Ferritinophagy‐mediated ferroptosis facilitates methotrexate‐induced hepatotoxicity by high‐mobility group box 1 (HMGB1).

Background and Aim: Hepatotoxicity is a well‐defined reaction to methotrexate (MTX), a drug commonly used for the treatment of rheumatoid arthritis and various tumours. We sought to elucidate the mechanism underlying MTX‐induced hepatotoxicity and establish a potentially effective intervention strategy. Methods: We administered MTX to liver cells and mice and assessed hepatotoxicity by cell viability assay and hepatic pathological changes. We determined ferroptosis and ferritinophagy by detecting ferroptosis‐related markers and autophagic degradation of ferritin heavy chain 1 (FTH1). Results: We have shown that hepatocytes treated with MTX undergo ferroptosis, and this process can be attenuated by ferroptosis inhibitors. Interestingly, NCOA4‐mediated ferritinophagy was found to be involved in MTX‐induced ferroptosis, which was demonstrated by the relief of ferroptosis through the inhibition of autophagy or knockdown of Ncoa4. Furthermore, MTX treatment resulted in the elevation of high‐mobility group box 1 (HMGB1) expression. The depletion of Hmgb1 in hepatocytes considerably alleviated MTX‐induced hepatotoxicity by limiting autophagy and the subsequent autophagy‐dependent ferroptosis. It is noteworthy that glycyrrhizic acid (GA), a precise inhibitor of HMGB1, effectively suppressed autophagy, ferroptosis and hepatotoxicity caused by MTX. Conclusion: Our study shows the significant roles of autophagy‐dependent ferroptosis and HMGB1 in MTX‐induced hepatotoxicity. It emphasizes that the inhibition of ferritinophagy and HMGB1 may have potential as a therapeutic approach for preventing and treating MTX‐induced liver injury. [ABSTRACT FROM AUTHOR]