| 1 BackgroundSyndrome of stagnation of liver qi and spleen deficiency is common in traditional Chinese medicine, which involves more than 40 diseases, including liver and gallbladder diseases, intestinal diseases, digestive system diseases and mental illness such as depression etc. Xiaoyaosan is a classic formula for treating syndrome of stagnation of liver qi and spleen deficiency. However, the biological basis of this syndrome is not yet clear, and the mechanism of Xiaoyaosan in treating this syndrome is not fully understood too.Stress is body’s unspecific adaptive responding to all kinds of stimulation. This response has its processes and stages, with a high degree of relevance with the formation of syndrome of stagnation of liver qi and spleen deficiency, so we can establish syndrome model by chronic immobilization stress to simulate the etiology and pathogenesis of the formulation process.Recent research results suggested that there was a wide range of pathophysiological changes, involving multiple systems, mainly including nerve-endocrine system, immune system, digestive system, brain gut axis, the hypothalamus-pituitary-adrenal axis, etc. Synapse plays an indispensable role in the transmission of neural information. Neurons can generate processes or synaptic connections, and synapses and other tiny structures can be modified by various factors, which is the synaptic plasticity of the nervous system. Studies have indicated that stress response can lead to changes in the synaptic plasticity of the nervous system, especially in the hippocampus, which may affect learning and memory, the structure of the brain and various physiological processes.N-Methyl-D-Aspartate receptors, calmodulin protein kinase, Kalirin, Rac are closely related with the synaptic plasticity changes. In 2007, Zhong Xie and Deepak P. Srivastava and their group first proposed NMDAR/CaMK Ⅱ/Kalirin/Rac pathway. Their research suggested that activation of this pathway could lead to rapid enlargement of existing spines, thus played the role of synaptic plasticity.Therefore, we can use chronic immobilization stress to establish rat model of syndrome of stagnation of liver qi and spleen deficiency, then, using NMDAR/CaMK Ⅱ/Kalirin/Rac signaling pathway as the breakthrough point, from the aspects of synaptic plasticity to study biological basis of syndrome of stagnation of liver qi and spleen deficiency and the treatment mechanism of Xiaoyaosan.2 Objective(1) To observe the changes of hippocampal NMDAR/CaMK Ⅱ/Kalirin/Rac pathway in rats with syndrome of stagnation of liver qi and spleen deficiency, so as to explore the biological basis of it, and to explore the relationship between this syndrome and the synaptic plasticity changes.(2) To investigate the treatment mechanism of Xiaoyaosan against syndrome of stagnation of liver qi and spleen deficiency, so as to provide experimental basis for treating stress-induced diseases in clinical.3 MethodsThis experiment mainly consists of four parts.In the first part, the rat model was established by immobilization stress for 21 days. With Xiaoyaosan as drug intervention, with fluoxetine as positive control, the model was recognized as model of syndrome of stagnation of liver qi and spleen deficiency from animal characterization, food intake, bodyweight, open field test, novelty suppressed feeding test, sucrose preference test, as well as effect of Xiaoyaosan.In the second and third part, based on successful model establishment, molecular biology techniques such as RT-PCR, Western blot method were used to investigate the change of NMDAR/CaMKII Kalirin/Rac pathway in hippocampus in the model rats, and the regulation effect of Xiaoyaosan was also investigated, so as to study the synaptic plasticity in rat models and adjustment of Xiaoyaosan.In the fourth part, ELISA methods was used to detect concentration of synaptic related proteins, and confirmed that Xiaoyaosan can adjust hippocampal synaptic plasticity in rats with syndrome of stagnation of liver qi and spleen deficiency.4 Mainly results(1) Through the chronic inmmobilization stress for 21 days, food intake of rats in all groups had no statistical significance. Compared with the control group, body weight of rats in the model group increased slowly (P< 0.01), but those of rats in fluoxetine group and Xiaoyaosan group were significantly different compared with the model group (P< 0.05). Open field test results showed that total distance moved in 5 minutes and number of square cross were significantly decreased in the model group (P< 0.05), and those of Xiaoyaosan group and fluoxetine group increased significantly, with significant difference compared with the model group (P< 0.05). Sucrose preference test showed that sucrose consumption rate of the model group was obviously lower than that of the control group (P<0.05), and that of the fluoxetine group was significantly higher than the model group (P< 0.05). All the results above suggested that rat model of syndrome of stagnation of liver qi and spleen deficiency was successfully established.(2) Western blot results showed that:Compared with the control group, the hippocampal protein expression of NR1, NR2B, CaMKⅡ, and Rac decreased significantly (P< 0.05 or P< 0.01). After treatment with fluoxetine, protein expression levels of NR2B, CaMKⅡ, and Rac increased (P< 0.05), NR2B and CaMKⅡ protein expression in Xiaoyaosan group increased too (P< 0.05), with statistically significant difference compared with the model group. There was a decrease trend of NR2A and Kalirin protein expression, but there was no significant difference among the four groups (P> 0.05).(3) Real-time PCR results showed that:Compared with the control group, NR2B, CaMKⅡa, Kalirin gene expression levels decreased (P< 0.05). After treatment with fluoxetine, CaMKⅡa, Kalirin gene expression levels increased (P< 0.05), and after treatment with Xiaoyaosan, NR2B and CaMKⅡa gene expression levels increased (P< 0.05), with significant difference compared with the model group. Gene expression of NR2A and Racl was not significantly different among the four groups (P> 0.05).(4) ELISA results showed that:Compared with the control group, SYP, MAP-2 content in model group in the hippocampus was significantly decreased (P<0.05), Nogo-A and F-actin level increased (P< 0.05). After treatment with fluoxetine, the MAP-2 levels increased (P<0.05), Nogo-A and F-actin content reduced (P<0.05), SYP level in Xiaoyaosan group increased (/)<0.05), and Nogo-A and F-actin level reduced (P<0.05), the difference was statistically significant. GAP-43 level had a lower trend in model group, but there was no statistically significant difference among the groups (P> 0.05).5 Conclusions(1) Rat model of syndrome of stagnation of liver qi and spleen deficiency was successfully reproduced through chronic immobilization stress for 21 days.(2) Stress could cause decrease of protein and gene expressions of NMDAR/CaMKⅡ /Kalirin/Rac pathway in rat hippocampus, which indicated the inhibition of signal pathway transmission by stress.(3) Synaptic related factors SYP, MAP-2, Nogo-A, GAP-43, F-actin were increased or decreased in rat hippocampus after chronic inmmobilization stress, which suggested that the synapse might change after stress.(4) Protein and gene expression of hippocampal NMDAR/CaMK Ⅱ/Kalirin/Racl pathway could be raised by Xiaoyaosan in rats with syndrome of stagnation of liver qi and spleen deficiency. Synapse related proteins such as skeleton protein F-actin could also be adjusted by Xiaoyaosan, leading to change of the synaptic structure, further might change its function. This might be therapeutic mechanism of Xiaoyaosan for treating syndrome of stagnation of liver qi and spleen deficiency.
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