Human antigen R -mediated autophagy-related gene 3 methylation enhances autophagy-driven ferroptosis in Crohn's disease colitis

Background and aims: Crohn's disease (CD) is a chronic inflammatory disorder that can affect any part of the gastrointestinal tract, with the exact etiology remaining unclear. Recent studies have implicated the role of human antigen R (HuR) in the pathogenesis of various inflammatory diseases, including CD. However, the role of HuR in the modulation of CD remains underexplored. Therefore, this study aimed to investigate the mechanistic involvement of HuR in CD.

Methods: We established colitis models using human intestinal epithelial cells and lipopolysaccharide and dextran sulfate sodium-induced mice. Additionally, by knocking out HuR in both cell and animal models, we validated the role of HuR in Autophagy and Ferroptosis. The role of HuR in regulating Ferroptosis accompanied by Autophagy activation in CD was detected using ELISA, flow cytometry, immunofluorescence, transmission electron microscopy, Western blot, and RT-qPCR. The demethylation level of ATG3 and the stability of ATG3 mRNA regulated by HuR were detected using immunofluorescence, RIP, and MeRIP-qPCR. The effect of HuR on DSS-induced colitis was evaluated using DAI score, H&E staining, TUNEL staining, and immunohistochemistry.

Results: The results show that HuR expression is significantly increased in CD colonic inflammation. Compared with the control group, the model group mice exhibited decreased levels of lipid peroxidation markers glutathione and superoxide dismutase, elevated malondialdehyde and Reactive Oxygen Species levels, and reduced expression of iron-related proteins Glutathione Peroxidase 4, ferritin heavy chain protein 1, and solute carrier family 7 member 11. Additionally, the expression of autophagy-related proteins microtubule-associated protein 1 A/1B-light chain 3, beclin-1, and Autophagy related 3 (ATG3) was upregulated, while p62 expression was downregulated. In both in vitro and in vivo models, HuR knockout reversed these changes induced by lipopolysaccharide and dextran sulfate sodium, concomitant with improved tissue pathology. Mechanistically, HuR enhances autophagy-mediated Ferroptosis in CD colonic inflammation by regulating ATG3 methylation and mRNA stability.

Conclusion: HuR accelerates colonic inflammation in CD by regulating ATG3 methylation, which enhances autophagy-mediated Ferroptosis. Knockout of HuR alleviates Crohn's colitis. This finding provides a potential therapeutic target for the treatment of CD.

ATG3; Autophagy; Crohn's disease; Ferroptosis; HuR.