Establishment Of Rat Model Of Depression With Liver Depression And Spleen Deficiency And Its Biological Basis

1. ObjectiveTo explore the time window of rat depression model induced by chronic unpredictable mild strees(CUMS) and reserpine antagonisim, and prove it by treating the two kinds of model with TCM fomula. Explore the molecular mechanism of both protein and gene level of the syndrome of liver depression with spleen-qi deficiency.2. Methods(1) The rat depression model was established by applying CUMS or intraperitoneal injection of reserpine. The syndromes were distinguished in rats through ethological determination and equivalent transforming from clinical symptoms to macroscopic surface features. The content of serum5-hydroxytryptamine(5-HT) was detected by using enzyme-linked immunosorbent assay(ELISA) and content of5-HT in cerebral hippocampal CA2zone was detected by using immunohistochemistry technique.(2) Adjust the number of rats in each group according to the proportion of liver pression and spleen qi deficiency syndrome rats at each time point from experiment1. Still use CUMS and intraperitoneal injection of reserpine as modeling methods, syndrome differentiation methods same with the experimental one, according to the syndromes of time window of two kinds of model experiments of a draw, reserpine group liver stagnation and spleen deficiency syndrome in rats by intragastric injection drug powder in4weeks later, group CUMS with liver qi stagnation and spleen deficiency rat gastric perfusion in bulk stress6weeks later to take Xiaoyao powder, after syndromes occurring changes in rats to stop the use of Xiaoyao Powder, but the target detection and data statistics will be included. The influence of Xiaoyao Powder is messured through ethological determination and equivalent transforming from clinical symptoms to macroscopic surface features.(3) Using Elisa method to detect the content of serum vascular angiotensin II (Ang Ⅱ), norepinephrine (NE), homocysteine (HCY),5-hydroxytryptamine (5-HT), gamma aminobutyric acid (GABA), corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cyclic phosphate adenosine monophosphate (cAMP), Dobaamine (DA), beta endorphin, superoxide dismutase (SOD), testosterone (T), estradiol (E2), tumor necrosis factor alpha (TNF-α). Detecting NE, DOPAC, DA,5-HIAA, HVA and5-HT content in brain tissues by high performance liquid chromatography. CORT expression was detected by immunohistochemistry in adrenal glan.(4) Analysis the digital gene expression in the brain tissue and liver tissue of rats of liver stagnation and spleen deficiency. The first step is sample quality detection, the second step is to prepare DGE library, third step computer sequencing, fourth step is bioinformatics analysis.3. Results(1) According to the weight of rats in each group at each time point, sucrose preference, behavioral test, RGB worth of macro characterization of tongue color, CUMS group and reserpine group were both in the depression after2weeks of intervention, CUMS group had syndrome of liver depression and spleen deficiency6weeks after stress, reserpine group had syndrome of liver depression and spleen deficiency4weeks after injecting. After8weeks of intervention, the level of brain5-HT was significantly lower in CUMS group and reserpine group than that in normal group (P<0.05), the content of5-HT in serum and intestinal tissue continued to rise (P<0.05), and the degree of elevation was different between CUMS group and reserpine group in different periods.(2) For each group, liver depression with spleen deficiency rats intragastrically in Xiaoyao Powder, weight, their sucrose preference, the open field test in lattice frequency and total distance, the forced swimming test in immobility time were improved to some extent, compared with the experimental one. Rat tongue R values are of no significant difference between the two the experiment. At the same time, in this study we also take macro scale characterization of acquisition points in rats before and after the usage of Xiaoyao Powder, which increased gradually in rats after the usage of XiaoYao Powder, proving that Xiaoyao Powder is effective for syndrome of liver stagnation and spleen deficiency in depression.(3) When the syndrome of Spleen deficiency and liver stagnation occurs in stress depression and reserpine antagonist depression, those whose content rise in serum are:Ang II, HCY,5-HT, CRH, ACTH, those whose content decreased in serum are:GABA, cAMP, DA, beta endorphin, SOD, T, E2. The content of content of TNF-a in serum didn’t change; while for brain tisuue, NE, DOPAC, DA,5-HIAA, HVA and5-HT all decreased. CORT expression in adrenal gland increased. When stress induced depression of liver stagnation and spleen deficiency syndrome occurs, serum NE increased first and then decreased, while when spleen deficiency syndrome of stagnation of liver Qi reserpine antagonistic depression occurs, the content of serum NE decreased continuously.(4) For all rats with liver qi stagnation and spleen deficiency syndrome:In brain tissue,190genes were up-regulated,684genes were down regulated in CUMS group, compared with normal rats;155genes up-regulated,476genes were down regulated in reserpine group compared with normal rats. In liver tissue,18genes were up-regulated,508genes were down regulated in CUMS group, compared with normal rats;88genes up-regulated,420genes were down regulated in reserpine group compared with normal rats. Target genes differentially expressed mainly in5-, GABA synaptic dopamine serotonin synapses, synaptic, calcium signal transduction pathway, neurotrophic factor signaling pathway, gonadotropin releasing hormone signal transduction pathway, NO and glutamate signaling pathway, antigen processing and presentation process, phagocytosis, intestinal immune network.4. Conclusion(1) Time window of depression of liver stagnation and spleen deficiency syndrome in rats model. Time window of liver stagnation and spleen deficiency is6-8weeks after stress in CUMS rats model and4-8weeks after the injection in reserpine rats model. The test to confirm the time window after the formula is reliable.(2) The downstream biological mechanism model in rats with depression of liver stagnation and spleen deficiency syndromeCRH-ACTH-CORT pathway is activated after positive feedback regulation and participated in the process of forming stress depression liver stagnation and spleen deficiency syndrome;increase of serum Ang II, HCY,5-HT and decrease of GABA, cAMP, DA, SOD, T, E2as well as decrease of NE, DOPAC, DA,5-HIAA, HVA and5-HT content in brain tissue are the common mechanism of stress-induced depression and reserpine reversal type depression liver stagnation with spleen deficiency formation.(3) Upstream biological mechanism of rat model of liver stagnation and spleen deficiency syndrome with depressionThe two depression model of liver stagnation and spleen deficiency syndrome are both related to the abnormalities of5-HT synapses, GABA synapses and serotonin synapses. The PKC gene and PKA gene is a key gene on the expression of the opposite effect, while Gi/o gene cluster also plays an important role in the depression of liver stagnation and spleen deficiency. Considering the downstream biological mechanism of liver stagnation and spleen deficiency syndrome of depression, it can be speculated that central5-HT and peripheral5-HT may be two independent systems, while there may be a common pathway of the occurrence and development of central PKA and peripheral cAMP may in the participation in the depression of liver stagnation and spleen deficiency syndrome;The two types of depression of liver stagnation and spleen deficiency syndrome models are both related to the abnormal function of calcium signal transduction pathways.The two types of depression of liver stagnation and spleen deficiency syndrome models are both related to immunity, mainly because of the down regulated expression of major histocompatibility complex gene Ⅰ (MHCI) and major histocompatibility complex gene Ⅱ (MHCII). Considering the downstream biological mechanism of liver stagnation and spleen deficiency syndrome, it can be speculated that the inflammatory factor TNF-α may not participate in the immune related pathophysiological process of depression of liver stagnation and spleen deficiency syndrome.