Study On The Analysis Of Efficacy Components And Common Targets Of Bupleurum Chinense DC Based On "Origin-Component-Disease-Target" Interaction Network

Objective:In this study,we analyzed the composition of the extract of Bupleurum chinensis DC.(Bupleurum)and investigated the pharmacological basis and key targets of the antidepressant and anti-inflammatory effects of Bupleurum,respectively.To analyze the common targets of efficacy between the antidepressant and anti-inflammatory effects of Bupleurum.To screen the bio-efficacy components associated with herb component-drug efficacy and build a model for the evaluation of the grade of Bupleurum.This will provide a basis for the future improvement of the quality evaluation of Bupleurum and the study of various proprietary Chinese medicine dosage forms.Methods:1 The Plackett-Burman(PB)and Central Composite Design(CCD)methods were used to optimize the chromatographic conditions of the Ultra Performance Liquid Chromatography(UPLC)of Bupleurum,and the liquid-mass spectrometry technique was used to characterize on the chemical composition of Bupleurum using UPLC,and the preliminary identification of the chemical components contained in Bupleurum by comparing molecular weight,isotope distribution,molecular ion peak and fragment ion cleavage information,and the compositional analysis of Bupleurum herbs from 28 origins.2.The results of component characterization by UPLC/Q-TOF-MS,TCMSP and ETCM were used to integrate the components of Bupleurum and to collect the targets related to Bupleurum components and the disease(depression and inflammation).The composition-disease intersection targets were obtained by the venny platform for the antidepressant and anti-inflammatory properties of Bupleurum,respectively.The String platform was used to generate a protein-protein interactions(PPI)network of intersecting targets.To analyze the key targets of the antidepressant and anti-inflammatory effects of Bupleurum by constructing the "component-disease-target" interaction network through Cytoscape software respectively.The ClusterProfiler package in R language was used to analyze the GO and KEGG pathway enrichment of the antidepressant and anti-inflammatory crossover targets of Bupleurum,respectively,to predict the signaling pathways through which Bupleurum exerts antidepressant and anti-inflammatory effects.3.To verify the prediction of key targets by network pharmacology techniques,firstly,we used the chronic unpredictable mild stress(CUMS)modeling method to establish a depression rat model of liver depression and qi stagnation.The inflammatory rat model was established by intraperitoneal injection of lipopolysaccharide(LPS).To quantify the changes of key metabolites with therapeutic effects after the intervention of Bupleurum using partial least squares(PLS)model fitting to analyze the differential metabolites between depressed rats,inflammatory rats model group and blank control group,The Joint-Pathway Analysis module in the Metaboanalyst 5.0 platform was used to correlate differential metabolites with component-disease intersection targets to locate the pathways where Bupleurum exerts more significant antidepressant and anti-inflammatory effects,The levels of key metabolites associated with therapeutic effects in each group were quantified by LC-MS technique to determine the modulating effect of Bupleurum on the levels of key metabolites in the enrichment pathway.We also used enzyme linked immunosorbent assay(ELISA),western blot(WB)and Real-time quantitative polymerase chain reaction(RT-PCR)to verify the key targets with more significant relevance to the antidepressant and anti-inflammatory effects of Bupleurum.4.In order to screen the antidepressant and anti-inflammatory bio-efficacy components of Bupleurum,this study was conducted to establish in vitro key target protein activity assays of Bupleurum from different origins using ELISA based on the association of major components with targets.Based on the significant anti-inflammatory effect of Bupleurum,therefore,LPS was used to induce inflammation in RAW264.7 macrophages,and the anti-inflammatory effect of northern Bupleurum of different origins was evaluated by detecting the changes in the concentrations of inflammatory factors IL-6,TNF-α and IL-1βafter the intervention.We analyzed the in vitro key target protein activity,anti-inflammatory effect and common components among different origins of Bupleurum by PLS,and selected compounds with variable importance in projection(VIP)greater than 1,OB≥ 30%or DL≥ 0.18 and correlation with pharmacophore by Simca-p software.The compounds with good correlation with the pharmacophore were selected as potential bio-efficacy components.Based on the methodology of UPLC-MS/MS,the bio-efficacy components were quantitatively determined and correlated with the biological activity by dichotomous logistic regression to establish a model for the evaluation for the