Citrus rhoifolin alleviated DSS-induced acute colitis by activating CEMIP/SLC7A11-mediated cystine uptake and inhibiting epithelial ferroptosis.

Abstract

Pharmacological inhibition of ferroptosis, a specific form of regulated cell death, has emerged as a promising therapeutic strategy for alleviating symptoms and enhancing endoscopic outcomes in patients suffering from ulcerative colitis. Rhoifolin, a prominent bioactive constituent abundant in the widely consumed fruit Citrus grandis (grapefruit), has garnered attention for its ability to diminish the levels of reactive oxygen species (ROS), which are key inducers of ferroptosis across diverse cellular contexts. In this study, we aimed to investigate whether rhoifolin exerts its beneficial effects on colitis by modulating the process of epithelial ferroptosis. Colitis model was successfully established in C57BL/6 mice through the administration of 2.5% dextran sulfate sodium (DSS) solution for a duration of 9days, which was freely accessible for drinking. RNA sequencing was conducted to delve into the mechanisms underlying the rhoifolin-mediated effects on colitis. To evaluate the impact of rhoifolin on ferroptosis in epithelial cells, several key indicators were measured, including mitochondrial morphology, colonic glutathione (GSH) levels, lipid peroxidation product contents, and ROS levels. The results indicated that rhoifolin exhibited profound anti-colitis properties and effectively curbs ferroptosis in epithelial cells of mice subjected to DSS treatment. The RNA sequencing analysis further revealed that rhoifolin stimulated a remarkable upregulation of colonic cell migration-inducing protein (CEMIP) expression by approximately 2.4-fold in colitis-affected mice. Notably, depletion of CEMIP significantly blocked the rhoifolin-induced increase in the cystine transporter solute carrier family 7 member 11 (SLC7A11, from 1.9-fold to approximately 1.1-fold), as well as the elevation of cystine uptake (from 1.72-fold to 1.2-fold) and glutathione (GSH) biosynthesis (from 2.1-fold to 1.2-fold), and the suppression of epithelial ferroptosis (from 0.51-fold to 0.94-fold) in mice with colitis. Molecular docking investigations have pinpointed crucial amino acid residues within CEMIP, specifically His267, His289, and Phe265, as the primary interaction sites (docking score: -7.8kcal/mol), facilitating the engagement of rhoifolin via hydrogen bonding interactions. Rhoifolin significantly mitigated DSS-induced colitis primarily through inhibiting epithelial ferroptosis. The activation of CEMIP by citrus-derived rhoifolin led to a notable upregulation of SLC7A11 expression, thereby enhanced cystine uptake and facilitated GSH biosynthesis, ultimately suppressed the occurrence of ferroptosis in epithelial cells.