT蛋白质水平在松果腺、下丘脑表现为模拟秋与自然秋无显著性差异(P>0.05),其余各组间均有显著性差异(P<0.05)；在垂体模拟与自然同季节之间均无显著性差异(P>0.05)。模拟与自然反季节相比,AANAT蛋白质水平在垂体模拟秋与自然春无显著性差异(P>0.05),其余各组间以及在松果腺、下丘脑各组间均具有显著性差异(P<0.05)。
3.2.6模拟四季气候对AANAT mRNA表达的影响
在模拟气候下,AANAT mRNA表达在松果腺秋与春、夏无显著性差异(P>0.05)。在下丘脑春与夏、秋与冬无显著性差异(P>0.05)；在垂体春和夏无显著性差异(P>0.05),其余各模拟季节组间均有显著性差异(P<0.05)。模拟与自然同季节相比,在松果腺均无显著性差异(P>0.05)；在下丘脑模拟春与自然春、模拟秋与自然秋之间有显著性差异(P<0.05)；在垂体模拟春与自然春、模拟冬与自然冬有显著性差异(P<0.05)。模拟与自然反季节相比,在松果腺模拟春与自然秋无显著性差异(P>0.05)；在垂体模拟秋与自然春无显著性差异,在其他各组间以及下丘脑各组间均具有显著性差异(P<0.05),AANAT mRNA表达在各组织中四季的变化趋势与自然气候变化趋势均一致。
4结论
4.1“肝应春”的适应性调控机制是以肝藏血主疏泄的功能为生理基础,MT在四季中的变化规律与肝主疏泄功能在季节上的强弱变化规律相反,与肝藏血功能在季节上的强弱变化规律一致,可以认为MT具有抑制肝主疏泄的功能,并促进肝藏血的功能,MT季节性变化可能是“肝应春”适应性调控机制之一。
4.2褪黑素合成限速酶AANAT在MT的合成与分泌的冬夏季节变化具有调节作用,而不存在明显的四季的调控作用,可能AANAT并不是血清MT季节性变化的限速酶。
4.3人工模拟箱较成功的模拟出四季气候的变化规律,但模拟气候与自然气候仍然存在差异。提示在模拟自然气候时只考虑光照时间、温度、湿度这些指标可能不能如实反映出自然气候的特点,人作为整个自然界的一份子感受着大自然的信息是各种各样的,利用人工气候模拟箱研究“五脏应时”理论,可能还需要在模拟参数上加以完善,使之更接近于自然气候的特点。
The theory of "five zang-viscera corresponding to four seasons" is an important part of the theory of "correspondence between man and nature" in TCM. It is the construction and development of the theory of viscera manifestation from the chronomedicine point of view. Therefore, based on the theory of "correspondence between man and nature", to understand the essence of viscera from the time structure has become an important way of the theoretical research of viscera manifestation. The theory of "the liver corresponding to spring " is an important part of the "five zang-viscera corresponding to four seasons". To correctly understand the essence of the liver, and understand the adaptive control mechanism of liver is an important part of the theoretical research on the theory of "liver corresponding to the spring. as well as key link of deepening and innovating the theory of liver viscus manifestation.
1.Objective
Based on the overall concept of the theory of "the corresponding between man and nature". this paper takes the timing factor of melatonin as the entry point, and adaptability regulation mechanism as the research object, and through the discussion of the connotation of the theory of the "the liver corresponding to spring" as well as its regulation and control mechanism with the melatonin and its synthesis rate-limiting enzyme seasonal correlative experiment of research, it observes the seasonal variations of the melatonin and its synthesis rate-limiting enzyme, and from the medical point of view, it reveals the essential connotation of the liver viscus in TCM and the biological basis of the adaptive regulation mechanism of "liver corresponding to spring" in TCM, which provides theoretical and experimental basis for the prevention and treatment of some seasonal liver disease.
2. Method
2.1Theoretical Research
By using philology and logic analysis, This paper sorts the theoretical origin of the theory of "liver corresponding to spring". It explores the scientific meaning of "liver corresponds to spring" from the liver" function of governing dispersion and raises the hypothesis of adaptability regulatory mechanism of "liver corresponding to Spring". It further carries out the logical analysis on the connotation of theory of the regulatory mechanism of liver corresponding to the spring and the regulation mechanism of melatonin in the neuroendocrine-immune network, and explores the biological basis of the regulatory mechanism of liver Spring.
