| Research Background: Syndrome of stagnation of liver qi and spleen deficiency was a summary for dysfunction of spleen and stomach and liver wood restricting spleen earth caused by stagnation of liver qi and liver dysfunction. The manifestations of this syndrome included negativity, poor resilience, depression, diarrhea, weight loss, appetite loss, and so on. The speeding of modern life rhythm, increasing of social competition, work stress, and inappropriate daily life could lead to the heavy psychological burden, emotional depression, qi-dynamic disorder, and lead to syndrome of stagnation of liver qi and spleen deficiency eventually. The people with syndrome of stagnation of liver qi and spleen deficiency became a common phenomenon. With the establishing of medical model as biology-psychology-society, it realized that the syndrome of stagnation of liver qi and spleen deficiency played an important role in the development of various diseases, so the researches on this syndrome had been paid further attention.For a long time, researchers have been trying to interpret the essence and clarify the material basis for syndrome of stagnation of liver qi and spleen deficiency from multisystem, multilevel, andmulti-dimension. Through years of exploration and research, the researchers had found that syndrome of stagnation of liver qi and spleen deficiency was closely related with stress, and hippocampus and prefrontal cortex(PFC) were the impressionable brain areas in the stress. Long-term exposure to stress condition, hypothalamus-pituitary-adrenal(HPA) axis derepression and high levels of glucocorticoid could damage hippocampus and PFC, lead to glial cells degeneration, pyramidal cells atrophy, low expression of postsynaptic density protein 95(PSD95) and synapsin Ⅰ, and these changes were closely related with cognitive impairment which correlated with PFC.Systermic stress response could increase the glucocotioid level, and also could enhancing the brain glutamate level simultaneously. If the concentration and effect time were beyond the physiological range, the glutamate-mediated excitotoxity was produced, and then lead to neuron damage. This was one of the mechanisms about mood disorder caused by social stress. This mechanism related with astrocytes(As) damage and excitatory amino acid transporters(EAATs) dysfunction which were caused by long-term stress. As undertook 80%-90% transport task by EAAT1 and EAAT2, so EAATs palyed an important role in maintaining the glutamate concentration of synaptic cleft, and avoiding the excitatory amino acids toxicity. In addition, the expression of EAAT1 and EAAT2 in hippocampus and PFC of rats could be decreased by acquired helplessness stress. All of the above research results showed that long-term stress could lead to As damage and EAATs dysfunction, so the concentration of glutamate in synaptic cleft increased, and the neuronal plasticity was damaged by excitatory amino acids toxicity eventually. Somestudies had found that chronic stress could lead to the reducing of density in PFC of animals, and the decreasing of GFAP protein expression in PFC of rats. The changes in number, morphology and functions of As might be interact as both as cause and effect, and what’s more, the stress could lead to the decreasing in expression of GFAP protein, which remaindered that the dysfunction of As in PFC might be involved in the occurrence of syndrome of stagnation of liver qi and spleen deficiency caused by chronic long-term stress, but there was no related reports about this mechanism of syndrome of stagnation of liver qi and spleen deficiency.Research Objective: Based on the above question, the changes in glutamate regulating function of As in PFC of mice with syndrome of stagnation of liver qi and spleen deficiency induced by chronic long-term complex stress and the treatment mechanism of Xiaoyao powder were researched in this study, in order to lay a theoretical foundation for the interpretation of the essence and biological mechanisms of syndrome of stagnation of liver qi and spleen deficiency, and find new drug targets for the treatment of syndrome of stagnation of liver qi and spleen deficiency.Research Methods: 1. 80 C57BL/6J mice with 10 weeks of age were randomly and averagely divided into control group, model group, Xiaoyao powder group and Fluoxetine group.Mice of control group were breeded in crowds, and mice in the other groups were breeded alone. 