Single-cell transcriptomic analysis reveals that Trametes robiniophila Murr. (Huaier) granules protect against ulcerative colitis by targeting macrophage-associated cGAS-STING signaling.
ETHNOPHARMACOLOGICAL RELEVANCE Trametes robiniophila Murr. (Huaier granules) is a medicinal fungus with over 1,600 years of historical use in traditional Chinese medicine. It has been traditionally applied to alleviate "intestinal wind" with bleeding, dysentery, and other inflammatory gastrointestinal disorders, in addition to its well-known role as an adjuvant therapy for various cancers. However, the scientific basis for its traditional use in intestinal inflammation remains poorly understood. AIM OF THE STUDY This study aims to investigate whether and how Huaier protects against ulcerative colitis (UC), a chronic inflammatory bowel disease, thereby providing a pharmacological rationale for its ethnopharmacological application in gut inflammatory conditions. MATERIALS AND METHODS Network pharmacology was used to predict the active compounds and potential targets of Huaier granules. A DSS-induced colitis mouse model was established to evaluate the therapeutic effects of Huaier, with 5-ASA as a positive control. Single-cell RNA sequencing (scRNA-seq) was performed to identify key cellular populations and signaling pathways affected by Huaier. In vivo and in vitro experiments were conducted in LPS-stimulated RAW264.7 macrophages and Caco-2 intestinal epithelial cells. Rescue experiments using the STING agonist cGAMP were performed to validate pathway dependency. Gut microbiota and short-chain fatty acids (SCFAs) were analyzed to assess microbial-metabolic remodeling. RESULTS Network pharmacology suggested that Huaier may regulate inflammatory and macrophage-related processes. ScRNA-seq revealed that Huaier prominently remodeled the myeloid compartment, increased monocyte abundance, decreased macrophage abundance, and restrained monocyte-to-macrophage differentiation. Huaier suppressed cGAS-STING-related inflammatory signaling in macrophages, which was validated in vivo and in vitro. Activation of STING with cGAMP partially reversed the protective effects of Huaier in both animal and cell models. Huaier also restored intestinal barrier-related molecules, improved gut microbial dysbiosis, and elevated several SCFAs, which were negatively correlated with colitis severity and *Tmem173/Cgas* expression. Furthermore, Huaier alleviated LPS-induced inflammatory injury and promoted repair-related functions in Caco-2 cells. CONCLUSION Huaier exerts significant protective effects against UC by remodeling myeloid cell populations, suppressing macrophage-associated cGAS-STING signaling, restoring intestinal barrier integrity, and modulating gut microbiota and SCFA metabolism. These findings provide a mechanistic basis for the traditional use of Huaier in treating inflammatory gastrointestinal disorders and support its development as a multi-target therapeutic candidate for UC.