Carvacrol ameliorates doxorubicin-induced human cardiomyocyte apoptosis, inflammation and ferroptosis by inhibiting histone deacetylase 6

Background Emerging evidence indicates that ferroptosis acts as a primary driver in doxorubicin (DOX)-triggered cardiomyocyte damage. Carvacrol, a natural monoterpenoid phenol, is recognized for its potent antioxidant and anti-inflammatory properties; however, its specific role and underlying mechanisms in modulating ferroptosis within DOX-injured cardiomyocytes remain unclear. This study aimed to elucidate the mechanism by which carvacrol attenuated DOX-induced injury in human cardiomyocytes, with a specific focus on the ferroptosis pathway. Methods An in vitro cardiomyocyte injury model was established by treating human cardiomyocytes (AC16) with DOX. Appropriate treatment conditions for carvacrol and DOX were initially established in AC16 cells using cell counting kit-8 (CCK-8) assays. Network pharmacology integrating predicted carvacrol targets with ferroptosis-related genes, alongside Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, was employed to identify potential molecular targets. Molecular docking and cellular thermal shift assays (CETSA) were employed to verify direct target binding. Functional rescue experiments were then performed in AC16 cells via target gene overexpression. Subsequently, cell apoptosis, inflammatory cytokine secretion, and ferroptosis-related biochemical indicators including lipid reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and Fe 2 + were systematically assessed. Finally, the expression of key proteins in the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) signaling pathway was evaluated by Western blotting. Results Carvacrol (1 μM, 24 h) effectively mitigated the viability reduction caused by DOX (1 μM, 24 h) in AC16 cells ( P < 0.05). Bioinformatic analysis identified 30 overlapping targets, with histone deacetylase 6 (HDAC6) identified as the core candidate due to its prominent enrichment in epigenetic acetylation processes. Molecular docking and CETSA confirmed the direct binding and enhanced thermal stability between carvacrol and HDAC6. Functionally, DOX exposure significantly induced apoptosis, elevated levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), and induced typical ferroptotic alterations characterized by the accumulation of lipid ROS, MDA, and Fe 2+ , as well as the depletion of GSH, SOD, glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) ( P < 0.05). Moreover, carvacrol administration robustly reversed these detrimental effects ( P < 0.05). Mechanistically, DOX upregulated HDAC6 protein expression and activated the MAPK/NF-κB pathway ( P < 0.05). Carvacrol suppressed HDAC6 expression and inactivated this pathway ( P < 0.05); however, these protective effects, including the suppression of apoptosis, inflammation, and ferroptosis, were partially abolished when HDAC6 was exogenously overexpressed ( P < 0.05). Conclusion Carvacrol exerted protective effects against DOX-induced cardiomyocyte injury by directly targeting HDAC6. Clinically, these findings provide experimental evidence for the future development of cardiac protectants.