2.2Experimental Research
2.2.1Experimental Design
This experiment takes the relationship between the functions of liver in storing blood and governing dispersion in "liver corresponding to spring" regulatory mechanism and the light in changing seasons as the starting point, it selected melatonin which has seasonal variation as the study object, and observes the seasonal changing law of the hamster serum melatonin (Mel) content in the physiological state, and observes the protein levels of AANAT which has the restrictive role on melatonin synthesis and secretion of the pineal gland, and mRNA expression in the hypothalamus and pituitary gland at different levels in the changing seasons, and analyzes their relevance. Meanwhile, it takes use of artificial simulated climate chamber to imitate the climates of four seasons to observe and imitate the influence of climate on the above indicators and compares it with the natural climates.
2.2.2Experimental animals:
Hamster (Syrian golden hamster), male,20-month-old,120-130g/only. They were purchased35days before the spring equinox, summer solstice, autumnal equinox and the winter solstice. After acclimatization for a period of one week, they were randomly divided into two groups:natural seasonal group and analog seasonal group (n=8). The natural seasonal group are equipped with natural lighting, room temperature, and humidity. Analog seasonal group use artificial climate modeling box to simulate the natural climate-season simulation. In order to exclude the impact of natural climate variability on climate modeling, it takes anti-season simulation, that is, in natural spring, it imitates autumn, in natural summer, it imitates winter, and in natural autumn, it imitates spring, and in natural winter, it imitates summer. Light, temperature, humidity are main simulated conditions. In lighting, it is according to the four Seasons circadian light duration variation with reference to flag raising and lowering time of each season at Tiananmen Square; in temperature, it is based on previous experiments experience on the average temperature of each season as a standard; in the humidity, it is based on the averaged humidity released in the Beijing Meteorological Observatory each month. Different seasonal groups of animals take free water and feed intake with common pellet feed for mice. Group of natural climate and climate modeling group are decapitated and sampling on the vernal equinox, summer solstice, autumnal equinox and the winter solstice.
2.2.3Test Method
Serum melatonin content determination takes125I mark radioimmunoassay method; melatonin synthesis rate-limiting enzyme AANAT protein levels were measured by using the Western blot method; rate-limiting enzyme AANAT mRNA expression takes quantitative real-time PCR.
2.2.4Experimental results statistical analysis methods
All data are expressed by x±S, the difference between the groups takes the statistical software SPSS17.0ANOVA test. Comparison between the two groups takes LSD method and t-test for statistical. P<0.05difference as significance.
3. Result
3.1Theoretical Research Result
3.1.1It raises the connotation of the theory of'"the liver corresponding to spring "is:in spring, the liver's function in dispersion enhances, its function in storing blood weakens, which plays an important role in regulating the system of liver and other four zang-viscera. In other seasons, the liver's function in storing blood and governing dispersion changes from time to time to promote or inhibit other four zang-viscera in order to maintain the body regulating the steady-state.
3.1.2The study, through modern biological research on the liver's function in storing blood and governing dispersion, found that the regulation mechanism "the liver corresponding to spring" has correlation with neuroendocrine system, and proposes research ideas on researching "the liver corresponding to Spring" regulation mechanism from the angle of the central regulation of the neuroendocrine system gland-melatonin.3.2Experimental Research Result3.2.1The influence of climate change on the hamster serum melatonin
During seasonal succession, the melatonin levels assayed in hamster serum in each season showed significant difference to others with a fluctuation mode as winter>spring>autumn>summer. Among them, the "summer" level fell to the lowest point while in winter it reached the peak.
3.2.2Influence of season alternation on the level of ANNAT protein
With the season alternation, the levels of AANAT protein in pineal gland showed no significant difference between hamsters in summer and autumn (P>0.05); those in hypothalamus manifested no significant differences between hamsters in winter and spring (P>0.05); and those in pituitary gland exhibited no significant differences between hamsters in autumn and spring (P>0.05). Significant differences can be observed between each pair of seasons other than the above mentioned (P<0.05). The level fluctuation of AANAT protein in pineal gland and/or hypothalamus were observed as winter>spring>autumn>summer, which was inconsistent with the level changes of serum melatonin along season alternation in spring and autumn. The level curve of AANAT protein in a given season was observed, from large to small, as hypothalamus, then pituitary gland and then pineal gland.
