| Background:Liver depression syndrome is a basic syndrome in Traditional Chinese Medicine (TCM), Which is characteristic of liver-qi stagnation because of the dysfunction and insufficiency of abreaction of liver-qi. It belongs to melancholia of TCM. The new concept of melancholia is proposed for the first time, and then the other theories of melancholia are proposed by later generations. Liver depression syndrome is evolved from the different theories of melancholia and it associates the stagnation of five qi with the stagnation of five zang organs. Clinical epidemiology survey showed that liver diseases in the viscera account for 40% of the overall disease. Liver depression syndrome is the basic pathological change of liver diseases and can be seen at the early stage of various kinds of diseases. Therefore, the nature of liver depression syndrome is always the hot topic in the area of TCM.The study of liver depression syndrome include clinical and animal experiment research. In respect of the essential of TCM syndrome, animal research has more advantages because of the unattainablility of some clinical samples. The establishment of animal model of TCM syndrome is an important approache to the study of the modernization of TCM. The method of chronic unpredictable mild stress (CUMS) had the highest recognition at home and abroad, in a variety of replica methods of animal model of liver depression syndrome. It commendably simulates the etiology of liver depression syndrome by the diversity, moderation and unpredictability of stressors, and can not damage the body of animals. The academic research on syndromes and prescriptions of liver depression syndrome is the important respect of its essential research. Treatment based on syndrome differentiation is the core of TCM. Theory, rule, prescription and drug are established according to it. The reference to each other of prescription and syndrome is the characteristic of TCM syndrome research. We can disprove the accuracy of treatment based on syndrome differentiation, according to the clinical symptoms before and after treatment with prescription and drug. In recent years, there are much study on essence of liver depression syndrome. It has initially been confirmed that there is pathophysiological basis in the etiology of liver depression syndrome. Studies suggest that liver depression syndrome generalize about the nervous, immune, digestive, sensory, sport and other systems of certain pathological changes depression, anxiety, grief, stress, premising on nerce center of the adjustment disorder. In recent years, there are some studies on Hypothalamic Pituitary Gonadal Axis (HPA) and hippocampal synaptic plasticity. The conclusion is that hippocampus plays an important role in the pathogenesis of liver depression syndrome.TCM syndrome is the reflection of internal and external environment which patients in the process of disease occurrence and evolution. TCM syndrome research is the critical question of theoretical research of TCM. The growing science of proteomics mybe the way to solve this problem. Proteomics is one subject which sthdy the expression and function of all proteins in cell. It understands biological phenomena at the integral level, which is similar to holistic concept of TCM. Proteins in cell will never be the same, like the proteomic, which is the integration of all proteins. TCM syndrome is changing at different stages of diseases and has developing laws. The material basis of change potentially reflect on the level of proteomic. If Proteomics is applied to the research of TCM syndrome, we can not only comprehend material basis of different symptoms, but also know variation of different proteins in the development of syndrome. The change mechanism of material basis of TCM syndrome could be studied according to comparison between proteomics research before and after the development of syndrome. Then, connotation of TCM syndrome will be revealed and lay a foundation for objectification of TCM.Therefore, the study select hippocampus as a starting point, to analyze differential proteins according to the methods of proteomics. And then, the potential key signal molecules and related signaling pathway related to liver depression syndrome are found by the analysis of differential proteins. Meanwhile, Xiao Yao San (XYS) is applied to interveneour animal model. Fluoxetine is selected as positive control drug. Therefore, we hope to provide further evidence for the correlation of liver depression syndrome and hippocampus and lay a solid foundation for the further study of essence of liver depression syndrome.Objective:CUMS and social isolation are applied to set up the rat model of liver depression syndrome. To judge the success of model, general conditions of rats, sucrose consumption rate and behavior tests are observed. And then analyze the differential proteins according to the method of proteomics. The potential key signal molecules and related signaling pathway related to liver depression syndrome are found by the analysis of differential proteins. We hope to find the specific proteins related to liver depression syndrome by the method of proteomics. And find material basis of TCM syndrome research and new idea for the treatment of liver depression syndrome. New targets will be found for its treatment. The differential proteins will be found by XYS and fluoxetine treated rats. The characteristic of TCM syndrome is used to study mechanisms of XYS action. And to provide new idea and method for the essential study of liver depression syndrome.Methods:This study established the rat model of liver depression syndrome by the method of CUMS and social isolation. XYS was used to treat it. Hippocampus were selected as the research object to analyze the differential proteins among the model group and the control group. The key signal molecules and related signaling pathway related to liver depression syndrome were found. Immunohistochemical (IHC), Western blot (WB) and real-time polymerase chain reaction (RT-PCR) were used to further research.1. Established the rat model of liver depression syndrome by the method of CUMS and social isolation.Groups and Model establishment:Forty male SD rats (200±20g) were randomly divided into four groups with 10 rats in each after one week’s adaptation