| Objective: Simiao Powder(Si Miao San,SMP),derived from Cheng Fang Bian Du Volume Three written by Zhang Bingcheng in the Qing Dynasty,has the effect of clearing heat and promoting dampness,relaxing tendons and activating collaterals.From ancient times to the present,traditional Chinese medicine doctors often use it to treat gouty arthritis(Gouty Arthritis,GA)and other joint inflammatory diseases.Modern studies have also shown that SMP can be used to treat GA.Although the clinical efficacy of SMP has been recognized by modern traditional Chinese medicine,the current research is still focused on clinical and chemical components.There are few reports on the material basis and molecular mechanism of SMP in the treatment of GA,which limits the further development and utilization of SMP.This paper is based on the research method of the combination of network pharmacology and experimental verification,to study the effective components and molecular mechanism of SMP in the treatment of GA.It is expected to provide a basis for the clinical application of SMP in the treatment of GA,and lay a foundation for the development and research of SMP and modern famous medical experience prescription-Tongfeng Qingxiao prescription.Methods:1.65 active components and 193 targets of SMP were identified by database,and 223 GA related targets were screened by Gene Cards and OMIM databases.In the Venny2.1.0 platform,the two targets were screened by constructing Wayne diagram,and 23 overlapping targets were identified as candidate targets,intersect the target genes of GA with the genes regulated by SMP active compounds;Input the common gene targets into STRING online platform for PPI network establishment and topology analysis,download the TSV format file of PPI network and import it into R software for visualization,and use the corresponding data packets to analyze the GO function and KEGG pathway enrichment of intersecting target proteins.Finally,the target-signal pathway network is constructed by Cytoscape3.7.2 software.2.Taking the GA model rats as the research model,the effects of SMP on the general behavior and histopathology of rats were studied by observing the ankle GA index score,ankle swelling degree and pathological changes of ankle synovium.3.GA model rats were used as the research model.The expression of inflammatory factors IL-1β,IL-6,TNF-α,IL-17 in rat serum was detected by ELISA,and the expression of MAPK protein and NF-κB protein in rat synovium was detected by Western Blot to explore the influence of SMP on IL-17 signal pathway.Results:1.A total of 65 SMP active ingredients and 193 top targets of pharmaceutical active ingredients were screened by TCMSP database,and 223 top GA-related targets were screened by Gene Cards and OMIM database,and in Venny2.1.0 platform,two targets were overlapped and screened by constructing Wayne diagram,and 23 overlapping targets were identified as candidate targets;and topological analysis showed that the key active components mainly included quercetin,rutaecarpine,wogonin,kaempferol,baicalein and so on.Through the construction of PPI network,the key targets of IL1-β,IL-6,PTGS2,CCL2,MMP9,PPARG and MMP3 in the treatment of GA were obtained,and the functional analysis of GO showed that BP,CC and MF were mainly related to oxidative stress,inflammation,MAP kinase activity and cytokine activity.KEGG pathway analysis showed that it was mainly related to IL-17 signal pathway,TNF signal pathway,AGE-RAGE signal pathway,NOD-like receptor signal pathway,fluid shear stress and atherosclerosis signal pathway,Chaga’s disease signal pathway and so on.Then the target-pathway network is constructed by Cytoscape3.7.2,and the visual analysis with R language shows that IL-17 ranks high.Therefore,in this study,SMP and IL-17 signal pathways are selected for subsequent experimental verification.2.Through the rat GA model,it was found that SMP could effectively improve the GA index score of rats(P<0.01),and the effect was similar to that of diclofenac sodium(P>0.05),and the joint swelling degree of GA model rats reached the peak at12 hours after modeling,and the therapeutic effect of SMP was better than that of diclofenac sodium 24 hours later(P<0.05).On the whole,SMP could effectively reduce the ankle swelling of GA rats,and the effect was similar to that of diclofenac sodium(P>0.05),and SMP could effectively improve the pathological changes of ankle synovial tissue or inhibit the further damage of inflammatory mediators to synovial tissue,and the effect was better than that of diclofenac sodium.3.The results of ELISA test showed that SMP could significantly inhibit the secretion of inflammatory factors such as IL-1β,IL-6,TNF-α and IL-17(P<0.01),and the inhibitory effect was significantly better than that of diclofenac sodium(P<0.01).Western Blot analysis showed that SMP could significantly inhibit the expression of MAPK and NF-κB protein(P<0.01),and the effect was better than that of diclofenac sodium(P<0.05).Conclusions: SMP can inhibit the secretion of inflammatory factors IL-1 β,IL-6,TNF-α and IL-17,and reduce the protein expression of MAPK and NF-κB,which may be the reason why SMP can relieve synovitis,pain,redness,swelling,heat and pain in patients with GA.IL-17 signal pathway may be one of the molecular mechani SMP of SMP in the treatment of GA. |
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