Single-cell and spatially resolved transcriptomics elucidate the therapeutic mechanism of Tripterygium wilfordii Polyglycosidium in ulcerative colitis.

Abstract

BACKGROUND: Tripterygium wilfordii polyglycosidium (TWP) can alleviate inflammation in ulcerative colitis (UC). However, the core disease-specific biomarkers and targets of UC and the therapeutic mechanism of action of TWP have not been fully elucidated. PURPOSE: A comprehensive strategy involving single-cell sequencing, spatial transcriptomics, and experimental validation was employed to investigate the potential targets of TWP in the treating UC. METHODS: The molecular mechanisms of action and therapeutic targets of TWP in the treating UC were investigated by integrating single-cell RNA sequencing with spatial transcriptomics. A CRISPR-Cas9 gene-edited animal model was established. Intestinal inflammation and mucosal damage were evaluated using Western blot analysis, hematoxylin‒eosin staining, and immunofluorescence to validate the target genes. The chemical constituents of TWP were characterized by UPLC-Q-TOF-MS, and interactions with the identified targets were assessed through molecular docking and surface plasmon resonance analyses. RESULTS: TWP significantly alleviated intestinal inflammation and mucosal damage in DSS-treated mice. Single-cell sequencing and spatial transcriptomic analyses identified S100A8, which facilitates signal transduction between neutrophils and their effector cells (macrophages), as a potential core target of TWP in treating UC. Multimodal validation revealed that S100A8 overexpression exacerbated colitis severity and intestinal mucosal disruption, whereas S100A8 knockout markedly attenuated intestinal inflammation and mucosal injury. We identified a total of 52 chemical components in TWP. Molecular docking indicated that 6 components could bind to S100A8. SPR analysis further confirmed that triptolide and demethylzeylasteral strongly bound S100A8. CONCLUSION: TWP may primarily alleviate inflammatory symptoms in UC by suppressing S100A8 expression, thereby protecting intestinal mucosal barrier function.