Network Pharmacology And Experimental Validation Of Polygonum Cuspidatum In The Treatment Of Rheumatoid Arthritis

Polygonum cuspidatum was first published in "Famous Doctors".Traditionally recorded that Polygonum cuspidatum can be used to treat rheumatic arthralgia.Modern research also shows that Polygonum cuspidatum can be used to treat rheumatoid arthritis.Its active ingredients and molecular mechanisms are still unclear.Network pharmacology is a method based on the theory of systems biology and multidirectional pharmacology,which analyzes biological systems through the construction of drug-gene-target-disease interaction network,so as to predict the active ingredients and molecular mechanisms of drugs.Based on network pharmacology and experimental verification,this paper studied the active ingredients and molecular mechanism of Polygonum cuspidatum in the treatment of rheumatoid arthritis,in order to provide a basis for the clinical application of Polygonum cuspidatum in the treatment of rheumatoid arthritis,and lay a foundation for the development and research of Polygonum cuspidatum.Experimental methods:1.TCMSP,TCMID,BATMAN-TCM and Phram Mapper databases were used to screen the active ingredients and predict the targets of the active ingredients.RA disease targets were collected from Drug Bank,TTD,Dis Ge NET,Gene Cards and OMIM databases.Then,Venny 2.1.0 software was used to screen disease targets of Polygonum cuspidatum in the treatment of rheumatoid arthritis.The network of drug-active ingredient-disease-target was constructed with Cytoscape 3.7.2 software.The STRING database was used to construct PPI network.The GO function and KEGG pathway enrichment analysis were performed using the DAVID 6.8 database,and the enrichment results were presented using the Omic Share platform.Finally,the drug-active ingredient-target-pathway network was constructed using Cytoscape 3.7.2 software.2.MH7 A cells were used as the disease research model,CCK-8 method was used to study the effects of different concentrations of quercetin,resveratrol,luteolin,emodin,polydatin and β-phytosterol on cell proliferation after treatment for 24 h and 48 h,and the IC50 values of different ingredients at different time were calculated.The influence of different ingredients on inflammatory factors TNF-α,IL-1β,IL-6,IL-8 and TLR4/NF-κB signaling pathway was explored by ELISA and Western Blot assay.Experimental results:1.A total of 14 active ingredients,346 active component targets and 690 disease targets of Polygonum cuspidatum were screened using the database;by constructing the Venn diagram 2.1.0,122 intersection targets were obtained;topological analysis showed that the key active components mainly included quercetin,resveratrol,luteolin,emodin,polydatin,β-sitosterol and so on;through the construction of PPI network,20 key targets were screened;GO function analysis showed that BP,CC and MF were mainly related to inflammatory response,extracellular space and cytokine activity;KEGG pathway analysis showed that it was mainly related to TNF signaling pathway,osteoclast differentiation,Toll-like receptor signaling pathway,T cell receptor signaling pathway,NF-κB signaling pathway,etc.Then the network of drug-active ingredienttarget-pathway was constructed.Therefore,six active ingredients,quercetin,resveratrol,luteolin,emodin,polydatin,β-sitosterol,and TLR4/NF-κB signaling pathway were selected for subsequent experimental verification.2.(1)Cell proliferation inhibition experiments show that these six active ingredients can inhibit the proliferation of MH7 A cells(P<0.01 or P<0.05),and are concentrationdependent.Except for emodin and polydatin,the IC50 of the other component at 48 h was lower than that at 24 h,which indicated that most of the components had stronger inhibitory effect on cell proliferation at 48 h.(2)ELISA assay showed that the six active ingredients could inhibit the secretion of inflammatory cytokines IL-1β and IL-8(P<0.01);quercetin could inhibit the secretion of TNF-α(P<0.01),the other five ingredients have a tendency to inhibit the secretion of TNF-α,but there was no statistical significance(P>0.05);the six active ingredients have a tendency to inhibit the secretion of IL-6,but there was no statistical significance(P>0.05).(3)Western Blot analysis showed that these six active ingredients have a certain tendency to inhibit the protein expressions of TLR4,NF-κB p65,p-p65 and IκB-α,but there was no statistical significance(P>0.05).Conclusion: The main effective ingredients of Polygonum cuspidatum for treating rheumatoid arthritis include quercetin,resveratrol,luteolin,emodin,polydatin,β-sitosterol,etc.It has the effect of inhibiting the proliferation of MH7 A cells,reducing secretion of inflammatory factors and having a certain tendency to inhibit the protein expression of related pathways.The molecular mechanism may be related to inhibition of TLR4/NF-κB signaling pathway.