3.2.3Influence of climate changes during season alterantion on AANAT mRNA
Along with season alternation, the expressions of AANAT mRNA in pineal gland showed no significant differences between hamsters in spring and summer, spring and autumn and summer and autumn (P>0.05); those in hypothalamus showed no significant differences between hamsters in spring and summer and between winter and autumn (P>0.05); and those in pituitary gland exhibited no significant differences between hamsters in spring and autumn, and spring and winter (P>0.05). Significant differences can be observed between each pair of seasons other than the above mentioned (P<005). The expressions of AANAT mRNA in pineal gland and/or pituitary gland showed a fluctuation mode as winter> spring>autumn>summer. This corresponded to how serum melatonin fluctuates along seasons. In hypothalamus, the expressions kept fluctuates by winter>spring>autumn>summer, which was inconsistent with how serum melatonin changes in spring and autumn.
3.2.4Influence of season models on serum melatonin
On the analog of climate changes, the serum melatonin presents no significant difference (P>0.05) in the spring and autumn, spring and winter, but it presents significant differences (P<0.05) between other seasons. The trend is winter> spring> autumn> summer, which is in accordance with the trend of natural seasonal variations. Compared to natural climate showed no statistically significant differences.In the comparisons between modeled seasons to their respective opposite seasons, there is significant difference (P<0.05) between the modeled summer and natural winter, modeled winter and natural summer.
3.2.5Influences of season models on the expression of AANAT protein
The level of AANAT protein in pineal gland, hypothalamus and pituitary gland presented significant seasonal variability (P<0.05). Compared hamsters in settings of natural autumn and the modeled one, the levels of AANAT in pineal gland or in hypothalamus exhibited no statistically significant differences (P>0.05); while the AANAT levels in the other comparisons between the hamsters in natural and model seasons showed statistically significant difference (P<0.05); in addition, the AANAT levels in the hamsters'pituitary gland in such nature-to-model comparisons showed no significant difference (P<0.05). In the comparison between hamsters in a modeled season to its opposite modeled season (e.g. spring is opposite to autumn and summer is to winter), there showed no significant difference of AANAT levels in pituitary gland between natural and modeled autumn (P>0.05), while all the other such comparisons of AANAT levels in pituitary gland, pineal gland or hypothalamus showed significant difference (P<0.05). The changing of AANAT levels in hypothalamus determined in the modeled annual succession of seasons was observed to be consistent with those in the natural ones, while the only exception-hypothalamus presented a level fluctuation of AANAT as winter>spring>autumn>summer.
3.2.6Influence of modeled seasons on the expression of AANAT mRNA
In the setting of modeled seasons, the expression of AANAT mRNA in pineal gland presented no significant seasonal variability between spring and autumn, and between summer and autumn (P>00);its expression in hypothalamus displayed no significant seasonal variability between spring and summer, and between winter and autumn (P>0.05); its expression in pituitary gland exhibited no significant seasonal variability between spring and summer; other than these, all the comparisons between modeled seasons showed significant differences (P<0.05). In the comparisons between modeled natural seasons, the expressions in pineal gland manifested no significant differences (P>0.05); the expressions in hypothalamus between modeled and natural spring/autumn showed significant differences (P<0.05); the expressions in pituitary gland between modeled and natural spring/winter exhibited significant differences (P<005). In the comparisons between modeled seasons to their respective opposite seasons, there showed no significant difference of the expression in pineal gland between modeled spring and natural autumn (P>0.05) and in pituitary gland between modeled autumn and natural spring; in all the other comparisons including those involved hypothalamus showed significant differences (P>0.05). The changing tendencies of expressions in AANAT mRNA in all three tissues were consistent with those in the setting of natural seasons.
