Exploring The Effect Of TR On MSU-Induced NETs Based On Network Pharmacology And In Vitro Experiments

Objective: Gout is a common and complex recurrent inflammatory disease that currently lacks a cure.Tongfengqingxiao Recipe(TR)contains many traditional Chinese medicines,which can treat Gouty Arthritis(GA)by reducing uric acid,anti-inflammatory,and improving human immunity through multiple targets and multiple pathways.Holistic,systematic and consistent with the core theories and methods of network pharmacology.In recent years,it has been newly discovered that regulating the generation and degradation of neutrophil extracellular traps(NETs)can also interfere with the process of GA disease.In this study,the network pharmacology method was used to explore the key targets and signaling pathways of TR in the treatment of GA,and to provide a reference for the selection of subsequent NETs-related inspection indicators.Effective research builds a solid experimental cornerstone.Methods: In the network pharmacology analysis,potential biologically active compounds in TR were selected using TCMSP and Pub Med databases.Obtain the "Homo sapiens" target gene name corresponding to the target protein of the active ingredient in the Uni Prot database.The target genes related to GA were obtained from the Gene Cards,Drug Bank,and Pharm GKB databases,and Wien analyzed the intersection of the above genes to obtain the potential targets of TR in the treatment of GA.Construction of protein-protein interaction(PPI)networks by STRING.GO analysis and KEGG pathway enrichment analysis were performed using David.Use Cytoscape to visualize TR-GA and pathway-target correlations.In the in vitro cell experiment,TR drug-containing serum was prepared in the diclofenac sodium group and different dose groups,and co-cultured with rat neutrophils and monosodium urate(Monosodium Urate,MSU)crystal suspension.Immunofluorescence specimens were prepared and combined with electron microscopy.The qualitative analysis of NETs was carried out by using fluorescence microplate reader,the DNA concentration in the supernatant was quantitatively detected,and the expression levels of TGF-β1,TNF-α,IL-1β and NF-κB were detected by ELISA kit.Results: There are 48 targets of TR in the treatment of GA,and PPI network analysis shows that the top ten key targets are IL-6,MMP9,HMOX1,PTGS2,CYP3A4,PPARA,PPARG,MAPK3,IL-1β,and CCL2.The function of GO mainly involves the positive regulation of apoptosis process,protein complexes,activated transcription factor binding,DNA binding,etc.TR treatment of GA involves TGF-β signaling pathway,as well as IL-17 signaling pathway,TNF signaling pathway,AGERAGE signaling pathway,NF-κB signaling pathway,etc.The results of the in vitro experiments showed that,compared with the model group,the production of IL-1β and TNF-α was significantly reduced after co-culture with the addition of drugcontaining serum,and the production of NF-κB in the TR high-dose group(High Dose,HD)was significantly decreased.The levels of TGF-β1 in the TR middle-dose group(Middle Dose,MD),HD and diclofenac sodium groups were significantly increased(P<0.05).The inhibition of NF-κB by MD group(Low Dose,LD)and MD,the promotion of TGF-β1,and the inhibitory effect of TR LD on TNF-α were not as good as those in the diclofenac sodium group(P<0.05).The formation of NETs in the TR and diclofenac sodium groups was inhibited,and the content of extracellular free DNA in neutrophils decreased.Conclusion: TR contains a variety of traditional Chinese medicine ingredients,showed a multimolecular,multi-target,multi-channel synergistic mechanism,and its treatment of GA is related to multi-channel regulation.Network pharmacology analysis and in vitro experiments showed that TR could reduce IL-1β,TNF-α,NF-κB through non-Smads pathway,up-regulate the production of TGF-β1,and then inhibit the production of NETs.