Hepatoprotection by Methylene Blue Against Doxorubicin Toxicity Through Coordinated Modulation of Oxidative Stress, ER Stress, and Apoptotic Pathways

<b>Background and Objectives:</b> Doxorubicin (DOX) is a potential chemotherapeutic whose clinical application is limited by hepatotoxicity mediated through apoptosis, endoplasmic reticulum (ER) stress, and oxidative stress (OS). This study aimed to assess the hepatoprotective impact of methylene blue (MB) against DOX-induced liver injury. <b>Methods:</b> Forty rats were arbitrarily divided equally into four groups: control, DOX (15 mg/kg, i.p., single dose), MB (4 mg/kg, i.p., daily for 7 days), and DOX + MB (same regimen, MB initiated 1 h post DOX). Serum ALT, AST, and γ-GT were measured, along with hepatic TAC and HO-1. ELISA quantified PERK, GRP78, and CHOP. Immunohistochemistry assessed Caspase-3, p53, NF-κB, and Nrf2. Histopathological evaluation was performed using H&E staining. <b>Results:</b> DOX administration significantly elevated ALT, AST, γ-GT, HO-1, PERK, GRP78, and CHOP while reducing TAC and Nrf2 expression. Strong Caspase-3, p53, and NF-κB immunoreactivity and severe histopathological damage were observed. MB treatment markedly reversed these changes, restoring antioxidant status, downregulating ER stress markers, preserving Nrf2 expression, and improving hepatic architecture. <b>Conclusions:</b> MB exerts significant hepatoprotection against DOX-induced injury, likely via attenuation of OS, ER stress, apoptosis, and inflammation.