4Conclusion
4.1The adaptive control mechanism "liver corresponding to spring" takes the function of liver in storing blood and governing dispersion as the physiological basis. The variation of melatonin in the four seasons is contrary to the variation of the function of liver in dispersion, but is in accordance with the function of liver in storing blood. It can be considered that melatonin has the function of suppressing the liver in dispersion, but promotes the function of the liver in storing blood. The seasonal change of melatonin may be one of the adaptive control mechanism of the "liver corresponding to spring"
4.2melatonin synthesis rate-limiting enzyme AANAT has a regulatory role in melatonin synthesis and secretion in the seasonal changes of the summer and winter, and there is no obvious regulating effect in the synthesis and secretion of four seasons, AANAT is not the rate-limiting enzyme of the seasonal changes of serum melatonin.
4.3Artificial simulation box successfully simulated climate changes of the four seasons, but there are still differences between the modeled climate and natural climate. It indicates that in modeled climate, these indicators such as the exposure time, temperature, humidity are only considered, which may not accurately reflect the characteristics of the natural climate. As a part ofthe whole of nature, people feel the information of the nature in a wide variety. To take use of artificial climate simulation box to study the theory of "five zang-viscera corresponding to seasons", adjustment on the simulation parameters may also needed to be made to be closer to the characteristics of the natural climate.
1目的
基于“天人相应”理论,以褪黑素(MT)为切入点,以中医“肝应春”适应性调控机制为研究对象,通过对“肝应春”理论内涵的探讨以及“肝应春”调控机制与MT及其合成限速酶AANAT季节相关性的实验研究,观察MT以及AANAT的季节性变化规律,从时间医学角度,揭示中医“肝应春”的理论内涵及其适应性调控机制的生物学基础,为某些季节性肝系疾病的防治提供理论和实验依据。
2方法
2.1理论研究
利用文献学和逻辑分析的研究方法,对“肝应春”理论源流进行梳理,从肝藏血主疏泄的功能探讨“肝应春”理论的科学内涵,提出“肝应春”适应性调控机制的理论假说。并进一步对“肝应春”调控机制的中医理论内涵以及MT在神经内分泌调控中枢中的作用进行逻辑分析,探讨“肝应春”调控机制的生物学基础。
2.2实验研究
2.2.1实验设计
本实验以肝藏血主疏泄在“肝应春”调控机制中的作用特点与光照在四季变化规律之间的关系为切入点,选择受光照调节具有季节性变化规律的MT为研究对象,观察在生理状态下,仓鼠血清MT含量四季的变化规律,并观察对MT合成与分泌具有限制性作用的AANAT在松果腺、下丘脑和垂体不同层次中的蛋白质水平以及mRNA表达的四季变化规律,分析其相关性。同时,利用人工模拟气候箱反季节模拟四季气候,对模拟气候与自然气候下上述指标进行比较,观察模拟气候与自然气候的差异性。
2.2.2实验动物
雄性仓鼠(叙利亚金黄地鼠),分别在春分、夏至、秋分、冬至前35天购入,适应性饲养1周,随机分为自然季节组和模拟季节组,每组8只。自然季节组采用自然光照、室温,湿度。模拟季节组利用人工气候模拟箱,模拟自然气候,为了排除自然气候变化对模拟气候的影响,采用反季节模拟的方式,即春季时模拟秋季,夏季时模拟冬季,秋季时模拟春季,冬季时模拟夏季。以光照、温度、湿度为主要模拟条件,在光照上,参照各季节天安门升降旗时间人工模拟昼夜变化；在温度上,根据前期实验经验以各季节的平均温度为标准；湿度是以北京气象台各月发布湿度,按季节取平均值。各季节自然气候组和模拟气候组分别至春分、夏至、.秋分和冬至断头处死,取样。
2.2.3检测方法
血清MT含量测定,采用125I标记放射免疫法；AANAT蛋白质水平测定,采用western blot法：AANAT mRNA表达,采用荧光实时定量PCR (Real-time PCR)法。
2.2.4实验结果统计学分析方法
所有数据以x±S表示,组间差异利用SPSS17.0统计软件进行ANOVA检验,两组间比较采用LSD法和t-检验进行统计分析,以P<0.05表示有显著性差异。
