Effect Of Xiaoyao San Against Chronic Stress-induced Injury On GABRD And Cacnb4 Gene Expression In Hippocampus Of Rats

Objective To observe effects of Xiaoyao San anti-chronic stress injury on transcription level of GABRD,Cacnb4 gene mRNA and translation products on the rat hippocampus,verify the results of the Agilent expression profile microarray analysis,In order to further explore the Xiaoyao San at the level of gene to anti-chronic stress injury mechanism.Method 45 SPF male Wistar rats were randomly divided into A.Normal control group;B.Chronic stress model group;C.Xiaoyao San intervention group,15 rats in each group.Continuous chronic stress stimulated for 21 days.Body weight test and open-field test were used to evaluate the behavior of each group of animals to test model is successful or not.The relative expression of GABRD mRNA and Cacnb4 mRNA was detected by Real-time fluorescence quantitative PCR.To detected GABRD and Cacnb4 protein transcription level by immunohistochemical staining and Western blot to verify the results of the Agilent expression profile microarray analysis.Results1.Weight testThere was no significant difference in body weight between the three groups before the experiment?P>0.05?;After the experiment,the body weight of rats in chronic stress model group was significantly lower than that in normal control group,and the difference was statistically significant?P<0.01?;Xiaoyao San intervention group rats were significantly higher body weight than the chronic stress model rats,and the difference was statistically significant?P<0.01?.2.Open-field test2.1 Horizontal movement scoreBefore the experiment,the horizontal movement scores of rats in each group were compared,the difference was not statistically significant?P>0.05?,so each group of rats is comparable.After the experiment,horizontal exercise scores compared to normal control rats,chronic stress model rats significantly reduced and the difference was statistically significant?P<0.01?;The scores of horizontal movement in Xiaoyao San intervention group were significantly higher than those in chronic stress model group,and the difference was statistically significant?P<0.01?,But lower than normal control rats,and the difference was statistically significant?P<0.01?.2.2 Vertical movement scoreBefore the experiment,the scores of vertical movement of rats in each group were compared,the difference was not statistically significant?P>0.05?,so each group of rats is comparable.After the experiment,vertical exercise scores compared to normal control rats,chronic stress model rats significantly reduced and the difference was statistically significant?P<0.01?;The scores of vertical movement in Xiaoyao San intervention group were significantly higher than those in chronic stress model group,and the difference was statistically significant?P<0.01?.3.Agilent expression profiling chip pre-study analysis resultsCompared to normal control rats,the relative expression of GABRD gene in hippocampus in chronic stress model group was significantly lower,and the difference was statistically significant?P<0.05?,Xiaoyao San intervention group has the tendency to up-regulate GABRD gene expression in hippocampus of chronic stress rats,but the difference was not statistically significant.Compared with the normal control group,the relative expression of Cacnb4 in the hippocampus of chronic stress model group was significantly lower,and the difference was statistically significant?P<0.01?,and after Xiaoyao San intervention,the difference was statistically significant too?P<0.05?.4.The transcription level of GABRD and Cacnb4 mRNA in hippocampus of rats in each groupCompared with the normal control group,the transcription level of GABRD mRNA in hippocampus of chronic stress model rats was significantly down-regulated,the difference was statistically significant?P<0.01?;Xiaoyao San dry GABRD mRNA transcription level was not up-regulated,and significantly lower than the transcription level of the chronic stress model group,the difference was statistically significant?P<0.01?,and compared with the normal control group,the difference was statistically significant?P<0.01?.Compared with the normal control group,the transcription level of Cacnb4 mRNA in hippocampus in chronic stress model group was significantly up-regulated,the difference was statistically significant?P<0.05?;Xiaoyao San dry Cacnb4 gene mRNA transcription level was significantly reduced,compared with chronic stress model group,the difference was statistically significant?P<0.01?,compared with the normal control group,the difference was statistically significant?P<0.01?.5.Immunohistochemical staining of GABRD and Cacnb4 protein in hippocampus of rats in each groupCompared with the normal control group,the relative expression of GABRD protein in chronic stress model group was significantly decreased,the difference was statistically significant?P<0.01?;Xiaoyao San dry GABRD protein expression was significantly up-regulated,compared with chronic stress model group,the difference was statistically significant?P<0.01?,but compared with the normal control group,the difference was not statistically significant.Compared with the normal control group,the relative expression of Cacnb4 protein in chronic stress model group was significantly higher,the difference was statistically significant?P<0.05?,Xiaoyao San dry Cacnb4 protein expression was significantly down-regulated,compared with chronic stress model group,the difference was statistically significant?P<0.01?,And lower than the normal control group,the difference was statistically significant?P<0.01?.6.The expression of GABRD and Cacnb4 protein in hippocampus of rats in each groupCompared with the normal control group,the relative expression of GABRD protein in chronic stress model group was significantly lower,the difference was statistically significant?P<0.01?;Compared with chronic stress model group,Xiaoyao San dry GABRD protein relative expression was significantly increased,the difference was statistically significant?P<0.01?,but compared with the normal control group,the difference was not statistically significant.Compared with the normal control group,the relative expression of Cacnb4protein in chronic stress model group was significantly higher,the difference was statistically significant?P<0.05?,compared with chronic stress model group,Xiaoyao San dry Cacnb4protein relative expression decreased significantly,the difference was statistically significant?P<0.01?,and lower than the normal control group,the difference was statistically significant?P<0.01?.Conclusion1.Chronic stress injury stimulates the down-regulation of GABRD gene expression,inhibit cell membrane chloride ion influx and membrane hyperpolarization,increased neuronal excitability,may be the one of the mechanisms for caused by a series of neurological and mental disorders.Xiaoyao San can fight this expression change of GABRD gene,effective regulation of anxiety,stress,depression,insomnia and other neurological and mental disorders caused by chronic stress.2.Chronic stress may stimulate the expression of Cacnb4 gene transcriptional and translation products,cause changes in the number or function of calcium channels or release of intracellular calcium stores,lead to Ca²⁺overload of intracellular,to making the function of hippocampal neurons disorder.Xiaoyao San may play a role in protecting hippocampal neurons by antagonizing Cacnb4 overexpression.