| ObjectivesTo observe the changes of acetylcholine metabolism of hippocampal of depression models on rats and the impact of Chaihu Shugan Powder on it. To further explore the function of the cholinergic nervous system to the occurrence and development of depression and the antidepressant mechanism of Chaihu Powder.Methods60 male Sprague-Dawley rats of 4～5 month old from which those with scores higher than 120 or less than 30 by open-field test had been removed were randomly divided into four groups and 15 rats in every group: normal control group, model control group, western medicine treatment control group and Chinese medicine group. The rats in control group were conventionally fed in the manner of 5 per cage without any stimulation and treatment. The Rats in model control group, western medicine control group and Chinese medicine group were fed in the manner of one per cage with mild chronic unpredictable stimulation for 21 days. Fluoxetine and Chaihu Shugan Powder were given respectively to the rats of western medicine treatment control group and Chinese medicine group during the course of model replication. The rats of model group were given normal saline in the same volume. After twenty-one days, behavior changes in rats were detected through open-field test, sugar consumption test and body weight; expression changes of acetylcholine transferase and acetylcholinesterase in hippocampus of rats brain were observed with immunochemistry staining; ChAT mRNA expression was detected with RT-PCR ; the activity of AChE in hippocampus of rats brain tissue was measured with colorimetric method; ultrastructural changes and apoptosis in hippocampal CA3 area of brain tissue were observed respectively with electron microscopy and flow cytometry.Results1. Chaihu Shugan Powder increased the body weight, sugar preference degrees, scores of horizontal activity and scores of vertical activity of depression model rats. On the 7th day of model replication, model group had the significantly lower scores of horizontal activity and vertical activity compared with normal control group (P<0.05), and preference degrees to sugar and weight showed a trend of decrease but there was no significant difference (P> 0.05); compared with model group, western medicine group and Chinese medicine group got higher scores of horizontal activity and vertical activity(P <0.05), and there was no significant difference in weight and sugar preference degrees (P> 0.05); On the 14th day of model replication, compared with normal control group, model group had the significantly lower scores of horizontal activity and vertical activity, weight and sugar preference degrees (P <0.05～0.01); compared with model group, western medicine and Chinese medicine group got significantly higher scores of horizontal activity and vertical activity(P <0.05～0.01), and weight and sugar preference degrees increased but there was no significant difference (P> 0.05 );On the 21st day of model replication, compared with normal control group, the scores of horizontal activity and vertical activity, weight and sugar preference degrees in model group continued to decrease significantly (P <0.01～0.001); compared with model group, western medicine group and Chinese medicine group got significantly higher scores of horizontal activity and vertical activity, weight and sugar preference degrees (P<0.05～0.001).2. Chaihu Shugan Powder reduced the protein and mRNA expression of ChAT of hippocampus of depression model rats .RT-PCR results showed that expression levels of ChAT mRNA in hippocampus of rats of model group were significantly raised compared with those of normal control group (P <0.05); compared with those of model group, expression levels of ChAT mRNA in hippocampus of rats of western medicine group and Chinese medicine group was significantly reduced (P <0.05); However, expression levels of ChAT mRNA in western medicine group were significantly lower than those of Chinese medicine group. The results of immunochemistry staining showed that the protein expression of ChAT of hippocampus of model group was significantly increased compared with that of normal control group (P <0.05); Compared with model group, western medicine group and Chinese medicine group had significantly lower ChAT protein expression of hippocampus of rats (P <0.05), and the protein expression of ChAT of western medicine group was significantly decreased than that of traditional Chinese medicine group.3. Chaihu Shugan Powder reduced the activity and protein expression of AChE of hippocampus of depression model rats. The results of colorimetric method showed that AChE activity in hippocampus of model group was significantly increased compared with that of normal control group (P <0.05); Compared with that of model group, AChE activity in hippocampus of western medicine group and Chinese medicine group was significantly decreased (P < 0.05). The results of immunochemistry staining showed that AChE protein expression was significantly increased in hippocampus of model group compared with that of normal control group (P <0.05); Compared with model group, western medicine group and Chinese medicine group had significantly . lower AChE protein expression in hippocampus (P <0.05).4. Chaihu Shugan Powder inhibited apoptosis in hippocampus of depression model rats. Flow cytometry test results show that model group had a marked increase in average number of apoptotic cells in hippocampal CA3 area compared with normal control group (P <0.05); Compared with model group, the average number of apoptotic cells in hippocampal CA3 area of western medicine group and that of Chinese medicine group decreased significantly (P <0.05). At the same time, electron microscopy results also showed that apoptotic cells in the hippocampus mainly concentrated in the model group, western medicine group and Chinese medicine.Conclusion1. Depression model rats established by the method of CMUS combination with being isolated can simulate the behavioral performance of depression; the method can be used to establish depression model.2. Chaihu Shugan Powder can improve the depressive symptoms of experimental animals.3. Cholinergic nervous system is closely related to the occurrence and development of depression.4. Antidepressant function of Chaihu Shugan Powder may be related to its inhibition to the protein and mRNA expression of ChAT, and to reducing the activity and protein expression of AChE and neural cell apoptosis in hippocampus of the brain tissue.
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