3结果
3.1理论研究结果
3.1.1提出“肝应春”理论的内涵是
在当旺的春季,肝的疏泄功能增强,藏血功能减弱发挥着对自身肝系统及其他四脏重要的调控作用。在其他季节肝藏血主疏泄的功能应时而变，促进或抑制其他四脏以维持机体应时而变的调节稳态。
3.1.2提出“肝应春”调控机制与神经内分泌系统密切相关
本研究通过对肝藏血主疏泄功能的现代生物学研究分析,发现“肝应春”调控机制与神经内分泌系统具有相关性,并提出了从与神经内分泌系统的调控中枢松果腺(MT)-下丘脑-垂体的角度来研究“肝应春”的调控机制的研究思路。
3.2实验研究结果
3.2.1四季气候变化对仓鼠血清褪黑素的影响
在自然四季变化下,仓鼠血清MT在各季节之间均有显著性差异,在四季呈现出冬>春>秋>夏的变化规律。
3.2.2四季气候变化对AANAT蛋白质水平的影响
在自然四季变化下,AANAT蛋白质水平在松果腺夏与秋之间无显著性差异(P>0.05),在下丘脑冬与春之间无显著性差异(P>0.05),在垂体秋与春之间无显著性差异(P>O.05),其他各季节之间均有显著性差异(P
3.2.3四季气候变化对AANAT mRNA表达的影响
在自然四季变化下,AANAT mRNA表达在松果腺春与夏、春与秋、夏与秋无显著性差异(P>0.05),在下丘脑春与夏、冬与秋之间无显著性差异(P>0.05),在垂体春与秋、春与冬之间无显著性差异(P>0.05),其它各季节之间均有显著性差异(P<0.05),AANAT mRNA表达在松果腺、垂体中的四季变化趋势呈现冬>春>秋>夏,与血清MT的季节变化趋势一致,在下丘脑中四季呈冬>秋>春>夏的变化趋势,与血清MT的季节变化在春、秋的变化趋势不一致。
3.2.4模拟四季气候对血清褪黑素的影响
在模拟气候变化下,血清MT春与秋、冬之间无显著性差异(P>0.05),其它各季节之间均有显著性差异(P<0.05),四季变化趋势与自然季节变化冬>春>秋>夏一致。与自然气候同季节相比,各组间无显著性差异。模拟与自然反季节相比,模拟春与自然冬、模拟冬与自然夏之间有显著性差异(P<0.05)。
3.2.5模拟四季气候对AANAT蛋白质水平的影响
在模拟气候变化下,AANAT蛋白质水平在松果腺、下丘脑、垂体各季节之间均有显著性差异(P<0.05)。在松果腺四季呈冬>春>夏>秋的变化趋势,与自然气候在夏、秋两季变化趋势不一致；在下丘脑呈冬>秋>春>夏的变化趋势,与自然气候在春、秋两季变化趋势不一致；在垂体呈冬>秋>春>夏的变化趋势,与自然气候变化趋势一致。模拟与自然同季节相比,AANAT蛋白质水平在松果腺、下丘脑表现为模拟秋与自然秋无显著性差异(P>0.05),其余各组间均有显著性差异(P<0.05)；在垂体模拟与自然同季节之间均无显著性差异(P>0.05)。模拟与自然反季节相比,AANAT蛋白质水平在垂体模拟秋与自然春无显著性差异(P>0.05),其余各组间以及在松果腺、下丘脑各组间均具有显著性差异(P<0.05)。
3.2.6模拟四季气候对AANAT mRNA表达的影响
在模拟气候下,AANAT mRNA表达在松果腺秋与春、夏无显著性差异(P>0.05)。在下丘脑春与夏、秋与冬无显著性差异(P>0.05)；在垂体春和夏无显著性差异(P>0.05),其余各模拟季节组间均有显著性差异(P<0.05)。模拟与自然同季节相比,在松果腺均无显著性差异(P>0.05)；在下丘脑模拟春与自然春、模拟秋与自然秋之间有显著性差异(P<0.05)；在垂体模拟春与自然春、模拟冬与自然冬有显著性差异(P<0.05)。模拟与自然反季节相比,在松果腺模拟春与自然秋无显著性差异(P>0.05)；在垂体模拟秋与自然春无显著性差异,在其他各组间以及下丘脑各组间均具有显著性差异(P<0.05),AANAT mRNA表达在各组织中四季的变化趋势与自然气候变化趋势均一致。
4结论
4.1“肝应春”的适应性调控机制是以肝藏血主疏泄的功能为生理基础,MT在四季中的变化规律与肝主疏泄功能在季节上的强弱变化规律相反,与肝藏血功能在季节上的强弱变化规律一致,可以认为MT具有抑制肝主疏泄的功能,并促进肝藏血的功能,MT季节性变化可能是“肝应春”适应性调控机制之一。
4.2褪黑素合成限速酶AANAT在MT的合成与分泌的冬夏季节变化具有调节作用,而不存在明显的四季的调控作用,可能AANAT并不是血清MT季节性变化的限速酶。
4.3人工模拟箱较成功的模拟出四季气候的变化规律,但模拟气候与自然气候仍然存在差异。提示在模拟自然气候时只考虑光照时间、温度、湿度这些指标可能不能如实反映出自然气候的特点,人作为整个自然界的一份子感受着大自然的信息是各种各样的,利用人工气候模拟箱研究“五脏应时”理论,可能还需要在模拟参数上加以完善,使之更接近于自然气候的特点。
The theory of "five zang-viscera corresponding to four seasons" is an important part of the theory of "correspondence between man and nature" in TCM. It is the construction and development of the theory of viscera manifestation from the chronomedicine point of view. Therefore, based on the theory of "correspondence between man and nature", to understand the essence of viscera from the time structure has become an important way of the theoretical research of viscera manifestation. The theory of "the liver corresponding to spring " is an important part of the "five zang-viscera corresponding to four seasons". To correctly understand the essence of the liver, and understand the adaptive control mechanism of liver is an important part of the theoretical research on the theory of "liver corresponding to the spring. as well as key link of deepening and innovating the theory of liver viscus manifestation.
