| Part 1 Effect of Huazhuo Jiedu Shugan Decoction on Anti-depression by Intervening Hippocampal Neurogenesis in MiceObjective: To observe the effects of Huazhuo Jiedu Shugan Decoction on depression-like behavior and hippocampal neurogenesis in CUMS mice.Methods:45 male ICR mice were used to establish the depression model of CUMS,which were randomly divided into normal group,CUMS model group,western medicine fluoxetine group,Chinese medicine high-dose and low-dose groups.Depression-like behavior of mice was observed by sugar water preference test,tail suspension test and elevated cross maze behavior test.The tissue morphology and nerve cell status of mouse hippocampus dentate gyrus were observed by Neptune staining.The expression of neuronal cell proliferation marker Ki-67 and neuronal differentiation marker DXC in the dentate gyrus of the hippocampus of mice was detected by immunofluorescence assay.Results: 1.Compared with the normal group,the sugar water preference index in the model group was significantly decreased,and it was increased in the western medicine group and the high and low dose Chinese medicine group.In the tail suspension experiment,the immobility time of the model group was significantly prolonged,and it was significantly improved after intervention in each dose of Chinese medicine group and western medicine group.The elevated cross maze showed that compared with the normal group,the visits and residence time of the open arm in the model group were significantly reduced,and the western medicine group and the high and low dose Chinese medicine groups were improved.2.The results of Nissl staining showed that compared with the normal group,the synthesis and number of cells in the model group were decreased and the arrangement of cells was loose.After treatment with traditional Chinese medicine and fluoxetine,the hippocampal neurons were large and abundant,with neat and compact arrangement,no obvious rupture of Nissolith and no loss of neuronal staining were observed.3.Ki-67 and DCX positive cells in the dentate gyrus of the hippocampus of the model group were significantly reduced compared with the normal group,and were significantly improved after intervention in each dose of Chinese medicine group and the western medicine group.Summary: 1.Huazhuo Jiedu Shugan Decoction can improve the depression-like behavior of mice induced by CUMS.2.Huazhuo Jiedu Shugan Decoction Decoction can restore normal tissue morphology and cell survival of hippocampal dentate gyrus of CUMS mice.3.Huazhuo Jiedu Shugan Decoction can promote the proliferation and differentiation of cells in the dentate gyrus hippocampus of CUMS mice,and promote hippocampal neurogenesis.Part2 Proteomic screening protein related to the anti-depressive effect of Huazhuo Jiedu Shugan Decoction though hippocampal neurogenic in miceObjective: Based on proteomics,to discover the related biological process of Huazhuo Jiedu Shugan Decoction regulating hippocampal neurogenesis in CUMS mice,and to further explore the molecular mechanism of the anti-depressive effect of Huazhuo Jiedu Shugan Decoction.Methods:Labfree proteomics was used to detect the differential proteins expressed in the hippocampus of the normal group,the model group and the traditional Chinese medicine treatment group(high dose),and the molecular mechanism related to the antidepressant effect of Huazhuo Jiedu Shugan Decoction was explored through GO analysis and KEGG-Pathway bioinformatics analysis.The candidate differentially expressed proteins were verified by Western blot.Results: 1.A total of 5080 proteins were identified by liquid/mass spectrometry,of which 4384 proteins contained quantitative information.22 proteins were up-regulated and 24 proteins were down-regulated in the CUMS vs Control comparison group,while 32 proteins were up-regulated and 17 proteins were down-regulated in the Chinese medicine treatment group vs CUMS comparison group.2.The distribution of differentially expressed proteins in the Chinese medicine treatment group compared with the CUMS model group in the GO secondary annotation: in the cell composition,it mainly involves cells,organelles and cell membranes;in the molecular function,it mainly involves protein binding,catalytic activity and molecular regulation function;Biological processes mainly involve cellular processes,single biological processes and biological regulation.3.GO enrichment analysis results show that: after the Chinese medicine treatment,the enrichment of mouse hippocampal differentially expressed proteins in biological processes is mainly involved in the metabolism of serotonin,regulation of protein activation cascade,regulation