living, which are control, model, XYS and fluoxetine group. Five rats per cage in the control group were housed. Rats in other three groups underwent CUMS procedures and social isolation. The CUMS procedures were continued for three weeks.Drug intervention:About 19g/kg/d XYS,2mg/kg/d fluoxetine, and an equivalent volume of distlled water (for model and control groups) were administrated by gavage using a tube twice a day.Model evaluation:Model success will be evaluated by the observation of general conditions of rats, sucrose consumption rate and behavior tests.2. HE staining to observe the morphological changes in rat hippocampus and the changes of Nissl bodies by Nissl staining.3.2-DE separation of hippocampus and image analysis.The samples were pair-matched. We made the 2-DE separation for all the groups and repeated for three times. The gels were scanned by optical density transmission scanners and the images were got. We used the software of PDquest7.0 to analyze the images of gels and got the quantity of each protein expression, soelectric point and the molecular weight of proteins and so on. The protein spot whose expression has more than two times difference was defined to differentially-expressed protein, the results were automatically generated by the software and then were verified manually.4. The mass spectrometry identification and database confirm of differentially expressed protein spots.MALDI-TOF-MS was applied to make the detection of peptide mass fingeprint (PMF) for differentially expressed protein spots. And the protein-related database on the web http://www.uniprot.org/was used to verify the proteins. 5. Expression of β-arrestin2 and CRHR in hippocampus and hormone in serum and cerebrospinal fluid (CSF) detected by WB, IHC and enzyme-linked immuno sorbent assay (ELISA).6. Expression of β-arrestin2-mediated PP2A-Akt-mTOR signaling pathways by WB and RT-PCR.7. Expression of β-arrestin2-mediated ERK-BDNF signaling pathways by WB and IHC.8. Statistical analysis.All data were expressed as mean ± SD and analyzed using an SPSS statistical package (version 13.0). ANOVA of repeated measures was applied for the weight changes. For the other indicators, the mean comparison of multiple samples used One Way ANOVA to analyze. When the variance was homogeneous, Least-significant difference (LSD) was applied for multiple comparison and when the variance was heteroscedastic, Games-Howell was applied for multiple comparison. The mean comparison before and after modeling used paired-sample T test. The value of P<0.05 would mean significant difference.Results:1. The rat model of liver depression syndrome was established successfully. During the 3 weeks of modeling, the model rats first were anxious and then displayed the typical appearance such as low activity, slow reaction, poor appetite and lusterless fur. Rats from XYS group and fluoxetine group also showed some above abnormalities, but they were not obvious. Rats in control group lived well. The rats’ weight increase was significantly different between groups. It showed rats in model group grow slowest, while rats in XYS group and fluoxetine group grow more than them. The weight of control group is always highest. The number of grids crossed with all paws, and the number of rearing responses significantly decreased in the model group. XYS and fluoxetine significantly increased the number of crossed grids, and XYS increased the rearing responses. The percentage of sucrose consumption significantly decreased in the model group. Moreover, food consumption significantly decreased except the control group. XYS significantly increased food consumption, whereas fluoxetine failed to increase food consumption unlike the model group.2. HE staining showed that neuronal cells in the hippocampus were smaller with irregular cell shape. Neuronal cell bodies and Nissl’s bodies decreased in the model group. The neuronal cell numbers improved and Nissl’s bodies was increased by XYS and fluoxetine.3. Based on the reproducible 2-DE, two downregulated and eight upregulated spots were observed in the model group unlike the control group. The related information of the protein spots were verified.4. Expression of P-arrestin2 and CRHR in hippocampus and hormone in serum and cerebrospinal fluid (CSF) showed:expression ofβ-arrestin2 decreased in the model group and CRHR2 just the opposite. They were both regulated by XYS and fluoxetine. But expression of CRHR1 had no differences among all the four groups. The serum CORT significantly increased in the model group, serum and CSF Urocortin-2 also increased. XYS and fluoxetine significantly decreased the CORT level as well as the serum and CSF Urocortin-2 levels. No significantly difference was observed in the serum and CSF levels of CRH and serum ACTH.5. Expression of β-arrestin2 mediated PP2A-Akt-mTOR signaling pathways showed: expression of PP2A b was increased in the model group. On the contrary, expression of p-mTOR and p-Akt are decreased. No significantly difference was observed in expression of PP2A c, mTOR and Akt. RT-PCR also showed that PP2A b, not PP2A c, was downregulated by XYS and fluoxetine.6. Expression of P-arrestin2 mediated ERK-BDNF signaling pathways showed: expression of p-ERK, BDNF and TrkB are decreased in the model group. All of them were upregulated by XYS and fluoxetine. No significantly difference was observed in expression of ERK.Conclusion:The rat model of liver depression syndrome was established successfully. Ten differential proteins related to liver depression syndrome were obtained and verified. And the key signal molecule β-arrestin 2 is found. Expression of P-arrestin 2 and CRHR in hippocampus and hormone in serum and CSF, β-arrestin 2-mediated PP2A-Akt-mTOR and ERK-BDNF signaling pathways were detected. We conclude that β-arrestin2 function by affect hippocampal synaptic plasticity in the pathogenesis of liver depression and syndrome. And XYS improves liver depression syndrome through modulation of P-arrestin 2-mediated pathways in hippocampus. The research has original innovation for its starting point of the results of proteomics and the key role of P-arrestin 2 in synaptic plasticity of hippocampus which mediate PP2A-Akt-mTOR and ERK-BDNF signaling pathways. |
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