Ginsenoside Rb1 Can Improve LPS-induced Acute Depression-like Behavior In Mice By Regulating Inflammation And Research On Its Active Ingredients In Vivo

Depression is a common mental disease.Severe cases can lead to disability or suicide At present,the pathogenesis of depression is unclear,and there are many hypotheses of depression.Existing antidepressant drugs have a single target,and there are obvious adverse reactions after long-term use.Therefore,it is very important to find antidepressant drugs that work on multiple targets and are safer and more effective.Traditional Chinese medicine has the characteristics of multiple targets,multiple pathways and integrity,and is an important source of antidepressant drug developmentGinseng(Panax ginseng C.A.Meyer)is a precious traditional Chinese medicinal material,which has the effects of nourishing vital energy,calming the mind and nourishing the mind,and can be used to treat insomnia,palpitation,and mood disorders.Ginsenoside is the main active ingredient of ginseng,including protopanaxadiol(PPD),protopanaxatriol(PPT)and oleanolic acid.Ginsenoside Rb1(Rb1)and ginsenoside Rg1(Rg1)are the representative components of PPD and PPT,respectively.Compared with PPT-type ginsenoside,PPD-type ginsenoside has higher concentration and half-life in plasma.The representative ingredient ginsenoside Rbl has various pharmacological activities such as anti-inflammatory,neuroprotection,prevention and treatment of cardiovascular and cerebrovascular diseases,and is also one of the main ingredients with high distribution in the brainGinsenosides,monomers Rbl,Rgl and Rg3 all showed good antidepressant activity Rbl with oral administration has the characteristics of poor bioavailability and uneven distribution in the brain.For example,the oral availability of ginsenoside Rb1 is only about 1%,but the deglycosylated metabolites Rg3,C-K,Rh2 and PPD of Rb1 have higher permeability and bioavailability,and also have antidepressant activity.Rbl shows an antidepressant effect in a chronic unpredictable mild stress model.The main mechanism is to upregulate monoamine neurotransmitters,regulate BDNF and its downstream protein TrkB,and affect the levels of adrenocorticotropic hormone and cortisol.Since neuroinflammation is one of the major mechanisms of depression,the effect and molecular mechanism of Rbl on acute depression caused by lipopolysaccharide neuroinflammation model has not been reportedNetwork pharmacology can analyze the action mechanism of Chinese herbal medicines.and independently present the "component-target-pathway" related to a specific disease,effectively revealing the material basis and molecular mechanism of Traditional Chinese medicines,and provide a theoretical basis for experimental verification.In this study,UPLC-QTOF-MS was used to analyze and identify the metabolic components of ginsenoside Rb1 in vivo,and the antidepressant mechanism of Rb1 and its metabolites was analyzed and predicted by network pharmacological methods and experimentally verified.Theoretical prediction and verification experiments have discovered the role of Rb1 anti-depression GR,p38 MAPK and NF-κB p65,as well as the potential interaction mechanism of neuroactive ligand-receptor interaction,MAPK and NF-κB signaling pathway;first discovered ginsenoside F2 has stronger than Rb1 in anti-inflammatory activity,similar in antidepressant-like activity,and ginsenoside Rd has anti-inflammatory and antidepressant activities similar to Rb1.Although ginsenosides F2 and Rd are also present in ginseng,but the content is relatively small.This discovery enriches Rbl’s antidepressant substances and their potential targets and possible pathwaysThis experiment combines network pharmacology and efficacy verification to study the antidepressant activity and target pathways of Rb1 and metabolites in vivo,and explore the antidepressant substances and mechanism of action of ginsenoside Rb 1(1)A lipopolysaccharide-induced mouse depression model was used to test the antidepressant effect of Rb1 through behavioral experiments of forced swimming and tail suspension.The results showed that Rbl significantly improved the prolongation of immobility time caused by lipopolysaccharide,exhibited antidepressant-like behavior in a dose dependent manner.(2)Using UPLC-QTOF-MS establish a qualitative analysis method for Rb1 and its metabolites to clarify the metabolites in normal and depressed model mice.The results showed that the metabolites of Rb1 in normal and model mice were the same.The components absorbed into the blood,brain and jejunum are Rbl,Rd and PPD.Metabolites in urine and feces are Rbl,Rd,Rh2,Rg3,F2 and C-K.The metabolic pathway of Rb 1 in plasma and brain is mainly by removing one molecule of glucose to generate Rd,and then losing 3 molecules of glucose to generate PPD.The metabolic pathway of Rb 1 in urine and feces is mainly to remove one molecule of glucose to produce Rd,and then lose one molecule of glucose at different positions to produce Rd and Rh2,and further lose one molecule of glucose to produce F2 and C-K.Deglycosylation is the main metabolic pathway of Rb1 in mice(3)Predict the antidepressant