Rapid Identification Of Chemical Constituents Of Xitong Preparation By LC-MS And Its Anti-Rheumatoid Arthritis Mechanism

Xitong preparation includes Xitong pill,Xitong capsule and Fengshi Xitong tablet,in which the former two are recorded in the 2020 edition of the Chinese Pharmacopoeia.Its prescription is composed of two herbs,Siegesbeckiae Herba of Asteraceae and Clerodendrum trichotomum of Verbenaceae.The prescription is simple,symptomatic and has excellent effects.It is widely used in the treatment of rheumatoid arthritis in clinic,but its pharmacodynamic material basis and mechanism of action are still not very clear.A comprehensive analysis and study of its chemical composition,and mechanism of anti-rheumatoid joint and pharmacodynamic material basis are of great significance for the quality control and clinical application of Xitong preparations.Firstly,an ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry method was established to rapidly analyze the chemical constituents in the Chinese medicine compound Xitong preparation for anti-rheumatoid arthritis.ACQUITY UPLC (?) HSS T3 column(2.1 mm × 150 mm,1.8 μm)was used with 0.1 % formic acid aqueous solution-0.1 % acetonitrile as the mobile phase for gradient elution at a flow rate of 0.3 ml / min.The detection was carried out in positive and negative ion modes,respectively.Peakview software combined with medicinal material compound database was used for analysis.According to the accurate relative molecular weight obtained,combined with comparison with reference substances,characteristic fragments of secondary mass spectrometry and literature reports,the chemical constituents were determined.A total of 78 chemical components were identified,including 20 phenylpropanoids,33 terpenoids,19 flavonoids and 6 lipid oxides,of which 2 may be new compounds.This method can comprehensively and quickly identify the chemical constituents in Xitong preparation,and provide reference for the quality control and pharmacodynamic material basis research of Xitong pill and Xitong capsule.Secondly,the pharmacodynamic material basis and molecular targets of Xitong preparations were studied by network pharmacology and molecular docking.The Swisstarget and targetnet databases were used to predict the targets of chemical components identified by liquid chromatography-mass spectrometry(LC-MS).Based on the rheumatoid arthritis-related chip data in the GEO database,the key differential genes of rheumatoid arthritis were obtained.After intersection,64 targets of Xitong preparation for rheumatoid arthritis were obtained.Cytoscape software was used to draw the Xitong preparation-target network diagram and perform topological analysis.Finally,four active components including apigenin,caffeic acid,aesculetin and clerodenoid A and eight target proteins such as BCL2A1,PLIN1,PTGS2,ALOX5,CA2,MMP1,HSD11B1 and MAOA were screened out.Further,molecular docking was used to verify the active components and target proteins screened by network pharmacology.The molecular docking results showed that PTGS2,ALOX5,MMP1 and HSD11B1 had excellent docking activity with apigenin,caffeic acid,aesculetin,clerodenoid A,kirenol and darutoside,indicating that they had high importance in the treatment of rheumatoid arthritis with Xitong preparations.At the same time,the combination of apigenin with each target was the best,followed by caffeic acid and clerodenoid A,indicating that they may play a greater role in the compound.Finally,the anti-rheumatoid inflammatory effect in vitro and mechanism of the water extract of Siegesbeckia pubescens Makino(SPW)were investigated in the IL-1β-induced MH7 A synovial cell.The effects of SPW on the m RNA levels of inflammatory factors(TNF-α,IL-6,IL-1β)in IL-1β-induced MH7 A cells were determined by RT-qPCR.The results showed that SPW could inhibit the m RNA levels of TNF-α,IL-6,IL-1β in IL-1β-induced MH7 A cells(P<0.01).Furthermore,RNA-seq technology was used to analyze the transcriptome of IL-1β-induced MH7 A treated with SPW.Differentially expressed m RNAs were screened with fold change |FC| > 1.5 as the standard,and metascape database was used for functional and signaling pathway enrichment analysis.GSEA analysis was used to predict the key gene subset and its core genes in the SPW treatment group compared with the model group.Metascape database was used to analyze the function and signal pathway enrichment of core genes.Cytoscape was used to screen Hub genes,and TRRUST database was used to predict the transcription factors regulating these Hub genes.The results showed that SPW treatment of IL-1β-induced MH7 A cells involved ’positive regulation of leukocyte migration’,’positive regulation of response to external stimulus’,’leukocyte activation’.The enriched pathways included ’IL-17 signaling pathway’,’PI3K-Akt signaling pathway’,’p53signaling pathway’,’TNF signaling pathway’,’ NF-κB signaling pathway’.The Hub genes of SPW acting on IL-1β-induced MH7 A cells were IL-6,CD28,FOXP3,CCR7,CCL5,IL2 RG,VEGFA,MAPK3,IL4 R,JAK3 and GATA3.According to their expression level,six genes including IL-6,IL2 RG,VEGFA,MAPK3,IL4 R and GATA3,were selected to be verified by qPCR The qPCR detection showed that SPW could reduce the mRNA expression level of these genes,suggesting that SPW could exert anti-inflammatory effects by affecting these genes.