of endocytosis cycle,etc.Among them,there are also a lot of enrichment in the biological processes related to neurogenesis,such as stem cell differentiation and development,cell tissue growth,etc.,which will be focused on.4.Cluster analysis based on KEGG pathway enrichment found that: Differentially expressed proteins upregulated in mouse hippocampus after treatment with traditional Chinese medicine involved in biological processes associated with depression include,cAMP signaling pathways,chemokine signaling pathways,cholinergic synapses,synapses dopamine,serotonin synapses,among them,cAMP signaling pathway and chemokine signaling pathway related to neurogenesis and synaptic plasticity would be further studied.5.Western blot results confirmed that compared with the control group,The key protein AC in upstream and middle of cAMP signaling pathway was significantly decreased in the CUMS group,and was significantly increased after 21 days of Chinese medicine treatment;Phosphorylation levels of PKA and CREB were significantly decreased in the CUMS group,and were significantly increased after 21 days of Chinese medicine treatment.Compared with the control group,the levels of PI3 K and Akt protein phosphorylation in the chemokine signaling pathway were significantly decreased in the CUMS group,and were significantly increased after 21 days of Chinese medicine treatment.Summary:1.Based on the results of proteomics analysis,the mechanism of Huazhuo Jiedu Shugan Decoction regulating hippocampal neurogenesis in depressed mice may involve cAMP signaling pathway and chemokine signaling pathway.2.Huazhuo Jiedu Shugan Decoction can improve the expression of the upstream key protein Ac in the cAMP signaling pathway in the hippocampus of CUMS mice,and the phosphorylation of the downstream PKA and CREB proteins as well as the phosphorylation of the key proteins PI3 K and ATK in the chemokine signaling pathway.The relevant mechanism of Huazhuo Jiedu Shugan Decoction with hippocampal neurogenesis needs to be further studied.Part 3 Study on the mechanism of Huazhuo Jiedu Shugan Decoction regulating hippocampal neurogenesis in mice through BDNF/GSK-3β /β-Catein signaling pathwayObjective: To explore the possible internal mechanism of Huazhuo Jiedu Shugan Decoction regulating BDNF/GSK-3β/β-catenin signaling pathway,improving hippocampal neurogenesis in CUMS mice,and playing an antidepressant role.Methods : Thirty male ICR mice were randomly divided into normal group,model group,western medicine group,high-dose and low-dose Chinese medicine groups.Western blot was used to detect the expression of BDNF protein,GSK-3β phosphorylation,β-catenin protein expression and nuclear translocation in the hippocampus of mice in each group.The expression levels of β-catenin downstream target genes Cyclin D1,c-myc,Neuro D1 and NGN2 were detected by Realtime PCR.Results: 1.Western Blot results showed that compared with the normal control group,the expression of BDNF protein and β-catenin total protein in the hippocampus of mice in CUMS model group was significantly decreased,the phosphorylation of GSK3β protein and the nuclear translocalization ofβ-catenin were significantly decreased,and the expression of β-catenin was significantly increased after treatment with high and low dose of traditional Chinese medicine and Western medicine.2.Q-PCR results showed that the mRNA levels of cyclin D1,c-myc,neuro D1 and NGN2 in the model group were significantly lower than those in the normal group,and the mRNA levels of cyclin D1,c-myc,neuro D1 and NGN2 in the model group were increased compared with those in the model group.Summary: 1.Chronic unpredictable stress can successfully induce depression-like behavior in mice,accompanied by changes in hippocampal BDNF/GSK-3β/β-catenin signaling pathway proteins and related target genes of downstream neurogenesis.2.Huazhuo Jiedu Shugan Decoction promotes hippocampal neurogenesis and plays an anti-depressive role by regulating hippocampal BDNF/GSK-3β/β-catenin signaling pathway proteins and downstream target genes.Conclusion:1.Huazhuo Jiedu Shugan Decoction can promote hippocampal neurogenesis and play an antidepressant role in CUMS mice.2.Proteomic studies have found that the biological process of Huazhuo Jiedu Shugan Decoction improving hippocampal neurodevelopment in CUMS mice may involve changes in cAMP signaling pathway,chemokine signaling pathway and related proteins.3.Huazhuo Jiedu Shugan Decoction promotes hippocampal neurogenesis and plays an antidepressant role in CUMS mice by regulating hippocampal BDNF/GSK-3β/β-catenin signaling pathway proteins and downstream target genes. |
PDF Full Text Request