| Depression as a common mental illness,seriously affect and damage people's lives,work and health.However,because of the side effects of antidepressant medicine and the effect of poor efficacy after treatment,the treatment of depression until today still is World research hot spots.Xiaoyao San' s mechanism of antidepression need to study and explore,as a classic compound of emotional disease treatment.OBJECTIVETo evaluate whether the expression of FKBP51 and FKBP4 in hippocampal neurons of chronic stress-induced hippocampal neurons can increase the expression of BDNF and improve the hippocampal neural plasticity.To reveal the Antidepressant mechanism of Xiaoyao powder treatment for regulating the neurotic plasticity.MethodsExperiment 1(Animal experiment)Adult male WISTAR rats were randomly divided into contral group,model group,Xiaoyao Powder low dose group,Xiaoyao Powder middle dose,Xiaoyao Powder high dose group,mifepristone group,Chronic mild unpredictable stress to establish rat model of depression.The effect of Xiaoyao Powder on behavior and sugar consumption in depression rats were observed.The morphological structure of hippocampal CA3 pyramidal cells was observed by HE staining.Serum corticosterone was detected by ELISA.The effect of Xiaoyao Powder on the expression of BDNF,FKBP4,FKBP51 and GR protein in hippocampal CA3 pyramidal cells of depression rats was studied by immunohistochemistry.The expression of GR,BDNF and FKBP4/FKBP51 mRNA in hippocampus of depressive model rats was observed by RT-PCR.The total protein of GR,BDNF and FKBP4/FKBP51 in hippocampus of depressive model rats were studied by Western blotting.Experiment 2(vitro experiments)The hippocampal neurons were cultured and the damage of corticosterone and the protection of mifepristone concentration were observed at the same time.2,the cultured neurons were divided into control group,cerebrospinal fluid group,corticosterone group,corticosterone + low dose of cerebrospinal fluid group,corticosterone + medium dose of cerebrospinal fluid group,corticosterone +high dose of cerebrospinal fluid Group and corticosterone + RU-38486 group.The effect of Xiaoyao Powder on the proliferation and apoptosis of neurons was observed by flow cytometry.The expression of GR,BDNF and FKBP4/FKBP51 mRNA in Hippocampal Neurons was observed by RT-PCR.The effects of Xiaoyao Powder on the expression of GR,BDNF and FKBP4/FKBP51 in rat hippocampal neurons were studied by Western blotting.The effects of Xiaoyao Powder on the expression of GR protein in rat hippocampal neurons were studied by Western blotting.FKBP4 and FKBP51 and GR combination were observed by immunoprecipitation.The effect of FKBP4 and FKBP51 on GR projection elements was observed by immunofluorescence.Experiment 3(Xiaoyao Powder Compound Research)To observe the separation of high-efficient liquid phase and the similarity of Xiaoyaosan samples.ResultsExperiment 1(Animal Experiment)There were significant differences in autonomic and investigative behavior between the model group and the control group(P<0.05).(P<0.05).Conclusion:The results showed that there was significant difference between the two groups(P<0.05),and the activity was significantly higher than that of the control group(P<0.05).The preference of the rats in the model group decreased,compared with the blank control group(P<0.05).(P<0.05).Conclusion:There is a significant difference between the two groups(P<0.05).Compared with the control group,the model control group was significantly lower than that of the normal control group(P<0.05).The number of pyramidal cells in the hippocampal CA3 region of the depressed rats increased significantly after the use of high dose Xiaoyaosan and mifepristone,and the number of pyramidal cells was significantly higher than that of the model control group(P<0.05).4,immunohistochemistry and HE staining positive cells showed that compared with the blank control group,depression group BDNF positive cells were significantly reduced,and Xiaoyao Powder and mifepristone significantly improved the situation,BDNF positive cells significantly increased(P<0.05).The positive cells of GR and FKBP4 in model group were significantly lower than those in blank control group,while high dose Xiaoyaosan and mifepristone significantly increased the number of positive cells(P<0.05).The number of FKBP51 positive cells in the model group was significantly higher than that in the blank