2. All the mice received complex stress for 21 days to establish the mouse models with syndrome of stagnation of liver qi and spleen deficiency after feeding for 7 days adaptively except mice in control group. Mice in model group received gavage administration with normal saline, and mice in Xiaoyao powder group and Fluoxetine group received gavage administration with Xiaoyao powder and Fluoxetine respectively. 3. The bodyweight, food comsumption, and macroscopic character of mice in each group were observed and recorded. 4. The changes in behavioristics of mice were evaluated by using open field test, elevated plus-maze test, new environment restrain feeding experiment, forced swimming test, and sucrose preference test. 5. The D-xylose concentration in mice serum was tested by using colorimetric method. 6. The pathologic changes in mice PFC were observed by using HE staining method. 7. The concentrations of b FGF, TGF-β1, BDNF and NGF in PFC of mice were tested by using ELISA method. 8. The Glu concentration in mice PFC was tested by using colorimetric method. 9. Western Blot and immunohistochemical methods were used to test the expression of proteins as GFAP, EAAT1, EAAT2 and Neu N. 10. Quantitative real-time PCR(q PCR) method was used to test the m RNA expression of GFAP, EAAT1, EAAT2 and Neu N. 11. The changes in numbers and morphology were observed by using Nissl’s staining.Research Results: 1. The comparison of general condition: mice in model group were irritability at the beginning, then turn to depressed, and the manifestations of mice included messy and aphotic hair, clustering, gavage resistance weakened, and loose stool. The above manifestations of mice in Xiaoyao powder group and Fluoxetine group were lighter, and there was no abnormal manifestation in control group. There was significant difference in bodyweight growth among the 4 groups(P<0.01), the bodyweight of mice in model group and Xiaoyao powder group increased slowly(P<0.01), and the bodyweight growth rate of mice in Fluoxetine group was between control group and model group. The difference in food consumption among all the groups was significant(P<0.01), the food consumption of mice in model group decreased significantly(P < 0.01), and the food consumption of mice in Xiaoyao powder group and Fluoxetine group increased significantly(P<0.05).2. The comparison of behavioristics: The results of FST showed that the immobility time of mice in model group increased significantly, and the immobility time of mice in Xiaoyao powder group and Fluoxetine group decreased significantly compared with model group(P<0.05). The results of OFT showed that the number of entries into the central zone and retention time in the central zone of mice in model group decreased significantly compared with control group(P<0.01), the retention time in the central zone of mice in Xiaoyao powder group and Fluoxetine group decreased significantly compared with model group(P<0.05), and the total moved distance of mice in the two group increased significantly compared with model group(P<0.05). The results of EPMT showed that the retention time in open-arm of mice in model group reduced significantly(P<0.01), and retention time in open-arm of mice in Xiaoyao powder group increased significantly compared with model group(P<0.05). The difference in sucrose preference of mice among all the groups was significant(P<0.01), the sucrose preference of mice in model group was significantly lower than that of the other three groups(P<0.05).3. The comparison of concentration of D-xylcose in serum: the serum D-xylcose concentration of mice in model group reduced significantly compared with control group(P<0.05), the serum D-xylcose concentration of mice in Xiaoyao powder group increased significantly compared with model group(P < 0.01), and the D-xylcose concentration of mice in Fluoxetine group decreased significantly compared with Xiaoyao powder group(P<0.05).4. The result of HE staining showed that pathologic changes in Pr L region in PFC of mice in model group included irregular arrangement of cells, contractible cells, condensation and erythro-stained cytoplasm, indistinct boundary of karyotheca and nucleolus, abundant heterochromatin, karyopyknosis and so on, and the above changes in Xiaoyao powder group and Fluoxetine group were lighter.5. The results of ELISA test showed that the concentration of BDNF and NGF of mice in model group reduced significantly(P<0.05), the b FGF concentration of mice in model group and Fluoxetine group increased significantly(P<0.01), and the NGF concentration of mice in Xiaoyao powder group and Fluoxetine group increased significantly