ranking of Bupleurum.Results:1.We analyzed the chemical composition of Bupleurum by liquid mass spectrometry and identified 37 components of Bupleurum,including 16 pentacyclic triterpenoids,11 flavonoids and 10 others.2.The results predicted by network pharmacology showed that Bupleurum on antidepressant effects included a target cluster of 39 key targets,mainly involving relevant pathways that are involved in regulation and transport of neurotransmitter levels,modulation of presynaptic and postsynaptic membrane transmission,regulation of cAMP pathway,dopamine and serotonergic synapses,which may be the key mechanistic pathways Bupleurum to exert antidepressant effects.There were 149 anti-inflammatory disease-component intersection targets of Bupleurum,and the more significant mechanism pathways were NF-κB signaling pathway,tumor necrosis factor,interleukin-17 and PI3K-Akt.3.The results of body weight change,behavioral evaluation,serum biochemistry and ELISA assay of the depressed rat model showed that the depression model was successfully established.According to the results of metabolomics data processing,a total of 405 differential metabolites were screened in positive and negative ion mode.Joint-Pathway Analysis enrichment results showed that dopaminergic synapses,cAMP,etc.might be the main pathways for the antidepressant effect of Bupleurum.The results of the quantitative assay of key metabolites using Liquid Mass Spectrometry showed significant differences in 5-hydroxytryptamine(5-HT)compared to the model group after the administration of the intervention.We analyzed the key targets on the signaling pathway predicted by network pharmacology using ELISA,WB and RT-PCR techniques,and compared with the model group,after the intervention of Bupleurum administration,we found that Bupleurum significantly regulated the CUMS-induced activation of the catalytic subunit α(PRKACA)of protein kinase cyclic adenosine monophosphate(cAMP),cyclic adenosine monophosphate response element binding protein(CREB)expression levels.By quantitative differential metabolite and validated target synthesis,the antidepressant of Bupleurum may be through the regulation of cAMP signaling pathway.The quantitative detection of inflammatory cells by blood cell analyzer,and inflammatory factors measured by ELISA indicated that the inflammation model was successfully established.A total of 38 differential metabolites were screened in positive and negative ion mode based on the results of metabolomics data processing.The enrichment results of Joint-Pathway Analysis showed that the tumor necrosis factor signaling pathway,interleukin 17 signaling pathway,PI3K-Akt signaling pathway and NF-κB signaling pathway were significantly correlated with the anti-inflammatory effects of Bupleurum.We analyzed the key targets on the more significant anti-inflammatory pathways of Bupleurum by ELISA,WB and RT-PCR experiments,and found that Bupleurum could show anti-inflammatory effects by regulating the expression levels of key targets CREB,NF-κB and TNF-α compared with the model group.4.The LPS was used to induce inflammation in RAW264.7 macrophages,and according to the analysis of the NO assay results,Bupleurum had a significant inhibition of cellular NO production at 40 micrograms,further proving that Bupleurum has anti-inflammatory effects,and the higher anti-inflammatory effect of Bupleurum from various origins was determined to be S8.The higher effect on CREB activity by ELISA was S23,on PRKACA activity was S16,on NF-κB activity was S5,and on TNF-α activity was S21 for different origins of Bupleurum.The data of anti-inflammatory effect and key target protein activity of Bupleurum combined with the components of Bupleurum from different origins were imported into Simca-p 14.1 software for PLS analysis,and the final screening of Bupleurum potent components were:Saikosaponin a,Saikosaponin d,rutin and quercetin.A binary logistic was used to establish an evaluation model for the grade of Bupleurum,which was divided into four grades of excellent,good,medium and poor for the 28 origins of Bupleurum.Conclusion:This study found that Bupleurum extract has good antidepressant and anti-inflammatory effects.Combining the results of network pharmacology prediction,metabolomics and WB,ELISA and RT-PCR analysis,Bupleurum can exert antidepressant effects by upregulating the expression of PRKACA and CREB in cAMP-PKA-CREB.Through regulating CREB,NF-κB and TNF-α to exert anti-inflammatory effects,the main pharmacological antidepressant and anti-inflammatory effects of Bupleurum were preliminarily screened to the group of Bupleurum pharmacological components by comprehensive analysis of Bupleurum’s main pharmacological effects,and a rank evaluation model was established to evaluate the good and bad of Bupleurum.The results showed that the quality standard of Bupleurum herbs and the research of its proprietary dosage forms are well established.