targets of Rb1 and its metabolites through network pharmacology.Network pharmacology was used to construct "component-target-disease",and mechanism prediction was realized through protein-protein interaction(PPI)analysis,GO function enrichment analysis,KEGG pathway analysis.Ninety-one key targets and 20 main biological enrichment functions and pathways were found.Neuroactive ligand-receptor interaction is the most relevant signalling pathway analyzed.In addition,MAPK and NF-κB inflammation signaling pathways frequently appear in other major pathways.Among the key targets,GR and 5-HT1A are important targets in the Neuroactive ligand-receptor interaction;MAPK 14 is an important target in the MAPK pathway.The network pharmacology results indicate that the Neuroactive ligand-receptor interaction and inflammatory response-related pathways may be the main mechanism for Rbl and its metabolites to exert antidepressant effects.(4)Perform target-pathway verification experiment.The experiment used enzyme-linked immunoassay(ELISA)to determine the inflammatory factors interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the serum,and found that Rb1 significantly reduced both due to lipopolysaccharide.The resulting content increases and exerts anti-inflammatory effects;HPLC-MS was used to determine serotonin(5-HT),tryptophan(TRP),kynurenine(KTN),and γ-aminobutyric acid(GABA),glutamate(Glu)and glutamine(Gln),Rb1 can regulate the content of 5-HT,TRP,KYN,GABA,but the action trend of Glu and Gln is inconsistent with the literature;use qPCR method to explore Rb 1 on mice.The expression levels of 5-HT1A and Nr3cl mRNA in hippocampus tissues were examined by Western Blotting to investigate the expression of 5-HT1A protein by Rb1.The results showed that Rb1 can significantly enhance the expression of 5-HT1A mRNA and protein levels,and increase the expression of Nr3c1 mRNA levels,indicating that Rb1 has a regulatory effect on Neuroactive ligand-receptor interaction.Using qPCR technology to investigate the expression of MAPK14 and NF-κB p65 mRNA in hippocampus tissues using Rb1,and Western Blotting to investigate the effect of Rbl on the phosphorylation level of NF-κB p65 protein.The results showed that Rb1 can significantly reduce the expression of p38 MAPK and NF-κB mRNA levels,and reduce the phosphorylation level of NF-κB,indicating that Rb1 has an inhibitory effect on the activation of MAPK and NF-κB signaling pathways.At the same time,the prediction of the influence of Rb1 in network pharmacology on neuroinflammation and neural function was also verified.(5)Newly discovered antidepressant activity of Rd and F2.In this study,a lipopolysaccharide-induced depression mouse model was used to conduct behavioral measurements on mice given Rd and F2 and found to have a significant antidepressant-like effect;serum levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α),and the high-dose F2 group showed more significant anti-inflammatory effects than Rb1.Conclusion:This study established the UPLC-QTOF-MS identification method of Rb1 metabolites in the LPS depression mouse model,and predicted "component-target-disease" of the antidepressant effect of Rbl and its metabolites is verified by experiments in vivo.The results show that Rb1 can dose-dependently increase 5-HT content and 5-HT1A receptor mRNA and protein expression;increase GR gene expression and regulate tryptophan metabolism.Meanwhile,Rb1 reduces the increase of LPS-induced proinflammatory cytokines and IDO activity,which may affect the MAPK and NF-κB pathways by inhibiting the phosphorylation of p38 MAPK and NF-κB p65.This result validates the prediction of network pharmacology and indicates that the neuroactive ligand-receptor interaction pathway may be related to the antidepressant effect of Rb1.Under the guidance of network pharmacology,it was discovered and verified firstly that Rd and F2 have antidepressant-like effects,which has not been reported in previous studies,and F2 is superior to Rb1 in anti-inflammatory effect.This study reveals for the first time that Rb1 may be involved in the neuroactive ligand-receptor interaction pathway and the potential regulation of related targets GR and 5HT1A,and has been experimentally verified to find that Rbl and its metabolites have multiple targets for antidepression,many pathway mechanisms.It provides a reference for further clarifying the substance basis and pharmacological mechanism of Rb1 pharmacodynamics,and also provides a theoretical basis for Rb1 and Rd and F2 which are metabolites in ginseng to become potential antidepressants.Innovation:This study explored the antidepressant-like effect and molecular mechanism of Rb1 on lipopolysaccharide-induced neuroinflammation model,and found that Rb1 can modulate the target of GR in Neuroactive ligand-receptor interaction,affects the MAPK and NF-κB pathways by inhibiting p38 MAPK and Phosphorylation of NF-κB p65,and illustrates the correlation between Rb1 metabolites and antidepressant efficacy.The potential new antidepressant-like activity of Rd and F2 were discovered for the first time,which enriched the material basis of anti-depression of ginseng.