1.Objective
Based on the overall concept of the theory of "the corresponding between man and nature". this paper takes the timing factor of melatonin as the entry point, and adaptability regulation mechanism as the research object, and through the discussion of the connotation of the theory of the "the liver corresponding to spring" as well as its regulation and control mechanism with the melatonin and its synthesis rate-limiting enzyme seasonal correlative experiment of research, it observes the seasonal variations of the melatonin and its synthesis rate-limiting enzyme, and from the medical point of view, it reveals the essential connotation of the liver viscus in TCM and the biological basis of the adaptive regulation mechanism of "liver corresponding to spring" in TCM, which provides theoretical and experimental basis for the prevention and treatment of some seasonal liver disease.
2. Method
2.1Theoretical Research
By using philology and logic analysis, This paper sorts the theoretical origin of the theory of "liver corresponding to spring". It explores the scientific meaning of "liver corresponds to spring" from the liver" function of governing dispersion and raises the hypothesis of adaptability regulatory mechanism of "liver corresponding to Spring". It further carries out the logical analysis on the connotation of theory of the regulatory mechanism of liver corresponding to the spring and the regulation mechanism of melatonin in the neuroendocrine-immune network, and explores the biological basis of the regulatory mechanism of liver Spring.
2.2Experimental Research
2.2.1Experimental Design
This experiment takes the relationship between the functions of liver in storing blood and governing dispersion in "liver corresponding to spring" regulatory mechanism and the light in changing seasons as the starting point, it selected melatonin which has seasonal variation as the study object, and observes the seasonal changing law of the hamster serum melatonin (Mel) content in the physiological state, and observes the protein levels of AANAT which has the restrictive role on melatonin synthesis and secretion of the pineal gland, and mRNA expression in the hypothalamus and pituitary gland at different levels in the changing seasons, and analyzes their relevance. Meanwhile, it takes use of artificial simulated climate chamber to imitate the climates of four seasons to observe and imitate the influence of climate on the above indicators and compares it with the natural climates.
