| Objective: The purpose of this study is to analyze the medication regularity of gouty arthritis(GA)with traditional Chinese medicine(TCM),and explore the combination of the core herbs.Then,the pharmacological mechanism of the core herbs and the effect of key compound were analyzed and verified by experiments in vitro/in vivo,so as to provide theoretical support for mechanism research and clinical treatment on GA.Methods:(1)Firstly,data mining technology was used to collect the relevant literature on GA treatment with TCM.After screening,prescriptions were picked out and a TCM prescription database was constructed.Then,the frequency analysis,property,taste,and channel tropism analysis,systematic cluster analysis were used to explore the rules of GA treatment with TCM.Finally,a core herbs combination was obtained by association rule analysis(2)Network pharmacological technology was used to analyze the mechanism of the core herbs combination on GA.Firstly,pharmacokinetic parameters were used to screen the active ingredients and potential targets.Then,a Drugs-Compounds-Targets network was constructed by the Cytoscape software,and key compounds were screened out according to the degree value.Meanwhile,the GA-related targets were predicted by the Gene Card,OMIM,and NCBI databases,and the protein-protein interaction(PPI)network was established based on Bisogenet to screen out core targets.Moreover,the common targets between GA and drugs were obtained by Venn analysis.Conclusively,merge the core targets and common targets as the interaction targets between herbs and GA,and import them into the DAVID and Metascape databases respectively for GO and KEGG enrichment analysis.(3)Experimental verification was carried out both in vivo and in vitro.According to the results above,quercetin was selected as the key compound.In vitro,human myeloid leukemia mononuclear cells(THP-1)were selected as the study object,besides,and BALB/c mice were selected as the study object in vivo.Firstly,the cell model of GA was obtained by stimulating the THP-1 cells with monosodium urate(MSU),and then a cytotoxicity assay was performed with cell counting kit-8.Subsequently,cells were treated with different doses of quercetin,and the expression of inflammatory factors and proteins related to the Interleukin-17(IL-17)signal pathway was detected by immunofluorescence.In vivo,the GA model was obtained by intragastric administering of hypoxanthine(0.1 g/kg)and joint injection of MSU(20～50 μL,0.1 g/m L)into BALB/c mice.Then,quercetin was gavaged for GA treatment,which contained a low-dose group(50 mg/kg)and a high-dose group(200 mg/kg)respectively.After the experiments,hematoxylin-eosin staining and Masson staining were used to observe the joint injury of mice,and the enzyme-linked immunosorbent assay was used to detect the level of inflammatory factors in serum.Moreover,immunohistochemistry and western blot were used to detect the expression level of inflammatory factors and proteins related to the IL-17 signaling pathway in synovial tissue.Results:(1)A total of 18 043 papers were retrieved.After screening,there were 246 papers which contained 252 prescriptions and 312 herbs collected for research.The total drug frequency was 2 846.After the property,taste,and channel tropism analysis,it was found that cold herbs were the main drugs in the treatment of GA,and the drug flavor was mainly bitter and sweet.Moreover,the main tropism contained liver,stomach,and spleen channels.In addition,the high-frequency herbs were divided into 5 categories by cluster analysis,corresponding to the effect of clearing away heat and toxic material,warming yang to promote diuresis,activating blood circulation and removing blood stasis,nourishing,and relieving pain.Finally,a core herbs combination contained Smilax glabra Roxb.,Semen Coicis,Phellodendron chinense Schneid,Rhizoma Atractylodis,and Glycyrrhiza uralensis Fisch.,was obtained by association rule analysis.(2)After database predicting and screening of the 5 core herbs,a total of 128 active compounds and 263 potential targets were obtained.Meanwhile,245 GA-related targets were predicted.Then,a DrugCompounds-Targets network was constructed,and 10 core active compounds were screened according to the degree value,including quercetin,stigmasterol,naringin β-sitosterol,kaempferol,viscidin,skullcap,7-methoxy-2-methylisoflavone,isocorydrine,and anthocyanin.Quercetin has the highest degree value which was considered as key compound.Moreover,a total of 59 drug-disease interaction targets were obtained through PPI and Venn analysis.And it showed that many biological processes and signal pathways were involved,including transcription,translation,protein binding,and other processes,as well as the IL-17 signal pathway,mitogen-activated protein kinase(MAPK)signal pathway,and tumor necrosis factor(TNF)signal pathway through GO and KEGG enrichment analysis.Finally,it was found that the IL-17 signaling pathway occupied the most important position,which was screened by P value and pathway-target network.(3)To verify the role of quercetin and IL-17 signaling pathway in the treatment of GA,cell experiments and animal experiments were carried out respectively.The results of the cell experiments showed that quercetin can effectively inhibit the inflammatory reaction of cells,reduce the level of IL-1 β,IL-6,and TNF-α,and inhibit the expression of IL-17 pathway-related proteins such as IL-17,p-p65,activator 1(ACT1).As for animals experiment,it found that quercetin can reduce joint swelling,and decrease synovial tissue damage and fibrosis in GA mice.Additionally,quercetin can reduce the level of inflammatory factors in serum and down-regulate the level of IL-1 β,IL-6,TNF-α in synovial tissue as well as the protein expression of IL-17,p-p65,and ACT1.Conclusion: The results of data mining shows that Smilax glabra Roxb.,Semen Coicis,Phellodendron chinense Schneid,Rhizoma Atractylodis,and Glycyrrhiza uralensis Fisch.make up the core herbs combination of GA treating.Quercetin is a key compound of the 5 core herbs.Experientially,quercetin is a potential candidate compound for GA treating and can alleviate GA by regulating the IL-17 signaling pathway. |
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