Background: Ferroptosis in colonic epithelial cells has been implicated in the development of ulcerative colitis (UC) and the accompanying gut leakage. Scutellaria baicalensis Georgi (Scu) is widely used herb medicine for alleviating UC.

Objective: We aimed to clarify the therapeutic effect of Scu on UC by inhibiting intestinal epithelial cell ferroptosis and explore its regulatory mechanisms on lipid peroxidation and the GPX4/ACSL4 pathways.

Methods: UPLC-Q-TOF/MS was employed to analyze chemicals in the herbal extract and the colonic exposure of prototypes in Scu-treated mice. Additionally, the main compounds were quantified using HPLC-UV. The ameliorative effects of Scu were comprehensively explored in a UC mouse model established by feeding with dextran sulfate sodium (DSS). HPLC-MS based metabolomic studies were conducted to identify the differential metabolites in colon tissues from Scu or vehicle treated UC mice. Network pharmacology was conducted for target prediction and potential pathway analysis. In conjunction with these bioinformatic analyses, we performed RT-qPCR, immunofluorescence, immunohistochemistry and immunoblotting to elucidate the regulatory mechanisms of Scu on ferroptosis-related pathways in both in vivo and in vitro models.

Results: 78 chemical constituents in Scu were characterized, with 42 detected in the colonic tissues of Scu-treated mice. Scu could alleviate UC related symptoms in mice, including increased colon length and decreased pathological score. Furthermore, Scu inhibited pro-inflammatory cytokines and mediators, while improving gut barrier function by increasing the expression of ZO-1 and Occludin at both mRNA and protein levels. Based on metabolomic studies, a total of 71 differential metabolites exhibited a reversal trend following Scu administration. These findings, combined with results from network pharmacology, suggest that arachidonic acid (AA) metabolism and ferroptosis may serve as potential pathways for Scu intervention in UC. Further experiments indicated that the amelioratory actions of Scu on ferroptosis partially contributed to its modulation on lipid peroxidation and its regulatory influence on the GPX4/ASCL4 axis to ameliorate UC. When AA was administered at the same time as concurrently with Scu, the regulatory effects of Scu on ferroptosis, GPX4/ASCL4 axis, and its protective effects against UC were significantly reduced. Moreover, the inhibitory effect of Scu on ferroptosis was weakened when we knocked down GPX4 or overexpressed ACSL4 in vitro.

Conclusions: The ameliorative effect of Scu in UC is closely related to the regulation of lipid peroxidation and GPX4/ASCL4 mediated intestinal epithelial ferroptosis.