Network Pharmacology-Based Approach To Investigate The Mechanism Of Kaixin Jieyu Granule For Treating Depression By Inflammatory Signaling Pathway

Depression is currently one of the most prevalent mental health problems and is associated with an increase in mortality,disability,and healthcare costs.Epidemiological studies show that more than 350 million people of all ages suffer from depression worldwide,of which 9%-18%were the elderly people.In addition,the burden of depression carries very high medical,financial and psychosocial costs,with US costs alone estimated at $210.5 billion annually.At present,there are a lot of side effects of western medicines that have been reported for the treatment of depression,such as insomnia,nausea,increased weight gain,drowsiness,agitation,and high rate of relapse.Therefore,it is critically important to develop more natural,safe,and effective drugs for depression treatment.Kaixin Jieyu Granule(KJG)is an expert experience prescription for the treatment of depression that our research group has been devoting to research for many years.Our previous studies have shown that KJG can regulate cerebral cortical homeostasis,protect hippocampal neurons,repair white matter damage,and increase cerebral blood flow supply.However,the molecular mechanisms of the active ingredients in KJG against depression have not been studied.Neuroinflammation plays a vital role in the pathogenesis of depression,in which TLR4-mediated inflammation is the critical nexus.In this research,network pharmacology and molecular docking were first used to explore the specific molecular mechanism of KJG in the treatment of depression.On the basis of the above results,a series of experiments were further conducted in vivo and in vitro to verify the possible mechanism of KJG against depression.This research was divided into three parts:1.Study on the antidepressant mechanism of Kaixin Jieyu Granule based on network pharmacology and molecular dockingObjective:To clarify the specific molecular mechanism of KJG in the treatment of depression.Methods:The TCMSP and TCM-BATMAN databases were used to screen the effective compounds and targets of KJG,and the GEO,Genecards,OMIM,PharmGkb,TTD and DrugBank databases were used to screen differential genes for depression.Through the intersection of KJG and depression targets,the active compounds and targets of KJG against depression were obtained,then the drug-disease-target network was drawn;the screened intersection genes were substituted into cytoscape software to perform the protein-protein interactions(PPI)network;the screened intersection genes were used to perform KEGG enrichment analysis;finally,the screened compounds and corresponding targets of KJG against depression were used for molecular docking simulation.Results:A total of 82 active compounds and 404 important genes in KJG related to depression were screened from the above databases.From the drug-disease-target network,it was concluded that ginsenoside Rg1(GRg1)and saikosaponin d(Ssd)had the highest degree values,which showed that they were the main components of KJG against depression;36 key target genes of KJG related to depression were extracted according to the twice median of PPI network parameters;the PI3K-AKT,FoxO,and Toll-like receptor pathways were the main pathways of KJG related to depression in the KEGG enrichment analysis,and the key genes enriched in the pathways were TLR4,PI3K(PIK3R1),AKT(AKT1)and FoxO1;molecular docking simulation showed that ginsenoside Rgl and saikosaponin d had good binding degree with TLR4,PI3K(PIK3R1),AKT(AKT1)and FoxO1,which indicated that they had the molecular basis for interaction.Conclusion:The main effective components of KJG against depression were GRg1 and Ssd,and KJG can exert antidepressant effects by regulating TLR4 through PI3K-AKT-FoxO1 pathway.2.KJG can exert antidepressant effects by regulating TLR4 through PI3K-AKT-FoxO1 pathway in vivo.Objective:To verify that KJG can exert antidepressant effects by regulating TLR4 through PI3K-AKT-FoxO1 pathway.Methods:1.Chronic unpredictable mild stress(CUMS)method was used to establish the depressive-like mice model.Then the behaviors tests,Nissl and immunofluorescence staining,pro-inflammatory cytokines were detected to evaluate the antidepressant effects of KJG and the correlation between KJG and TLR4;2.Lipopolysaccharide(LPS)method was used to establish the inflammation mice model,in which TLR4 antagonist(TAK-242)was used to block the expression of TLR4.Then the behaviors tests,Nissl and immunofluorescence staining,pro-inflammatory cytokines,TLR4 and FoxO1 expression were detected to evaluate the antidepressant effects of KJG and the correlation between KJG and TLR4.3.The CUMS method was used to establish the depressive-like mice model,in which PI3K antagonist(LY294002)was used to block the PI3K-AKT