| Defensive qi, which is circulating outside the pulse, is an important part of qi in human body. Defensing the exogenous evil, warming and nourishing the body, and regulating the sweat are its functions according to Chapter Benzang in Lingshu. With the development of science and technology,the researches on the substance of defensive qi mainly involved with mmunity and heat metabolism,stressed its single-function and deviated form the concept of holism in TCM and theories of defensive qi in Huangdi Neijing.Metabolomics, a new branch of systematic biology after the rise of genomics, transcription group and proteomics, is a technology by which researching metabolic network of living things system through metabolite map and their dynamic changes before and after stimulus of living things system. It skips the complicated regulation of life entity, obtains final and integral results through analysis on metabolite. Therefore, as a technology revealing dynamic changes of organism on the whole,metabolomics avoids the defects of researching physiological and pathological changes by single or few index in the past,has feature of gathering entirety,moving,composition,analysis in one in methodology,is in conformity with the principle of globality of TCM, and is technology suitable for research of TCM.Rhythmicity is the important feature of defensive qi. At present, the modern medicine research on rhythm of defensive qi mainly focus on circannian rhythm and circadian rhythm, lunar rhythm research is rerely involved. However, 2000 years ago, Neijing had mentioned and illuminated the lunar rhythm of defensive qi. Moreover, the modern medicine have discovered that biothythm is controlled by inner clock, which have its own ring circuit of regulation and feedback. In mammal, regulation of clock is related to mutual combination of some clock genes,clock-related genes, clock control genes and their protein products through transcription- translationevents after translation. The break or changes of expression level on some one gene could result in disorder of rhythm. Therefore, observing the changes of clock gene on the level of transcription and translation is the direct way of researching regulative mechanism of biothythm.Our research group had established the rat model of deficient defensive qi by the methods of the cold and hot stimulus,and conducted research on physiologic lunar rhythm of defensive qi by serum metabolomics. Based on the early research,we will establish the rat model of deficient defensive qi by the methods of the stimulus of fatigue combined with coldness and hotness,select 3 time points:the beginning of month, the middle of month and the end of month, measure the metabolin in the control and model rats by thymus metabolomics, and measure the expression of clock genes in lymphocyte and thymocyte of the control and model rats. Furthermore, we will speculate the complex regulation by researching the lunar rhythm of defensive qi from the two sides “the level of metabolin”and“the level of clock genetic expression”.This paper mainly includes the theory and experiment two parts: PART 1 THEORETCAL RESEARCHObjective:To explore the theories of the lunar rhythm of defensive qi and its mechanism, and make in-depth, all-sided exploration of the lunar rhythm of defensive qi using t he new technology from the ancient and modern literature.Method:Discussing the genetic regulation mechanism of biorhythm, the research status of rhythm of defensive qi(the key point is lunar rhythm)from the view of traditional Chinese and western medicine, and the feasibility of lunar rhythm of defensive qi using the technology of metabolomics and clock gene in detail.Conclusion:At present, researches of defensive qi only focus on a single or few index, rather than research it as a whole. Moreover, the rhythmical researches on the immunity of defensive qi mainly focus on circannian rhythm and circadian rhythm, rather than the lunar rhythm. Having researches by the technology of metabolomics characterized with globality and dynamics and the techniques of gene is significant. PART 2 EXPERIMENTAL RESEARCH EXPERIMENT Ⅰ Establishment of Rat Model with Deficient Defens-ive qi and Effects of Jade-screen PowderObjective:To probe the method by which established the rat model with deficient defensive qi,and observe the apagogic effects of Jade-Screen Powder on the model.Methods:The SD rats were made by the stimulus of fatigue combined with coldness and hotness,namely FCH group, and were compared with the normal group,the CY group,the cold and hot stimulus group(CH group) and the Chinese crude drug intervention gourp(DI gourp). We observed the changes of the rats on behavioristics,weight,thymic index,spleen index,contents of cytokine in spleen, and evaluated the scientificalness and reasonableness of this modeling method.Results:Through the observation of behavior changes of rats,we discovered that the modeling method of FCH group was closer to the evaluation criteria of deficient defensive Qi.Through the statistical