| As society develops, the accelerating pace of life has increased pressure among everyone in working and studying. Fatigue has become a common problem to modern people; it includes physical fatigue and mental fatigue, which shows symptoms of tiredness, mood disorder, slow responses, and decline of learning and memory ability. As memory effects on learning performance and work efficiency, our main research goal is to study the relationship between fatigue and memory under the concept of Traditional Chinese Medicine, and also to search for methods to improve memory loss. Since the coexistence of physical and mental fatigue and affect between each other, our study underlies the theory of the effect on heart, liver and kidney pathology, which led by fatigue and memory loss, and combine experimental researches to have a better perspective on the mechanism of how soothing liver and tonifying kidney with nourishing heart can improve memory loss induced by fatigue.1. Theory ResearchThere was no difference between physical or mental fatigue in ancient Chinese medicine. Symptoms were described as "slack", "laziness", "limb fatigue", which all refers as "fatigue". With great amount of physical and mental wear out will cause physical and psychological dysfunction. Excessive physical fatigue and mental fatigue will interact, and cause damage to the normal physiological manifestations on liver and kidney, showing symptoms such as stagnation in liver Qi or lack of kidney essence, therefore essence, Qi, blood and the spirit will be affected. In TCM theory, learning and memory are being categorized in "Mind". The five spirits are in charged by the five viscera. Heart masters "God", and takes charge of all mental activities. In the five spirit cognitive theory, "liver soul" extracts the long-term memory in "kidney will" and delivers the message to "heart" along with the decision information made in "spleen intent". This implies the key role of heart, liver and kidney in the recognition process.Therefore, according to the relationship between liver, kidney, heart and brain. and the connection with the pathological mechanism of fatigue induced memory loss, we propose the theory of recover fatigue to block the occurrence of learning and memory ability decline by soothing liver and tonifying kidney with nourishing heart, and also to improve memory loss directly by soothing liver and tonifying kidney with nourishing heart. 2. Experimental ResearchAt first, we applied ATR-FTIR (Attenuated Total reflection-Fourier transform infrared spectroscopy) technique to exam the herbs we use. In fatigue animal experiment, combine swimming and multiple platform method in order to imitate the realistic pathological state of fatigue. And in behavior experiment, it has been proven that memory loss can be induced by fatigue. Learning and memory is underling synaptic plasticity in neurology, and is activated by different protein kinase activity and regulate by protein phosphorylation on synapse. Therefore our research focus on the study of Neurogranin (Ng), Protein Kinase C (PK.C) and Ca2+-CaM-dependent protein kinase (CaMKâ…¡). application of Western Blot and Real-time PCR technique to observe the level of protein and mRNA in the Hippocampal CA1 region of fatigue rats. And the expression difference after applying soothing liver and tonifying kidney with nourishing heart formulas.ATR-FTIRBy using ATR-FTIR technique screen all of the single herbs in Si Ni San and Sheng Hui Tang. Experimental herbs were proven to be reliable. And we speculate polysaccharide and amino substance are contained in these formulas, as also relevant to signal transmit in neurons.Combined fatigue animal modelCombine swimming and multiple platform sleep deprivation methods as composite fatigue animal model on sleep deprivation rats, which is an ideal method to avoid additional stress, inflammation and other occurred error. Animal model is proved by animal's weight, coat color and other general observations, tests of CK and LDH in serum, and other common indicators of fatigue testing. Use soothing liver Qi formula:Si Ni San, and tonify kidney and nourishing heart formula:Sheng Hui Tang has also proved to be effective to reduce fatigue and improve memory.Evaluation of learning/memory in fatigue ratsEvaluate learning/memory changes through Y maze. Results show, comparing model group and normal group, the correct response rate was significantly lower (p< 0.01), numbers of error increased (p< 0.01); numbers of time to learn have increased (p< 0.01). Comparing soothing liver group, tonify kidney and nourishing heart group, combined formula group and model group have all increased correct response rate (p< 0.01), decreased in error rate (p< 0.01). Indicate that comparing with model group, soothing liver group, tonify kidney and nourishing heart group and combined formula group can elevate the correct respond rate and decline error rate. The combined formula group has the most significant learning ability among all.The expression of Ng, PKC, CaMKâ…¡in the Hippocampal CA1 region of fatigue ratsAfter the behavioral experiment,6 rats were randomly selected in each group, obtain the left hemisphere of the brain for Western Blot test, the right half for Real time PCR experiments, examine the protein and mRNA expression levels of Ng, PKC and CaMKâ…¡in the Hippocampal CA1 region of fatigue rats.Ng is the brain-specific protein, is the substrate of PKC, and is also the binding protein of calmodulin. which is involved in the process of learning and memory by regulating Ca2+ and CaMKâ…¡. Results show, both Ng protein and mRNA expressions in the Hippocampal CA1 region of fatigue rats decrease in the model group than in control group. According to the expression of Ng was positively related to learning and memory, we speculate that fatigue can reduce learning and memory ability. Comparing to model group the Ng protein and mRNA expression level increased after intake soothing liver and tonifying kidney plus nourishing heart formulas. Among the three treatment groups, the combined formula has the most effective influence, followed by tonifying kidney and nourishing heart formula, and then soothing liver formula. Indicates combining soothing liver and tonifying kidney and nourishing heart has the most effective influence than methods only applying soothing liver or tonifying kidney and nourishing heart.The level of PKC protein and mRNA expression in the Hippocampal CA1 region of fatigue rats, decrease significantly in the model group than in control group. Base on the decrease level of PKC. might indicate damage occurred to nerve cells in the hippocampus. Both PKC protein and mRNA expression level increased after intake soothing liver and tonifying kidney formulas. In the result of Western blot, tonify kidney group and combined lormula group perform better than soothing liver group. Although, the results of western blot and PCR in combined formula group are just slightly higher than tonify kidney and nourishing heart group, but they indicate, with combining soothing liver and tonifying kidney with nourishing heart has the most effective influence.The detection of Thr286 phosphorylation CaMKâ…¡in Western blot experiments showed that the model group was significantly lower than the control group, results are the same based on gene expression level. CaMKâ…¡Thr286 autophosphorylation is cruial to induce LTP, thus indicates there might be damge in hippocampus while animal model experiment, lead to affect CaMKâ…¡experession. In three treatment groups. the Thr286 phosphorylation of CaMKâ…¡in soothing liver group and in combine formula group were significant upregulation comparing to model group. And there's only slightly upregluate in tonifing kidney and nourishing heart group, but there are not difference. On the gene level, the combined formula group has significant upregulation on CaMKâ…¡comparing with the model group. But there are no difference with soothing liver group and tonifying kidney and nourishing heart group. Plus, the regulation comaring between the protein and gene espression are different, maybe because the formulas don't effect on CaMKâ…¡genes, and directly effect on CaMKâ…¡phosphorylation.ConclutionOur project focus on the mechanism of the regulation of memory loss induced by fatigue with the methods of using soothing liver and tonifying kidney. Our theory is to use heart, liver and kidney as a breakthrough to regulate memory loss lead by fatigue, according to the impact between fatigue and memory loss, and the connections within heart, liver and kidney. With the experimental research, by ATR-FTIR. behavioral experiment, and by Western blot and PCR technique examing the expression of Ng, PKC and CaMKâ…¡, we discover that the regulation of soothing liver and tonifying kidney and nourishing heart formulas can improve memory loss induced by fatigue. Thus, the method of soothing liver and tonifying kidney with nourishing heart can regulate memory loss by recovering fatigue or to improve memory directly.
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