Targeting carbonic anhydrase IX-mediated ferroptosis by atractylenolide I alleviates DSS-induced colitis in mice

While the involvement of ferroptosis in the pathological progression of chronic inflammatory bowel disease (IBD) is recognized, the specific regulatory capacity of the natural derivative Atractylenolide I (ATT-I) within this metabolic framework is not yet fully elucidated.

To investigate how ATT-I alleviates IBD by modulating ferroptosis via targeting carbonic anhydrase IX (CA9).

To determine the therapeutic potential of ATT-I, a murine model of colitis was established via dextran sulfate sodium (DSS) administration. Mice were treated with different doses of ATT-I. At the end of the experiment, colon tissues and serum were collected for histological analysis, inflammatory cytokine measurement, and ferroptosis marker evaluation. Erastin (ferroptosis inducer) and adenovirus associated virus 9 (AAV9)-mediated CA9 silencing was applied to assess its role in ATT-I efficacy.

Medium and high doses of ATT-I significantly alleviated DSS-induced colitis symptoms by reducing histological damage, preventing colon shortening, decreasing spleen index and cytokine levels, and improving epithelial integrity. ATT-I inhibited ferroptosis by upregulating GPX4 and SLC7A11 and downregulating COX-2 and ACSL4. Notably, co-administration of Erastin reversed the protective effects of ATT-I. Network pharmacology and molecular docking suggested CA9 as a putative binding target of ATT-I which was confirmed by CETSA demonstrating increased thermal stability of CA9 upon ATT-I treatment. To investigate the functional necessity of CA9, AAV9 was employed to silence its expression. In a murine model of DSS-induced colitis, the therapeutic benefits of ATT-I, specifically its capacity to prevent splenic hypertrophy, alleviate leukocyte infiltration, and preserve colonic morphology, were entirely abolished following the genetic knockdown of CA9. Furthermore, the capacity of ATT-I to inhibit ferroptosis in the colon was obviated when CA9 was silenced. For the in vitro experiments, silencing CA9 abrogated ATT-I-mediated Claudin-1, Occludin, GPX4 and SLC7A11 upregulation in Erastin-treated Caco-2 cells, indicating that CA9 is an indispensable mediator for ATT-I to restrain Erastin-induced ferroptosis in Caco-2 cells.

ATT-I alleviates IBD by modulating ferroptosis mechanisms through targeting CA9. This indicates that ATT-I, as a natural compound, holds potential for regulating ferroptosis, providing a novel therapeutic strategy and approach for IBD treatment.