analysis of the above indexes,we discovered the following situations:compared with the control group,the indexes of the FCH group dramatically declined(P<0.05﹚; compared with the CY group,the indexes of the FCH group were no significant differences(P>0.05﹚; compared with the cold and hot stimulus group,the indexes of the FCH group dramatically declined(P<0.05﹚; the indexes of the Chinese crude drug intervention gourp were significantly higher than the FCH group(P<0.05﹚.Conclusion : The model by the stimulus of fatigue combined with coldness and hotness was an ideal model of deficienct defensive qi.EXPERIMENT Ⅱ The research on physiologic lunar rhythm of defen-sive qi by thymus metabolomicsObjective:Using the technology of metabolomics, we try to seek the symbolic compound which could reflect the changes of physiologic lunar rhythm of defensive qi below the partial microenvironment-thymus.Methods:The experiment was divided into 3 groups: the early group, medium group and late group. The moon month in this experiment was fixed according to the day of autumnal equinox in. At 12:00,August 1,15 and 30,2013 in the lunar calendar, we respectively selected 10 normal SD rats, taken the thymus, measured metabolin by liquid chromatograph-mass spectrometer, analyzed the data by PCA and PLS-DA of SIMCA-P software,and certained the potential marks.Results:Through the PCA analysis of the above 3 groups and the PLS-DA analysis of medium group and early group, medium group and late group, we discovered that the medium group was obviously classified by late group and early group, and it had different metabolins. The contents of Thiamine, Guanosine and Cortisol in thymus had tendency of highness in medium group, lowness in late group and early group, which was in accordance with the variation tendency of defensive qi.Conclusion:The SD rats had physiologic lunar rhythm of defensive qi. Thiamine, Guanosine and Cortisol were the symbolic compound which could participate in or regulate the changes of it. EXPERIMENT Ⅲ The research on lunar rhythm of deficiency syndrome of defensive qi by thymus metabolomicsObjective:Using the technology of metabolomics, we try to seek the symbolic compound which could reflect the deficiency syndrome of defensive qi and the changes of lunar rhythm of this syndrome below the partial microenvironment-thymus.Methods:The experiment was operated in 3 time points: the beginning of month, the middle of month and the end of month. Before the experiment every time, 30 rats were divided into 3 groups: the control group, the model group and the medication group. The rats of the control group were fed conventionally. The rats of the model groups were given the stimulus of fatigue combined with coldness and hotness. The rats of the medication group were given Chinese crude drug intervention while given the above stimulus. We measured metabolin in the thymus by liquid chromatograph-mass spectrometer, analyzed the data by PCA and PLS-DA of SIMCA-P software, and made sure the potential marks.Results:(1)Through the PCA analysis of the 3 groups in 3 time points,we discovered that the model group was obviously classified by the control group, and it had different metabolins. The control group was incompletely classified by the medication group and they were partly syncretized.(2)There were incompletely same symbolic compound about model of deficiency of defensive qi in 3 time points.(3)Cortisol and c AMP in thymus were the symbolic compounds arisen in 3 time points,and had tendency of highness in the middle of month, lowness at the beginning of month and the end of monthConclusion:The SD rats model of deficiency of defensive qi had lunar rhythm. Cortisol and c AMP were the symbolic compound which could participate in or regulate the changes of it. EXPERIMENT Ⅳ The research on lunar rhythm of expression of Clock, Bmal, Per1 m RNA in lymphocyte and thymocyte of normal ratsObjective: Observing the lunar rhythm of expression of Clock, Bmal, Per1 m RNA in lymphocyte and thymocyte of normal ratsMethods: The experiment was divided into 3 groups: the early group, medium group and late group.The moon month in this experiment was fixed according to the day of autumnal equinox in. At 12:00,August 1,15 and 30,2013 in the lunar calendar, we respectively selected 10 normal SD rats, took blood by decapitation and took thymus,measured the expression of Clock, Bmal, Per1 m RNA in lymphocyte and thymocyte using the technology of RT-PCR.Results: Compared with the early group and late group,the expressions of Clock, Bmal, Per1 m RNA in lymphocyte and thymocyte of medium group dramatically increased(P<0.05﹚. The expressions in the early group and late group were no significant differences(P>0.05﹚.Conclusion: The lunar rhythm of the expression of Clock, Bmal, Per1 m RNA in lymphocyte and thymocyte of normal rats was in accord with the lunar rhythm of defensive qi in it,and probably was the important molecular biology basis of the lunar rhythm of defensive qi. |
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