| Objectives:1.A metabonomics-based method was used to study the intervene effect of Danggui Sini decoction on collagen-induced arthritis in rats,thus revealing the mechanism of action of Danggui Sini decoction in treating rheumatoid arthritis at the microscopic level of metabolic pathways.2.Using network pharmacology research methods to predict the potential role of Danggui Sini decoction in the intervention of rheumatoid arthritis,and to prove that Danggui Sini decoction has certain intervention effects on rheumatoid arthritis.3.Integrate the results of metabolomics and network pharmacology,aiming to elucidate the specific mechanism of Danggui Sini decoction in the intervention of rheumatoid arthritis at the metabolic level and on the genetic level based on computer simulation techniques.Methods:1.Validation of Network Pharmacology Findings,the rats were divided equally and randomly into four groups: the normal group(NG),model group(MG),dexamethasone group(DG)and DSD treated group(TG).After 1 week for acclimation,except the normal group,the rats were injected intradermally with 400 μl bovine type II collagen(CII)emulsified in an equal volume of incomplete Freund’s adjuvant(IFA)at the base of the tail(day 0).7 days after the primary immunization,rats were boosted intradermally with 100 μl CII in IFA for the second time.The progression of CIA was evaluated by measuring thickness of the right hind joint,body weight every seven days with a vernier calliper(GB/T21389-2008,Guilin)and an electronic balance(JSC-1000,Zhejiang).To assess the effect of DSD on the established CIA model rats,all drugs were administered to them once daily via gavage from day 20 to day 40 of the experiment.2.To evaluate the modeling results by discussing the weight,changes in the thickness of the ankle,and the histopathological changes in the joints,and to discuss the intervention effects of Danggui Sini decoction.3.NMR-based metabonomics analysis techniques and multivariate statistical analysis were used to look for differences in urinary metabolites between the model group and the blank group at the metabolic level.To analyze the possible relationship between the differential substances and rheumatoid arthritis,to identify potential biomarkers related to the treatment of rheumatoid arthritis,and to further characterize the changes of the endogenous metabolites in the urine of model rats.The law found that Danggui Sini decoction interfered with the metabolic pathways of rheumatoid arthritis and explained the mechanism of Angelica Sini decoction in the intervention of rheumatoid arthritis.4.Danggui Sini decoction(DSD)is a classic TCM formula consisting of seven herbs: Angelica sinensis,Cinnamomi cassia,Paeonia lactiiflora,Tetrapanax papyriferus,Asarum heterotropoides,Glycyrrhiza uralensis and Ziziphus jujuba Mill.Statistical analysis of chemical composition,screening of its active ingredients,and prediction of possible targets for the active ingredient.5.Query the relevant gene targets of rheumatoid arthritis through the database.6.Establish network of Danggui Sini decoction-chemical-target-disease relationship using network visualization software Cytoscape 3.2.1software to find related gene targets.7.Gene enrichment analysis was performed to determine the signal pathways of Danggui Sini decoction main intervention,and preliminary prediction of the intervention mechanism of Danggui Sini decoction on rheumatoid arthritis.8.Integrate the results of metabolomics and network pharmacology to discuss the specific intervention mechanism of Danggui Sini decoction on rheumatoid arthritis.9.Detection of serum biochemical markers of tumor necrosis factor(TNF-α)changes,preliminary validation of the research results.Results:1.Seven potential biomarkers were found in the urine of collagen-induced arthritis rats,in which the levels of acetone,succinate,and pyruvate were elevated,and the levels of taurine,urea,betaine and hippuric acid were decreased.It mainly affects amino acid metabolism,gut bacteria metabolism,energy metabolism and lipid metabolism pathways.2.Danggui Sini decoction can down-regulate acetone,succinate and pyruvate in the urine of CIA rats and up-regulate taurine,urea,betaine and hippuric acid.It is mainly responsible for the metabolism of taurine and hypotaurine caused by collagen-induced arthritis,metabolism of arginine and valine,metabolism of intestinal microflora,metabolism of pyruvate,TCA cycle,glycolysis and gluconeogenesis.Lipid metabolism plays a role.The results of the study provide new ideas for the treatment of rheumatoid arthritis in the later period and research on the mechanism of action of traditional Chinese medicine on diseases.3.The network pharmacology found that there were 138 active ingredients in Danggui Sini Tang and 185 diseases related to rheumatoid arthritis.A total of 19 related gene targets were found after using the Cytoscape software to establish the medicinal-chemical-target-disease network.4.After enrichment analysis,13 signal pathways were obtained,among which the highest enrichment score of TNF signaling pathway may be the main signaling pathway of Angelica Sini decoction in the intervention of rheumatoid arthritis.5.After combined metabolomics and network pharmacology studies,Angelica Sini decoction mainly plays a role by regulating amino acid metabolism,energy metabolism,and lipid metabolism disorders.Among them,Danggui Sini decoction can regulate the expression of GAD1,GGT1,and GAD2 genes in the metabolic pathways of taurine and hypotaurine to retreat the disorder of tumor necrosis factor signaling pathway.Conclusions:1.Danggui Sini decoction can improve the serum biochemical indexes,body weight,foot and ankle lesions,joint pathological changes in CIA rats.2.The CIA rat model can cause a significant change in the metabolic profiles of the urine of rats,significantly disturb amino acids and gut becteria metabolism,energy metabolism and lipid metabolism.Danggui Sini decoction has a corrective effect on these metabolic pathways.3.Danggui Sini decoction plays a role in interfering with the TNF signaling pathway in RA. |
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