Methylene Blue Mitigates Doxorubicin-Induced Cardiotoxicity via KEAP1/NRF2/GPX-4/Caspase3 Modulation
Shaimaa G. Ibrahim, Ahmed M. Abu‐Dief, Amany M. Gad, Elham A. Gad, Abdullah Yahya Abdullah Alzahrani, Alhafez M. Alraih, Ibrahim Omar Barnawi, Mona Mansour, Mohamed H. A. Gadelmawla, Ali Khames
Abstract
Doxorubicin (Dox) is a potent anthracycline antitumor drug whose clinical utility is significantly restricted by its dose-dependent, cumulative cardiotoxicity, driven by increased oxidative stress, impaired antioxidant defenses, and apoptosis-mediated cardiomyocyte loss. Methylene blue (MB), a phenothiazine derivative with well-documented redox-modulating properties, is being explored as a viable cardioprotective agent due to its antioxidant and anti-apoptotic effects. This study evaluated the protective role of MB against Dox-induced cardiotoxicity in rats by examining its impact on oxidative stress markers (Kelch-like ECH-associated protein 1; KEAP1, nuclear factor erythroid 2-related factor 2; NRF2, Glutathione peroxidase 4; GPX-4, 8-hydroxy-2'-deoxyguanosine; 8-OHdG), neurohormonal indicators (noradrenaline), cardiac injury biomarkers (troponin I), and apoptotic mediators (p53, Caspase-3). Forty male albino rats were divided equally into four groups: control, Dox (15 mg/kg, i.p.), MB alone (4 mg/kg/day, p.o. for 7 days), and Dox plus MB. Dox administration significantly increased serum troponin I and noradrenaline levels, elevated cardiac KEAP1 and 8-OHdG, and reduced NFE2L2, NRF2, and GPX-4 expression. It also upregulated p53 and Caspase-3 and caused marked myocardial degeneration, necrosis, and inflammatory infiltration. MB co-treatment significantly reduced troponin I and noradrenaline levels, restored KEAP1/NFE2L2 (NRF2)/GPX-4 pathway balance, decreased oxidative DNA damage, and attenuated p53 and Caspase-3 activation, preserving myocardial architecture with minimal inflammatory changes. These findings demonstrate that MB confers potent cardioprotection against Dox-induced cardiac injury by enhancing antioxidant defenses, limiting oxidative DNA damage, suppressing apoptosis, and normalizing neurohormonal imbalance, suggesting its promise as an adjunctive strategy to mitigate anthracycline-associated cardiotoxicity.