| "Bi Zheng" is belongs to the category of rheumatism in the modern medicine system,including rheumarthritis and rheumatoid arthritis(RA),etc.Wu-tou Decoction(WTD)was first recorded in "Jin Gui Yao Lve",which is an ancient book of traditional Chinese Medicine written by traditional Chinese medical sage Zhang Zhongjing in the Han dynasty.It is composed of five kinds of single herbs,namely Aconiti Radix Preparata,Ephedrae Herba,Astragali Radix,Glycyrrhiza Radix and Paeoniae Radix Alba.This formula has been used to treat arthritis for more than a thousand years and has excellent clinical effects.Taking "drug-target-pathway" as the main line,this paper uses ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS)to explore the potential pharmacodynamic substances and their in vivo process of WTD in the treatment of RA,and further study its action mechanism based on targeted network pharmacology,metabolomics and 16S rRNA gene sequencing technology.1.After oral administration of WTD,the absorbed ingredients were identified to clarify the pharmacodynamic constituents in vivo.The differences in absorption,metabolism and distribution of main pharmacodynamic substances between normal and adjuvant induced arthritis(AIA)rats were further identified by pharmacokinetic and tissue distribution study.The "compound-target-pathway-disease" interaction network was constructed by using the method of targeted network pharmacology.The results showed that 11 inflammatory or immune response related pathways and 10 core targets(including COX-2 and TNF-α)were the key of treating RA with WTD.Finally,RAW 264.7 cells were induced by lipopolysaccharide to establish inflammation model and the moderating effects of WTD on expression of COX-2 and TNF-α were further verified.2.A comprehensive analysis for the absorbable components from licorice and the corresponding metabolic pathways of these components in vivo were performed.14 prototype components from licorice and 33 metabolites were identified.The differences of pharmacokinetic characteristics of 7 key pharmacodynamic substances(6 prototype components and 1 metabolite)between normal rats and AIA rats were clarified.The regulatory effects of licorice intake on 21 endogenous metabolites were also studied.Through the correlation analysis between endogenous metabolites and exogenous drug components,5 key biomarkers from tryptophan metabolic pathway were closely related to the treatment of RA with licorice.11 key targets of licorice against RA were found by targeted network pharmacological analysis.The key pharmacodynamic substances,key biomarkers and key targets of licorice against RA were obtained.The material basis and potential mechanism of licorice in treating RA were systematically studied.3.16S rRNA gene sequencing technology was used to clarify the changes of gut microbiota in AIA rats and obtain the key types of gut microbiota affected by WTD.The content changes of 31 important microbial metabolites,including short-chain fatty acids,bile acids and tryptophan metabolites,were analyzed by targeted metabolomics method.The results indicated that WTD could relieve the gut microbiota dysbiosis and reverse the abnormal changes of microbial metabolites in AIA rats.The correlation analysis of "gut microbiota-microbial metabolitespharmacodynamic index" proved that WTD could regulate inflammation and intestinal barrier function partially by modulating gut microbiota and microbial metabolites.This part expounded the mechanism of WTD against RA from the perspective of gut microbiota and microbial metabolites.4.The pharmacodynamic effects of WTD,polysaccharides of WTD(PS)and small molecules of WTD(SM)were evaluated by pharmacodynamics experiment.16S rRNA gene sequencing technology was used to clarify the regulatory effects of WTD,PS and SM on intestinal flora disorder caused by RA.The regulation effects of PS on the pharmacokinetic characteristics of 23 pharmacodynamic constituents from SM were clarified by pharmacokinetic study.The results showed that the efficacy and regulating effect on intestinal flora disorder of WTD were better than PS and SM.PS could change the pharmacokinetic characteristics of 13 pharmacodynamic constituents by regulating the composition of intestinal flora.The rationality and superiority of WTD as a complex of PS and SM was emphasized. |
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