Thymol ameliorates ethanol-induced ferroptosis and calcium ion overload

The therapeutic effect of thymol against ethanol-induced injury has been reported. However, the mechanism by which thymol ameliorates ethanol-induced damage remains unclear. In this study, we found that thymol mitigated the fluctuations in GSH/GSSG ratio and Gpx4 mRNA abundance, and total cholesterol levels mediated by ethanol. Subsequently, differential expression analysis using RNA-sequencing identified 1748 and 2802 differentially expressed genes related to ethanol treatment and ethanol plus thymol treatment, respectively. Enrichment analysis revealed that peroxisome, glutathione metabolism, ferroptosis, lysosome, mTOR signaling pathway, and protein processing in endoplasmic reticulum participated in the ameliorating effect of thymol on ethanol-induced damage. Higher mRNA abundance of Aifm2 and lower abundances of malondialdehyde, Acsl4 mRNA and ferrous iron were involved in the suppression of thymol on ethanol-induced ferroptosis. Meanwhile, the impact of thymol on ferroptosis in ethanol-treated cells was associated with the elevation of lysosome number, down-regulation of Ncoa4 gene expression levels and reduced mTOR activity. These data indicate that lysosomes link iron metabolism with mTOR signaling pathway in cells treated with ethanol plus thymol. In addition, reduction of calcium ion content accompanied by down-regulation of Mcoln1 mRNA levels and up-regulation of Hspa8 mRNA levels were observed in cells stimulated with ethanol plus thymol. Finally, HSPA8 inhibitor VER-155008 significantly amplified ethanol-induced increases in both malondialdehyde content and calcium ion concentration, while intensified ethanol-induced reduction in lysosome abundance. These findings underscore the therapeutic mechanisms by which thymol ameliorates ethanol-induced ferroptosis and calcium ion overload.