Cumulative Immune Activity Of Ginsenoside Rb₁ And Its Structural Analogues Through Hitting Shared Targets

Chemical diversity of traditional Chinese medicine(TCMs)is featured by not only various structural types but also abundant analogues of one type.It is well recognized that TCMs belonging to different structural types exert synergistic actions by hitting different disease-related targets;it is not known,however,if and how structural analogues in TCMs work together.Based on the receptor theory,the interaction between ligands(drugs)and receptors(targets)is structurally selective,that is,compounds with similar structures are likely to act on the same targets.If so,the multiple hits on the target by multiple analogues could achieve cumulative,and thus significantly greater,bioactivity.Therefore,it is hypothesized that structural analogues in TCMs hit a shared target to achieve cumulative bioactivity.In this study,ginsenosides Rb1,Rd,Rg3 and F2,the main structurally similar components of ginseng,as well as their immunological activities and main shared targets,were taken as an example to verify the hypothesis.This paper is divided into four parts:(1)Design of structural analogues based on ginsenosides:Here we proposed a resolution by integrating pharmacodynamics,serum pharmacochemistry and pharmacokinetics approaches.We evaluated the dynamic immunomodulatory activity in vivo of orally administrated ginsenoside Rb1 for 72 h;we qualitatively and quantitatively characterized Rb1 and its intestinal metabolites absorbed into the blood,and then constructed their concentration-time curves in the serum.Taken together,the results allowed us to capture the serum profile of these ginsenosides at the time point with the strongest immunomodulatory activity,and such a combination consisting of Rb1 and its metabolites(analogues)was assumed to significantly interact with immune-related target(s)in the circulation.Therefore,this analogues combination was then selected as a model to further explore how the analogues jointly hit one target to achieve cumulative immunomodulatory activity.As a result,the oral dosage of Rb1 was determined as 160 mg/kg as it showed the strongest immunomodulatory activity in vivo among the dosages tested.The serum pharmacochemical and pharmacokinetic analyses then indicated that Rb1 and its deglycosylated metabolites(Rd,Rg3,F2,Rh2 and CK)varied qualitatively and quantitatively during the 72 h after the oral administration.The dynamic pharmacodynamic evaluation at the same duration showed that the best improvements on immunocompromised phenotypes were achieved at the 2nd h.At this time point,Rb1 and three metabolites,Rd,Rg3 and F2,were identified in the serum,whereas Rh2 and CK were not detected.Therefore,ginsenosides Rb1,Rd,Rg3 and F2 at the serum concentrations as determined at the 2nd h were selected as the combination investigated in this study.(2)Verification of cumulative immunoregulatory activity of ginsenosides structural analogues:RAW264.7 macrophage induced by LPS/ATP was used as a model to compare the immune activity of ginsenoside monomer and combination at the same dose.We observed that the combination treatment enhanced cell proliferation,migration and invasion,inhibited mRNA expressions of pre-inflammatory cytokines(iNOS,IL-1β,TNF-α),increased mRNA expressions of anti-inflammatory cytokines(IL-4,IL-10),decreased the contents of iNOS,IL-1β,TNF-α and increased the contents of IL-4 and IL-10.In individual-treated groups,the indicators were selectively improved,but none were equivalent to the combination-treated group.In other words,the combination showed more potential in enhancing macrophage function than any individual analogue at the same dosage,which supports the conclusion that the combination achieved cumulative immunomodulatory activity in vitro.(3)Screening and prediction of shared targets of ginsenosides structural analogues:Systems pharmacology was used to predict the potential targets whereby the ginsenoside analogues exert the immunomodulatory activity.Ginsenosides Rbi,Rd,Rg3 and F2 were imported into the PharmMapper,Swiss Target Prediction,SEA and STITCH databases for targets prediction.A total of 32 targets were obtained,then the functional analysis and protein-protein interaction analysis were performed,and 29 targets were finally retained.These targets imported to the DAVID website for GO function enrichment analysis and KEGG pathway analysis,and Cytoscape software was used to visualize the "component-target-pathway" network.The centrality algorithm analyses of the network screened out 7 key targets,and NLRP3 ranked at the top,suggesting that NLRP3 is a crucial target in mediating the immunomodulatory activity of the ginsenosides.Molecular docking was used to predict the binding affinity and sites between the ginsenosides and NLRP3.The results showed that the four ginsenosides can all bind to NLRP3 with an average docking score of-11.55 kcal/mol.Ginsenosides can form different numbers(1-3)of hydrogen bonds with different amino acid residues of NLRP3(NLRP3-Rb1 complex forms 1 hydrogen bond at Thr167 site,NLRP3-Rd complex forms 3 hydrogen bonds at Argl68,Argl65 and Lys375 sites,NLRP3-Rg3 complex forms 2 hydrogen bonds at Phe408 and Tyr441 sites,and the NLRP3-F2 complex forms 1 hydrogen bond at Tyr379).In order to more precisely validate that the ginsenosides directly target NLRP3,the equilibrium dissociation constant(KD)between the ginsenosides and NLRP3 were then determined by bio-layer interferometry.The KD of ginsenosides Rb1,Rd,Rg3 and F2 binding to NLRP3 were 9.96×10-5、5.01×10-5、9.61×10-6、6.16×10-5 mol/L,respectively,indicating that all the ginsenoside analogues can bind to NLRP3 directly with strong affinity.These results further suggested that ginsenoside analogues may have accumulated bioactivity by hitting the shared targets.(4)Confirmation of shared targets of ginsenosides structural analogues:The impacts on the activation of NLRP3 were examined by immunofluorescence and western blot.Cells stimulated with LPS/ATP had a higher fluorescence intensity after NLRP3 labelling,which was however weakened by the treatments,particularly the one with the combination.The result of western blot constituted evidence that combination treatment inhibited the activation of NLRP3 inflammasomes as well as the NLRP3-caspase 1-IL-1β/IL-18 signaling pathway,whereas the effects were not equivalently observed in the presence of individual analogues at the same dosages.This result met our expectations,i.e.in contrast to dispersed and weak actions delivered by each analogue,the combined analogues cumulatively,significantly suppressed the shared target NLRP3.In order to confirm that NLRP3 mediates the immunomodulatory activity of the combination,RAW264.7 macrophage was transfected with lentiviral vectors to induce the overexpression of NLRP3.It was observed that the immunomodulatory activity of the combination on RAW264.7 macrophages was greatly counteracted in the NLRP3-overexpressed cells regarding cell proliferation,cell cycle,cell migration,cell invasion and cell pyroptosis.In a similar way,the overexpression of NLRP3 also impeded the suppression of NLRP3 inflammasome signaling(NLRP3,NEK,ASC,cleaved caspase1,cleaved IL-1β,IL-18)and inflammatory responses(iNOS,TNF-α,IL-1β,IL-4 and IL-10)by the combination intervention.Overall,the results demonstrated that NLRP3 is a crucial target by which the combination overcomes immunodeficiency,which further verifying our hypothesis that structural analogues of TCMs achieve cumulative effect by hitting the shared targets,thus exerting biological activity.In conclusion,this study proposed and verified the hypothesis that "structural analogues of TCMs exert cumulative bioactivity by hitting on the shared targets ".This new mechanism is complementary to the principle of "multiple components hitting multiple targets".The knowledge expands the scientific interpretation of the therapeutic rationale of herbal medicines,and additionally inspires us to shift the focus from single to combined compounds,including structural analogues,in herbal medicines-based drug discovery.