Experimental And Theoretical Study Of Spring Regulation Mechanism Should Liver

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.ObjectiveBased 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. Method2.1Theoretical ResearchBy 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 DesignThis 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 MethodSerum 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 methodsAll 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. Result3.1Theoretical Research Result3.1.1It 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 melatoninDuring 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 proteinWith 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 melatoninOn 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 proteinThe 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 mRNAIn 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.4Conclusion4.1The 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.