2.2.2Experimental animals:
Hamster (Syrian golden hamster), male,20-month-old,120-130g/only. They were purchased35days before the spring equinox, summer solstice, autumnal equinox and the winter solstice. After acclimatization for a period of one week, they were randomly divided into two groups:natural seasonal group and analog seasonal group (n=8). The natural seasonal group are equipped with natural lighting, room temperature, and humidity. Analog seasonal group use artificial climate modeling box to simulate the natural climate-season simulation. In order to exclude the impact of natural climate variability on climate modeling, it takes anti-season simulation, that is, in natural spring, it imitates autumn, in natural summer, it imitates winter, and in natural autumn, it imitates spring, and in natural winter, it imitates summer. Light, temperature, humidity are main simulated conditions. In lighting, it is according to the four Seasons circadian light duration variation with reference to flag raising and lowering time of each season at Tiananmen Square; in temperature, it is based on previous experiments experience on the average temperature of each season as a standard; in the humidity, it is based on the averaged humidity released in the Beijing Meteorological Observatory each month. Different seasonal groups of animals take free water and feed intake with common pellet feed for mice. Group of natural climate and climate modeling group are decapitated and sampling on the vernal equinox, summer solstice, autumnal equinox and the winter solstice.
2.2.3Test Method
Serum melatonin content determination takes125I mark radioimmunoassay method; melatonin synthesis rate-limiting enzyme AANAT protein levels were measured by using the Western blot method; rate-limiting enzyme AANAT mRNA expression takes quantitative real-time PCR.
2.2.4Experimental results statistical analysis methods
All data are expressed by x±S, the difference between the groups takes the statistical software SPSS17.0ANOVA test. Comparison between the two groups takes LSD method and t-test for statistical. P<0.05difference as significance.
3. Result
3.1Theoretical Research Result
3.1.1It raises the connotation of the theory of'"the liver corresponding to spring "is:in spring, the liver's function in dispersion enhances, its function in storing blood weakens, which plays an important role in regulating the system of liver and other four zang-viscera. In other seasons, the liver's function in storing blood and governing dispersion changes from time to time to promote or inhibit other four zang-viscera in order to maintain the body regulating the steady-state.
3.1.2The study, through modern biological research on the liver's function in storing blood and governing dispersion, found that the regulation mechanism "the liver corresponding to spring" has correlation with neuroendocrine system, and proposes research ideas on researching "the liver corresponding to Spring" regulation mechanism from the angle of the central regulation of the neuroendocrine system gland-melatonin.3.2Experimental Research Result3.2.1The influence of climate change on the hamster serum melatonin
During seasonal succession, the melatonin levels assayed in hamster serum in each season showed significant difference to others with a fluctuation mode as winter>spring>autumn>summer. Among them, the "summer" level fell to the lowest point while in winter it reached the peak.
3.2.2Influence of season alternation on the level of ANNAT protein
With the season alternation, the levels of AANAT protein in pineal gland showed no significant difference between hamsters in summer and autumn (P>0.05); those in hypothalamus manifested no significant differences between hamsters in winter and spring (P>0.05); and those in pituitary gland exhibited no significant differences between hamsters in autumn and spring (P>0.05). Significant differences can be observed between each pair of seasons other than the above mentioned (P<0.05). The level fluctuation of AANAT protein in pineal gland and/or hypothalamus were observed as winter>spring>autumn>summer, which was inconsistent with the level changes of serum melatonin along season alternation in spring and autumn. The level curve of AANAT protein in a given season was observed, from large to small, as hypothalamus, then pituitary gland and then pineal gland.
3.2.3Influence of climate changes during season alterantion on AANAT mRNA
Along with season alternation, the expressions of AANAT mRNA in pineal gland showed no significant differences between hamsters in spring and summer, spring and autumn and summer and autumn (P>0.05); those in hypothalamus showed no significant differences between hamsters in spring and summer and between winter and autumn (P>0.05); and those in pituitary gland exhibited no significant differences between hamsters in spring and autumn, and spring and winter (P>0.05). Significant differences can be observed between each pair of seasons other than the above mentioned (P<005). The expressions of AANAT mRNA in pineal gland and/or pituitary gland showed a fluctuation mode as winter> spring>autumn>summer. This corresponded to how serum melatonin fluctuates along seasons. In hypothalamus, the expressions kept fluctuates by winter>spring>autumn>summer, which was inconsistent with how serum melatonin changes in spring and autumn.