compared with model group(P<0.05).6. The results of colorimetry test showed that the Glu concentration of mice in model group increased obviously(P<0.01), and the Glu concentration of mice in Xiaoyao powder group and Fluoxetine group decreased obviously compare with model group(P<0.01).7. The results of immunohistochemical test showed that the positive cell number and mean optical density(OD) value of GFAP, EAAT1, EAAT2, and the OD value of Neu N of mice in model group decreased significantly(P<0.01); the positive cell number and OD value of GFAP, EAAT1, EAAT2, and the OD value of Neu N of mice in Xiaoyao powder group increased significantly compared with model group(P<0.05); the positive cell number and OD value of EAAT2, and the OD value of EAAT1, GFAP and Neu N of mice in Fluoxetine group increased significantly compared with model group(P<0.05).8. The results of Western Blot showed that the protein expression of GFAP, Neu N, EAAT1 and EAAT2 in PFC of mice in model group reduced significantly(P<0.01), the protein expression of GFAP, Neu N and EAAT1 of mice in Xiaoyao powder group increased significantly(P<0.05), and the protein expression of GFAP, EAAT1, EAAT2 and Neu N of mice in model group increased significantly compared with model group(P<0.05).9. The results of q PCR test showed that the m RNA expression of EAAT2 of mice in model group decreased significantly(P<0.01), and the m RNA expression of GFAP, EAAT1 and Neu N of mice in model group was falling, but the difference between control group and model group was not significant(P<0.05); the m RNA expression of GFAP, EAAT1, EAAT2 and Neu N of mice in Xiaoyao powder group increased significantly compared with model group(P < 0.05); the m RNA expression of GFAP, EAAT1, EAAT2 and Neu N of mice in Fluoxetine group increased significantly compared with model group(P<0.05).10. The result of Nissl’s staining showed that the nerve cell number of mice in model group decreased significantly(P<0.01), and the nerve cell number of mice in Xiaoyao powder group and Fluoxetine group increased significantly compared with model group(P<0.05); in additional, the manifestation of nerve cells in PFC of mice in model group included arranged in disorder, deletion, damage, irregular shapes, fuzzy nucleus boundary, maldistribution, and so on, there were only a small number of neurons with irregular form in Xiaoyao powder group, and there was no obvious abnormality in Fluoxetine group compare with control group.Research Conclusion: 1. It could be determined that the mouse models with syndrome of stagnation of liver qi and spleen deficiency had been established successfully according to the macroscopic characters, changes in behavioristics, concentration of serum D-xylose, and the theory of by means of prescriptions to speculate the symptoms.2. It could be determined that the function of astrocytes in PFC of mice with syndrome of stagnation of liver qi and spleen deficiency was damaged to some level according to the changes in PFC tissue morphology, reduced expression of GFAP protein and m RNA, and the changes in levels of relative neural factors as BDNF, NGF, TGF-β1, and b FGF.3. It could be concluded that the circulation of Glu in PFC of mice with syndrome of stagnation of liver qi and spleen deficiency was disordered, and this pathologic change was closely related with dysfunction of astrocytes in the PFC of mice according to the reduced expression of EAAT1 and EAAT2 proteins and genes, and the sharp increase of Glu in the PFC of mice with syndrome of stagnation of liver qi and spleen deficiency.4. It could be concluded that the functions and morphology of nerve cells in PFC of mice with syndrome of stagnation of liver qi and spleen deficiency were damaged to a certain extent, and the pathologic change was closely related with the sharp increase of Glu caused by astrocytes dysfunction in PFC according to the reduced expression of Neu N protein and gene, and the changes in numbers and morphology of nerve cells in PFC of mice with syndrome of stagnation of liver qi and spleen deficiency.5. Xiaoyao powder could treat syndrome of stagnation of liver qi and spleen deficiency by improving the pathological changes, up-regulating the protein and gene expression of GFAP, EAAT1, EAAT2, and Neu N, adjusting the concentration of relative neural factors, reducing the concentration of Glu, and improving the number and morphology of nerve cells in PFC of mice with syndrome of stagnation of liver qi and spleen deficiency. |
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