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

BACKGROUND

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.

OBJECTIVE

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

METHODS

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.

RESULTS

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.

CONCLUSION

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.