pathway.Then the behaviors tests,Nissl and immunofluorescence staining,pro-inflammatory cytokines,PI3K-AKT pathway related proteins expressions were detected to evaluate the antidepressant effects of KJG and the correlation between KJG and PI3K-AKT pathway.4.The CUMS method was used to establish the depressive-like mice model.Then the TLR4 and PI3K-AKT-FoxO1 pathway-related proteins expressions were detected to evaluate the antidepressant effects of KJG and the correlation between KJG and PI3K-AKT-FoxO1 pathway.Finally,the database was established,and Graphpad Prism software(9.0.0)was used for statistical analysis.Results:1.Compared with the CUMS group,KJG can significantly alleviate the depression-like behaviors of CUMS mice,increase the consumption of sucrose and the numbers of crossings in open-field test,and reduce the immobility time of tail suspension and forced swimming test;it can protect the hippocampal nerve cells and reduce the expression of TLR4 in the hippocampal CA1 area;it can also reduce the expression levels of serum TNF-α,IL-6,and IL-1β in CUMS mice.The high-dose group of KJG showed a more significantly antidepressant effect than the low-dose group.2.Compared with the LPS group,KJG can significantly alleviate the depression-like behavior of LPS-induced mice,protect hippocampal neurons,reduce the expression of TLR4 in the hippocampal CA1 area,decrease the levels of serum TNF-αand IL-1β,reduce the expression level TLR4 and FoxO1,and promote the expression of p-FoxO1 in LPS mice.Similarly,the above phenomenon can also be observed in the TAK-242 group.3.Compared with the CUMS group,KJG can alleviate depression-like behavior,reduce neuroinflammation,and reduce the levels of pro-inflammatory cytokines.In addition,KJG can also promote the expressions of PI3K,p-PI3K,Akt,and p-Akt,followed by the inhibition the TLR4 expression.But the above effects can be reversed by LY294002.4.Through western blot analysis,KJG can exert antidepressant effects by promoting the expression of PI3K,p-PI3K,Akt,p-Akt,p-PTEN,and p-FoxO1.whereas reducing the expression of FoxO1,which can further inhibit the expression of TLR4 to relieve neuroinflammation.Conclusion:1.KJG can exert antidepressant effects in CUMS mice,which depends on reduce of neuroinflammation in the hippocampus and is related to the TLR4 expression.2.KJG can significantly reduce the neuroinflammation induced by LPS in mice.This protective mechanism is achieved by downregulating the expression of TLR4,which is related to the expression of FoxO1.3.KJG can exert antidepressant effects by activating PI3K-AKT pathway to reduce neuroinflammation,which is regulated by TLR4.4.KJG can exert antidepressant effects by reducing neuroinflammation through the inhibition of TLR4 via PI3K-AKT-FoxO1 pathway.3.The main compounds of KJG can reduce neuroinflammation by regulating TLR4 through PI3K-AKT-FoxO1 pathway in vitro.Objective:To verify that the main components of KJG(GRg1,Ssd)can reduce neuroinflammation by regulating TLR4 through PI3K-AKT-FoxO1 pathway.Methods:The LPS-induced BV2 microglia model was established and divided into multiple groups,including blank group,model group,GRg1/Ssd group,GRg1/Ssd+LY294002 group.Then CCK8 method was used to determine the IC50 values of GRg1 and Ssd.Before incubation with LPS for 24h,the above groups were pretreated with GRg1,Ssd,and LY294002 for 1h,respectively.Then the pro-inflammatory cytokines(TNF-α,IL-6,and IL-1β)levels,PI3K-AKT-FoxO1 pathway related proteins expressions,FoxO1 expression between nucleus and cytoplasm,immunofluorescence staining of FoxO1 and TLR4 were detected to explore the mechanism underlying the anti-neuroinflammation effect of the main components of KJG.Finally,the database was established,and Graphpad Prism(9.0.0)software was used for statistical analysis.Results:Compared with the LPS group,GRg1 and Ssd group can significantly reduce neuroinflammation by reducing the expressions of pro-inflammatory factors(TNF-α,IL-6,and IL-1β)in LPS-induced BV2 cells,and promoting the expressions of PI3K,p-PI3K,Akt,p-Akt,and p-FoxO1,whereas reducing the expression of FoxO1,resulted in the inhibition of TLR4.The results of the expression of FoxO1 between nucleus and cytoplasm using the nuclear-plasma separation kit showed that GRg1 and Ssd can promote the nuclear efflux of FoxO1,reduce its transcriptional activity,promote the expression of p-FoxO1 in the cytoplasm,and reduce FoxO1 in the nucleus.In addition,this phenomenon had also been confirmed by the immunofluorescence staining of FoxO1.However,the above protective effects of GRg1 and Ssd can be reversed by LY294002.Conclusion:The main components of KJG(GRgl and Ssd)can regulate TLR4 through PI3K-AKT-FoxO1 pathway to reduce the levels of pro-inflammatory cytokines,promote FoxO1 nuclear efflux,and relieve neuroinflammation in LPS-induced BV2 cells.