control group,while the high dose Xiaoyaosan and mifepristone significantly reduced FKBP51 positive cells.The results of quantitative real-time PCR showed that the mRNA levels of GR,BDNF and FKBP4 were significantly decreased and the FKBP51 mRNA level was significantly increased in the model control group compared with the blank control group.Compared with the model control group,Xiaoyao Powder significantly increased the levels of GR,BDNF,FKBP4 mRNA and down-regulated FKBP51 mRNA(P<0.05).Western blotting showed that the average optical density of GR,BDNF and FKBP4 in the model control group decreased and the average optical density of FKBP51 increased compared with the blank control.Compared with model control group,Xiaoyao Powder significantly increased the mean optical density of GR,BDNF,FKBP4 and down-regulated FKBP51(P<0.05).Experiment 2(vitro experiment)By MTT method the best corticosterone concentration(IC50)223 ?mol/L and the best RU-38486 protective concentration of 2.078 ?mol/L were found out.The MTT results showed that the survival rate of neuronal cells was significantly lower than that of cerebrospinal fluid group(P<0.01)or(P<0.05).The hippocampal neurons exposed to corticosterone showed the characteristics of neurodegeneration,including loss of synapses.The expression of RU-38486 was significantly higher than that of the control group(P<0.05).And Xiaoyao powder can reduce synaptic damage.The results of flow cytometry showed that corticosterone significantly reduced cell viability,and Xiaoyao powder and RU38486 could increase the rate of corticosterone.The DNA content of GO/G1 and G2/M cells increased after corticosterone treatment,while the DNA content of S phase decreased after treatment.Xiaoyao Powder reduced the DNA content of GO/G1 and G2/M cells and increased the DNA content of S phase cells.Corticosterone significantly increased the apoptosis.Compared with corticosterone,Xiaoyaosan and RU38486 could decrease the rate of apoptotic cells The Immunofluorescence assay showed that there was no significant difference in the cytoplasmic translocation of GR(green fluorescence)between the cerebrospinal fluid group and the control group(P>0.05).Compared with the cerebrospinal fluid group,the GR in the corticosterone group was significantly increased.Compared with RU38486 group,Xiaoyaosan and RU38486 reduced the nuclear translocation of GR,and increased the number of GR in the cell membrane(P<0.05).The results of quantitative real-time PCR showed that the mRNA levels of GR,BDNF and FKBP4 were significantly decreased,and the levels of FKBP51 mRNA in hippocampal neurons of corticosterone group were significantly increased compared with the blank control group(P<0.05)The levels of GR,BDNF and FKBP4 mRNA were significantly increased and FKBP51 mRNA levels were significantly decreased(P<0.05)compared with that in RU38486 group.Western blotting showed that the protein levels of GR,BDNF and FKBP4 were significantly down-regulated and the expression of FKBP51 protein was significantly up-regulated in corticosterone group(P<0.05).Compared with the corticosterone group,the protein levels of GR,BDNF,FKBP4 and FKBP51 were significantly decreased in the Xiaoyaosan and RU38486 groups,and there was significant difference(P<0.05).Western blotting showed that GR expression in corticosterone group was significantly lower than that in blank control group,but Xiaoyaosan and RU-38486 significantly increased the expression of GR in cytoplasm and nucleus P<0.05).9,EMSA electrophoresis showed that FKBP4 and GR combined,and FKBP51 does not combine with GR.10,double luciferase gene analysis showed that FKBP4 could significantly increase GRE transcription activity(P<0.05 or P<0.01)in a dose-dependent manner,and FKBP51 significantly reduced GRE transcription activity(P<0.01).Experiment 3(Xiaoyao Powder Compound Research)(HPLC)to achieve excellent separation,in which the content of paeoniflorin,glycyrrhizin,ferulic acid and glycyrrhizic acid were 21.53mg/g,5.32mg/g,4.56mg/g and 6.41mg/g,and all similarity values for the nine samples are greater than 0.95.conclusionXiaoyaosan can improve the performance of stress rats and the stress-induced hippocampal neurons.Xiaoyaosan by reducing the concentration of blood GC,Improve the expression of GR,BDNF and FKBP4,reduce the expression of FKBP51,and realize the protection of hippocampal neurons. |
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