基于UPLC-Q-Exactive Orbitrap MS/MS、网络药理学及体内实验探究防风抗类风湿关节炎的活性成分及作用机制OA

Objective:To explore the active components and mechanism of action of Saposhnikoviae Radix in treating rheumatoid arthritis(RA).Methods:The chemical components of Saposhnikoviae Radix were analyzed by ultra-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive-Orbitrap-MS/MS).Network pharmacology was employed to screen the potential active components and targets for the treatment of rheumatoid arthritis(RA),and the key targets were verified by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot analysis.Results:A total of 59 compounds were identified in Saposhnikoviae Radix.Among them,5-O-methylvisamminol,decursin,phellopterin,imperatorin,divaricatol,ledebouriellol,11-hydroxy-sec-O-β-D-glucosylhamaudol,and divaricatacid were possibly the main anti-RA components of Saposhnikoviae Radix.Network pharmacology analysis predicted 185 common targets.Topological analysis of the protein-protein interaction(PPI)network identified 36 key targets including tumor necrosis factor(TNF),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),and prostaglandin-endoperoxide synthase 2(PTGS2).The key targets ranking top three in terms of degree value and those closely related to the main active components were nitric oxide synthase 2(NOS2)and PTGS2.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis mainly predicted classic signaling pathways such as TNF,mitogen-activated protein kinase(MAPK),and nuclear transcription factor-κB(NF-κB).The results of animal experiments showed that compared with the model group,each treatment group showed the body weight and spleen index gradually approaching the normal levels.The treatment decreased the arthritis index(P<0.001)and hind paw swelling in a dose-dependent manner(low-dose group:P<0.05;medium-dose group:P<0.01;high-dose group:P<0.001).Measurement of serum levels of inflammatory factors showed that the levels of interleukin(IL)-1β and IL-6 in the medium and high-dose groups were decreased(P<0.01 or P<0.001),and the level of TNF-α in each treatment group declined(P<0.001).The levels of prostaglandin E2(PGE2)became lowered in the medium-and high-dose groups(P<0.01 or P<0.05).RT-qPCR results showed that the mRNA levels of TNF-α and IL-1β in the synovial tissue of each treatment group were lower than those in the control group(P<0.001).The mRNA levels of IL-6 and PTGS2 in the low-dose group were lower than those in the model group(P<0.01 or P<0.05).The mRNA levels of IL-6 and PTGS2 in the medium-and high-dose groups were lower than those in the model group(P<0.001).The results of Western blotting indicated that the protein level of COX-2 in the synovial tissue of the model group was higher than that of the control group(P<0.001),and the protein level of COX-2 in the synovial tissue of the high-dose group was lower than that of the model group(P<0.001).Conclusion:Saposhnikoviae Radix may inhibit downstream inflammatory signal transduction and alleviate arthritic damage in the rat model of adjuvant-induced arthritis(AIA)by targeting the COX-2/PGE2 signaling axis via its active components such as 5-O-methylvisamminol and decursin,thereby exerting a therapeutic effect on RA.