Screening active small-molecule components from wutou decoction for rheumatoid arthritis treatment via intestinal...

Disruption of intestinal barrier integrity is increasingly recognized as an important contributor to the initiation and progression of rheumatoid arthritis (RA), making intestinal barrier protection a potential therapeutic strategy. Wutou Decoction (WTD), a traditional Chinese medicine prescription, has been reported to exert anti-inflammatory and immunoregulatory effects and to ameliorate RA-associated intestinal barrier dysfunction. However, the small-molecule constituents responsible for its intestinal barrier-protective effects remain largely unclear. In this study, the intestine was considered a key target organ, and an intestinal-retention-oriented strategy was employed to identify the active small-molecule constituents of WTD and investigate their potential mechanisms underlying intestinal barrier protection in RA. First, WTD aqueous extract and its small-molecule fraction (WS) were evaluated using an LPS-induced Caco-2 intestinal barrier injury model, including cell viability, transepithelial electrical resistance (TEER), FITC-dextran permeability, and tight-junction protein expression. WS exhibited significant protective effects against intestinal barrier damage. Subsequently, UPLC-Q-TOF-MS/MS was employed to characterize the chemical profiles of WS in vitro and in vivo. A total of 145 small-molecule constituents were identified in vitro, while 29 blood-entry components and 23 intestinal retention components were detected in rat serum and colonic contents, respectively. Unlike conventional approaches focusing primarily on blood-entry constituents, this study prioritized intestinal retention components as potential locally acting constituents at the intestinal target site. Network pharmacology was then integrated with molecular docking and molecular dynamics simulations to prioritize potential target-compound interactions, identifying GPX3 and TAP1 as predicted candidate targets and 13 intestinal retention components meeting the initial docking-score screening criterion. Finally, three representative intestinal retention components were experimentally evaluated in the LPS-induced Caco-2 model using barrier function, permeability, apoptosis, and related protein expression assays. The selected compounds significantly alleviated intestinal barrier damage, supporting their potential contribution to the intestinal barrier-protective effects of WTD. In conclusion, this study establishes an intestinal-retention-oriented strategy for screening potential active small-molecule constituents of WTD and integrates chemical profiling, network pharmacology, molecular docking, molecular dynamics simulations, and experimental validation to characterize their potential roles in intestinal barrier protection. These findings provide new insights into the pharmacodynamic material basis of WTD from the perspective of the gut-joint axis and provide a mechanistic basis for its potential relevance to RA.