Anti-depressive Effects Of Jiao-Tai-Wan On Depression By Inhibiting Neuroinflammation And Microglia Activation

Depression is a familiar mood disorder characterized with persistent depression,helplessness and hopelessness,with high rates of relapse,disability and suicide,placing a heavy burden on others and society.However,the cure rate of depression is low and the recurrence rate is high.Its pathogenic factors include biological,psychological and social environment as well as complex neurofeedback loops.Therefore,the pathogenesis of depression is very complex.Microglia-mediated neuroinflammation is an important pathological feature of depression.Exploring effective strategies to improve microglia-mediated functional phenotypes and inhibit neuroinflammation may be one of the breakthroughs in the development of drugs for depression.At present,single-agent therapy has limited effect on the pathological treatment of complex depression.Therefore,effective multi-target combined therapy is very necessary.Traditional Chinese medicine not only has a variety of chemical components and multiple targets,but also is significant clinical efficacy and safety in the therapy of depression.At the same time,transcriptomic can study the transcription of genes in cells and their regulation rules under specific conditions from the overall level,which may systematically and deeply reveal the material foundation and mechanism of the efficacy of Chinese medicine.Molecular docking can quickly discover the key pharmacodynamic ingredients,reveal the pharmacodynamic material basis of complex chemical substance systems,and improve the efficiency and pertinence of the screening process of traditional Chinese medicine.The traditional Chinese medicine formula has many unique advantages in the therapy of depression.According to the clinical manifestations of depression,it belongs to the category of "depression syndrome" at Traditional Chinese medicine,and the heart-kidney disagreement is its important pathogenesis.Jiao-Tai-Wan(JTW)is a representative prescription for the therapy of heart-kidney disagreement and is widely used in the treatment of anxiety,insomnia and depression.Clinical studies have shown that JTW is good in antidepressant effects on patients,including improving depressive behavior,reducing inflammation and regulating brain function.However,its biologically active ingredients and the exact antidepressant mechanism are still unclear.ObjectiveOur study intends to use a combination of transcriptomics technology,molecular docking analysis and pharmacological experiments,aiming to explore JTW by inhibiting the activation and pro-inflammatory response of microglia,and improving behavioral disorder of depression model mice,protecting the brain’s frontal cortex and hippocampus nerve cell,to reveal the antidepressant mechanism and key target protein of JTW in vivo and in vitro.So,it can provide new potential targets for clinical depression treatment and new drug development.Methods1.The corticosterone(CORT)was administered to induce depression mouse models.According to the random number table method,the mice were randomly divided into five groups:blank control group(Control),model group(CORT),JTW low-dose group(JTWL),JTW high-dose group(JTWH),positive fluoxetine group(FLX).The behavioral effects of JTW against depression were evaluated by open field test(OFT),elevated Cross Maze(EPM),suspended tail test(TST)and forced swimming test(FST)experiments.Nissl staining and β-galactose staining were used to observe the improvement of neuronal degeneration and damage in depressed mice,and the Image J software was used to quantify the related indicators.Enzyme-linked immunosorbent assay(ELISA)was used for examining serum serotonin(5-HT)and norepinephrine(NA)levels in mice.In this way,the effectiveness of JTW in treating depression mice was tested.2.Transcriptomics sequencing.Each group of 3 hippocampal tissue samples were subjected to RNA sequencing(RNA-seq).Transcriptome analysis was used to screen the potential therapeutic targets and signaling pathways of JTW in depression.3.Molecular docking analysis.The 8 active substances of JTW obtained by high performance liquid chromatography analysis results from the early stage of the research group were used to conduct molecular docking to predict the interaction between the active compounds and key targets,and screen out the potential key factors of JTW in the treatment of depression.4.Combining the analysis results of transcriptomics and molecular docking,the key targets and molecular pathways were verified in vivo and in vitro.(1)In vivo experimental verification.ELISA method was used to detect the level of scrum IL-6 and IL-1β in mice.Immunofluorescence staining was used to observe the activation and protein expression of microglia,astrocytes and colony stimulating factor 1 receptor(Csf1r)in hippocampal CA3 region and medial(HIPP)prefrontal cortex(mPFC)of mice.And the Image J software was used for quantifing the related indicators.(2)In vitro experimental verification.In this study,two cell model systems of macrophages(Raw264.7)and microglia cells(BV2)induced by lipopolysaccharide and interferon(LPS/IFNγ)were used to verify the results of transcripomics and molecular docking analysis for key targets and molecular pathways in vitro.MTT method was used to examine Raw264.7 cell viability and its morphological analysis.NO concentration in Raw264.7/BV2 cells was detected by nitric oxide(NO)assay,and nitric oxide synthase(iNOS)immunofluorescence staining was used to observe the protein expression of BV2 cells.