Ya Han Jie ameliorates adjuvant-induced arthritis by inhibiting the NF-κB/NETosis/inflammation axis

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that mainly affects the joints. Ya Han Jie (YHJ) is a Dai medicine that has significant therapeutic effects on RA. However, the bioactive compounds and potential mechanisms of YHJ in treating RA still need to be elucidated. PURPOSE: The aim of this study was to investigate the effects of YHJ on RA and to illuminate its underlying therapeutic mechanisms. METHODS/STUDY DESIGN: We injected Freund's complete adjuvant (FCA) into the joint cavity of mice to establish an adjuvant-induced arthritis (AA) mouse model and evaluate the therapeutic effect and safety of YHJ on RA. First, we measured the joint diameters of the mice and assessed the effect of YHJ on the degree of joint swelling. Second, we assessed the effects of YHJ on pathological changes in the joints of AA mice by performing Hematoxylin-eosin staining and Safranin O-fast green staining. Afterward, we used network pharmacology, molecular docking, and molecular dynamics simulations to identify the active components in YHJ that act on RA and explored the therapeutic mechanism of YHJ in treating RA with RNA sequencing. Moreover, we evaluated the effect of YHJ on neutrophil extracellular traps (NETs) using ankle joint tissue sections and purified neutrophils. Finally, we further evaluated the effects of YHJ on the gut microbiota of AA mice by 16S rDNA sequencing. RESULTS: YHJ significantly reduced joint swelling, arthritis scores, and synovitis-related pathological changes in AA mice. YHJ also inhibited the expression of TNF-α, IL-6, IL-17A, and IFN-γ. UPLC-Q-TOF MS/MS was used to characterize the chemical composition of YHJ, identifying 104 compounds mainly classified as amino sugars, glycosides, alkaloids, and terpenoids. Network pharmacology was employed to identify potential targets and signaling pathways of YHJ in the treatment of RA, and transcriptomic analysis revealed that YHJ downregulated genes associated with NET formation. In vivo and in vitro experiments further confirmed that YHJ significantly inhibited NET formation. CONCLUSION: YHJ has a significant therapeutic effect on AA mice by reducing the phosphorylation and activation of NF-κB, decreasing the expression of proteins related to NET formation, and inhibiting NET formation and the release of inflammatory factors in vitro and in vivo.