3.2.4Influence of season models on serum melatonin
On the analog of climate changes, the serum melatonin presents no significant difference (P>0.05) in the spring and autumn, spring and winter, but it presents significant differences (P<0.05) between other seasons. The trend is winter> spring> autumn> summer, which is in accordance with the trend of natural seasonal variations. Compared to natural climate showed no statistically significant differences.In the comparisons between modeled seasons to their respective opposite seasons, there is significant difference (P<0.05) between the modeled summer and natural winter, modeled winter and natural summer.
3.2.5Influences of season models on the expression of AANAT protein
The level of AANAT protein in pineal gland, hypothalamus and pituitary gland presented significant seasonal variability (P<0.05). Compared hamsters in settings of natural autumn and the modeled one, the levels of AANAT in pineal gland or in hypothalamus exhibited no statistically significant differences (P>0.05); while the AANAT levels in the other comparisons between the hamsters in natural and model seasons showed statistically significant difference (P<0.05); in addition, the AANAT levels in the hamsters'pituitary gland in such nature-to-model comparisons showed no significant difference (P<0.05). In the comparison between hamsters in a modeled season to its opposite modeled season (e.g. spring is opposite to autumn and summer is to winter), there showed no significant difference of AANAT levels in pituitary gland between natural and modeled autumn (P>0.05), while all the other such comparisons of AANAT levels in pituitary gland, pineal gland or hypothalamus showed significant difference (P<0.05). The changing of AANAT levels in hypothalamus determined in the modeled annual succession of seasons was observed to be consistent with those in the natural ones, while the only exception-hypothalamus presented a level fluctuation of AANAT as winter>spring>autumn>summer.
3.2.6Influence of modeled seasons on the expression of AANAT mRNA
In the setting of modeled seasons, the expression of AANAT mRNA in pineal gland presented no significant seasonal variability between spring and autumn, and between summer and autumn (P>00);its expression in hypothalamus displayed no significant seasonal variability between spring and summer, and between winter and autumn (P>0.05); its expression in pituitary gland exhibited no significant seasonal variability between spring and summer; other than these, all the comparisons between modeled seasons showed significant differences (P<0.05). In the comparisons between modeled natural seasons, the expressions in pineal gland manifested no significant differences (P>0.05); the expressions in hypothalamus between modeled and natural spring/autumn showed significant differences (P<0.05); the expressions in pituitary gland between modeled and natural spring/winter exhibited significant differences (P<005). In the comparisons between modeled seasons to their respective opposite seasons, there showed no significant difference of the expression in pineal gland between modeled spring and natural autumn (P>0.05) and in pituitary gland between modeled autumn and natural spring; in all the other comparisons including those involved hypothalamus showed significant differences (P>0.05). The changing tendencies of expressions in AANAT mRNA in all three tissues were consistent with those in the setting of natural seasons.
4Conclusion
4.1The adaptive control mechanism "liver corresponding to spring" takes the function of liver in storing blood and governing dispersion as the physiological basis. The variation of melatonin in the four seasons is contrary to the variation of the function of liver in dispersion, but is in accordance with the function of liver in storing blood. It can be considered that melatonin has the function of suppressing the liver in dispersion, but promotes the function of the liver in storing blood. The seasonal change of melatonin may be one of the adaptive control mechanism of the "liver corresponding to spring"
4.2melatonin synthesis rate-limiting enzyme AANAT has a regulatory role in melatonin synthesis and secretion in the seasonal changes of the summer and winter, and there is no obvious regulating effect in the synthesis and secretion of four seasons, AANAT is not the rate-limiting enzyme of the seasonal changes of serum melatonin.
4.3Artificial simulation box successfully simulated climate changes of the four seasons, but there are still differences between the modeled climate and natural climate. It indicates that in modeled climate, these indicators such as the exposure time, temperature, humidity are only considered, which may not accurately reflect the characteristics of the natural climate. As a part ofthe whole of nature, people feel the information of the nature in a wide variety. To take use of artificial climate simulation box to study the theory of "five zang-viscera corresponding to seasons", adjustment on the simulation parameters may also needed to be made to be closer to the characteristics of the natural climate.
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