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the inflammatory factor gene expression of M1 and M2 related to Raw264.7/BV2 cells.In the end,Image J software was used for quantitative analysis of related indicators.Results1.The therapeutic effect of JTW in depression mice(1)BehaviorBehavioral tests showed that CORT administration significantly induced depressive behavior of mice,while JTW significantly improved CORT-induced depressive behavior in mice.Specifically,in the OFT experiment,compared with the CORT model group,the total running distance of the JTWL and JTWH groups increased(P<0.05).Similarly,in the EPM test,the number of mice entering the open arm in the JTWL group was obviously higher than the mice in the CORT group(P<0.01).Although the number of mice entering the open arm in the JTWH group also increased compared with the CORT model group,there was not significantly statistical difference(P>0.05).Compared with CORT model group,the residence time of mice in JTWL and JTWH group also increased(P<0.05).Compared with the CORT model group in TST and FST test,JTWL and JTWH group might significantly reduce the immobility time of the depressed mice(P<0.01).(2)Neuropathology and the serum levels of 5-HT and NAThe results of Nissl staining showed that CORT significantly induced the damage of neurons in the HIPP area of mice,and the neuronal cell bodies showed different degrees of pyknosis,degeneration and necrosis.Compared to the CORT group,JTWH group(P<0.001)significantly increased the survival rate of neurons and improved the damage of neurons in HIPP region of depressed mice.However,in mPFC area,there was no significant statistical difference between JTW administration group and CORT model group(P>0.05).The results of β-galactosidase staining showed that CORT significantly induced the senescence of mouse neurons,accompanied by the color of neurons deepened blue staining.JTWH group significantly improved the senescence of neurons in HIPP(P<0.01)and mPFC(P<0.05).Compared with CORT model group,JTWH group significantly increased the levels of 5-HT and NA in serum of depressed mice(P<0.05).2.Transcriptome data analysisThe heat map of different gene expression analysis displays that there are 293 differential genes expression between the JTW group and the CORT group,of which 160 genes up-regulated and 133 genes down-regulated.Analysis of differential gene KEGG function enrichment shows that JTW mainly regulates the immune inflammatory and signal transduction pathways.PPI network interaction key driver gene analysis(KDA)showed that JTW regulated immune inflammatory response related to macrophages,dendritic cells and T lymphocytes in depressed mice,and 10 KDA genes(Csflr,Ctss,Pld4,C1qa,C1qb,Clqc,Cd14,Fcgr2b,Tlr7,Fyb)had the strongest correlation in regulating depression mice.3.Molecular dockingMagnoflorine,the main active component of JTW,has the strongest binding force to the colony stimulating factor 1 receptor(Csflr)of the signaling pathway on which microglia depend for survival.Magnoflorine hydrogen bonds with the residues of GLU-633,LYS-616,ASP-796 and LEU-699 of Csflr and binds in the active pocket,and combined in Csflr active pocket and the active pocket electrostatic potential energy is very low,which makes the energy lower,better binding effect,which makes the energy lower and the binding effect better.4.Experimental verification(1)In vivo experimentImmunofluorescence staining showed that the CORT model significantly induced the activation of microglia in the brain tissue of the depressed mice.Compared with the CORT model group,the JTWH group significantly reduced the number of microglia(P<0.05,HIPP;P<0.01,mPFC),and increased the number of branch ends(P<0.01,HIPP;P<0.01,mPFC)and branch length(P<0.01,HIPP;P<0.01,mPFC).The CORT also induced the activation of astrocytes at a certain extent,but there was no statistically significant difference in the number of astrocytes in HIPP and mPFC of mouse brain(P>0.05).Csflr immunofluorescence staining results showed that CORT modeling significantly induced the increase of Csflr protein expression.Compared to CORT group,JTWL and JTWH group significantly inhibited the overexpression of Csflr protein in HIPP(P<0.05)and mPFC(P<0.05)of mouse brain.The results of mouse serum ELISA showed that,compared with CORT model group,administration of JTWL and JTWH group significantly decreased the levels of IL-6(P<0.001)and IL-1β(P<0.001).(2)In vitro experimentThe results of NO detection showed that JTW significantly reversed the increase the concentration of NO in Raw264.7 cells(P<0.001)and BV2 cells induced by LPS/IFNy(P<0.05);improved the decrease cell viability(P<0.01)and cell circularity(P<0.01)of Raw264.7 cell.Immunofluorescence Intensity statistics showed that LPS/IFNy stimulation significantly increased the expression of iNOS in BV2 cell(P<0.01),while JTW decreased the expression of iNOS(P<0.01).RT-qPCR showed that both JTW and Csflr inhibitor(BLZ-945,MK-2206)significantly decreased the mRNA expression of IL-1,IL-6,TNFa,Ctss and Statl in LPS/IFNy induced Raw264.7 cell(P<0.01).Meanwhile,the mRNA expressions of iNOS,IL-1,IL-6 and TNFa in LPS/IFNγinduced BV2 cell were significantly inhibited by JTW administration(P<0.01).ConclusionOur experimental results show that the JTW has good antidepressive efficacy.Its underlying mechanism might be to ameliorate the damage of CORT-induced depression mouse neurons by inhibiting Csflr mediated microgliocytes Ml-type activation and neuroinflammatory reactions.Therefore,JTW provide a new potential candidate compound for clinical depression and new drug development.