| Pouzolzia Zeylanica(L.)Benn.Var.Microphyll,a traditional Chinese medicine commonly used in South China,has the effect of removing dampness,eliminating swelling,detoxification and anti-inflammation.In recent years,we have made a systematic study of this plant including chemical constituents,effective fraction,pharmacological activities and development of preparations.Some lignans and flavonoids have anti-inflammatory activity in vitro.Due to the influence of isolation and identification progress,and also of the content of compounds obtained,the anti-inflammatory activity and mechanism of those compounds were relatively scattered,and the research is not systematic and in-depth.Analysis the total components of the effective fraction of P.Zeylanica showed that the main components were flavonoids and lignans.Flavonoids accounted for about 60% in the effective fraction,and most of them were glycosides.The structures of lignans are special and novel,most of them are carbon reducing lignans,which are the unique components of P.Zeylanica.On the basis of previous work,seven lignans and seven flavonoids were selected to study their anti-inflammatory activity in the inflammatory model of monocyte macrophages in RAW264.7 mice.In order to clarify the anti-inflammatory activity and mechanism of lignans and flavonoids and look for synergistic anti-inflammatory effects,network pharmacology were used to predict the target and anti-inflammatory pathway of these two kinds of compounds.In order to verify the effect of key compounds on potential targets,the affinity of compounds for targets was evaluated by molecular docking technology,and analyzed the binding mode of compounds.The main contents and results are as follows:(1)LPS-induced RAW264.7 murine macrophages in vitro inflammation model was used to study the anti-inflammatory activity of seven lignans(P-3~P-7,P-9,P-10)and seven flavonoids(F-1~F-7).MTT assay showed that 14 compounds had no effect on cell viability in the concentration range of 6.25 ~ 100 μM.The content of NO was determined by Griess assay.The results showed that five lignans(P-3,P-5,P-7,P-9,P-10)and three flavonoids(F-3~F-5)could significantly inhibit the production of NO at five concentrations.F-1 and F-6 had no obvious inhibition on the production of NO at the concentration of 6.25 μM,but could inhibit NO production at the concentration range of 12.5 ~100 μM.The other four compounds(P-4,P-6,F-2,F-7)had no significant inhibitory effect on NO production.The production of inflammatory cytokines PGE2,TNF-α and IL-6 were detected by ELISA.The results showed that 10 compounds could inhibit the release of PGE2,TNF-α and IL-6.P-10,F-1,F-3 and F-6 had the best inhibition effect on PGE2.F-1,F-3,F-4 and F-6 had the best inhibition effect on TNF-α.P-5,P-7,P-10,F-5 and F-6 had the best inhibitory effect on IL-6.The i NOS gene expression of five lignans(P-3,P-5,p-7,P-9,P-10)was detected by fluorescence quantitative PCR.At the concentration of 25μM,all of those five compounds could inhibit the expression of i NOS,especially for P-9and P-10.The results showed that some lignans and flavonoids could inhibit the inflammatory reaction by inhibiting the release of NO and inflammatory factors PGE2,TNF-α and IL-6.Lignans played an anti-inflammatory role by inhibiting the expression of i NOS.(2)Based on the analysis method of network pharmacology,according to the "Lipinski rules" and experimental activity,23 active compounds were selected,including 12 lignans(P-1~P-12)and 11flavonoids(F-1~F-11).The potential action targets of 23 compounds were collected by Swiss Target Prediction server.Inflammation-related targets were collected by Gene Cards and OMIM and compared with compound targets to obtain the intersection between compounds and diseases.The results showed that there were 31 common targets between lignans and flavonoids,31 intersections among lignans and flavonoids and inflammation.Eight of them were common targets of lignans,flavonoids and inflammation.Protein interaction networks with high confidence were generated from the String 11.0 database,and PPI network analysis was performed on these intersections.The top 6 core targets with the most close connection were EGFR,IL-2,TNF,JAK1,PIK3 CD and JAK2.Four inflammation-related pathways,including NF-κB signaling pathways,chemokine signaling pathways,Jak-STAT signaling pathway and TNF signaling pathway,were selected for potential target labeling through GO enrichment and KEGG pathway by David 6.8 database.Some of the targets in these signaling pathways belong to the common targets of the two kinds of compounds,suggesting that the main active compounds in P.Zeylanic had multiple targets and had certain synergistic effects on the complex pathways of inflammation.(3)The molecular docking of six core targets and 13 key compounds was carried out for 23 times.Comparing with the binding energy of the original ligand in the target to verify the affinity of the compound to the target.The results showed that F-1,F-2 could bind to TNF,especially F-2,whose binding ability to TNF dimer surpassed the original.These results suggested that F-1 and F-2 may affect inflammation-related pathway by combining TNF,thus exerting anti-inflammatory effects.In the docking of JAK1,P-3,P-8 and P-12 had binding advantages,especially P-8,whose binding ability to JAK1 surpassed the original.These results indicated that lignans P-8 may act as inhibitors of JAK1 and displayed anti-inflammatory effect.P-12 and P-3 could bind to JAK2.(4)Based on the results of cell experiment and molecular docking,F-1,F-2 and F-2 showed good anti-inflammatory activity.They were both diglycosides and triglycosides with quercetin as the aglycone,which provided a great protein binding advantage by relying on the B ring of quercetin.Lignans,P-3,P-5,P-7,P-8 and P-10 had the best anti-inflammatory effect.Based on the analysis of structure-activity relationship,it was speculated that the number of-OCH3 group and the configuration of compounds might have some influence on the anti-inflammatory activity,but the presence or absence of-OAc group had little influence.This study has important scientific significance for elucidating the anti-inflammatory active compounds of Pouzolzia Zeylanic and their anti-inflammatory mechanism.It provides a new idea for the research and development of new anti-inflammatory drugs. |
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