Zhilining formula alleviates ulcerative colitis by restricting STAT3 activation and subsequent Th17 cell differentiation.

BACKGROUND

Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease with rising prevalence and limited therapies. Zhilining Formula (ZLN), a Traditional Chinese Medicine (TCM), shows clinical efficacy in UC, yet its core material basis and precise mechanisms remain obscure.

PURPOSE: To identify bioactive constituents and molecular targets of ZLN, and to uncover the pharmacological mechanisms underlying its anti-UC effects.

METHODS

DSS and TNBS-induced colitis models were established to assess ZLN's efficacy. Network pharmacology, KEGG/GO enrichment, Machine learning and Mendelian randomization were employed to identify core targets and pathways. IL-17A neutralization and AAV9-Stat3 were employed to validate involvement of Th17 cell differentiation and STAT3 signaling in ZLN's therapeutic effects. UHPLC-MS/MS was utilized to characterize chemical composition of ZLN, and surface plasmon resonance assay was applied to confirm direct interactions between core compounds and STAT3.

RESULTS

ZLN significantly alleviated DSS and TNBS-induced colitis. Network pharmacology identified 65 active compounds, 864 potential targets, and 2826 UC-related genes, with 275 overlapping targets as key mediators of ZLN's effects. Enrichment analysis and IL-17A neutralization highlighted Th17 cell differentiation as a central pathway. Machine learning and Mendelian randomization identified STAT3 as a crucial target. Mechanistically, ZLN inhibited Th17 cell differentiation by blocking STAT3 activation. Four core constituents including andrographin, panicolin, wogonin, and luteolin directly bound to STAT3, contributing to Th17 inhibition and therapeutic efficacy.

CONCLUSION

ZLN attenuates UC by curbing Th17 cell differentiation via STAT3 inhibition. These findings support ZLN's clinical application and provide new insights into TCM-based strategies targeting STAT3-Th17 axis for UC treatment.