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

Figures

• 

  Figure 1. MB effects on serum ALT (A), AST (B), and γ-GT (C) levels. Data are expressed as mean ± S.E.M (n = 10). a: significance towards control, b: significance towards DOX group at p < 0.05. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, gamma-glutamyl transferase; DOX, doxorubicin; S.E.M., standard error of the mean.

• 

  Figure 2. Effects of MB on total antioxidant capacity (TAC) (A) and HO-1 (B) contents. Data are expressed as mean ± S.E.M (n = 10). a: significance towards control, b: significance towards DOX group at p < 0.05. Abbreviations: TAC, total antioxidant capacity; HO-1, heme oxygenase-1; DOX, doxorubicin; S.E.M., standard error of the mean.

• 

  Figure 3. MB effects on PERK (A), GRP78 (B), and CHOP (C) contents. Data are expressed as mean ± S.E.M (n = 10). a: significance towards control, b: significance towards DOX group at p < 0.05. Abbreviations: DOX, doxorubicin; PERK, protein kinase RNA-like endoplasmic reticulum kinase; GRP78, glucose-regulated protein 78; CHOP, C/EBP homologous protein; S.E.M., standard error of the mean.

• 

  Figure 4. The photomicrographs show the liver tissue of all groups. The control group displayed normal hepatic architecture with well-arranged hepatocyte cords radiating from the central vein, intact cytoplasm, and clearly defined nuclei. The MB group revealed apparently normal hepatocytes in the centrilobular and periportal areas, indicating no morphological alterations. In contrast, the DOX group exhibited marked hepatic injury characterized by diffuse hepatocellular vacuolation, cytoplasmic degeneration, and focal necrosis accompanied by mononuclear inflammatory cell infiltration (arrows). The DOX + MB group demonstrated a remarkable improvement in hepatic architecture, showing nearly normal hepatocytes around the central and portal areas, with restoration of cellular integrity and minimal vacuolar degeneration (100 and 400×, H&E). Abbreviations: DOX, doxorubicin; H&E, hematoxylin and eosin.

• 

  Figure 5. Protective effect of MB on hepatic immunoreactivity of p53 and NRF-2 in DOX-evoked hepatic damage in rats. Photomicrographs of liver tissues from all groups. The immunoreactivity of P53 and NRF-2 were visible in the tissues as a brown color generated by DAB chromogen (DAB, ×400). The control group exhibited normal hepatic histoarchitecture with faint p53 and moderate NRF-2 staining. The MB-alone group showed mild NRF-2 nuclear localization and negligible p53 expression. The DOX-treated group revealed intense p53 immunostaining in hepatocyte nuclei and cytoplasm, accompanied by a marked reduction in NRF-2 expression. In contrast, the DOX + MB group displayed attenuated p53 staining and enhanced NRF-2 immunoreactivity. Quantitative analysis of immunostaining intensity for p53 and NRF-2 is expressed as mean ± S.E.M. (n = 10). a: significant difference compared with the control group; b: significant difference compared with the DOX group at p < 0.05. Abbreviations: NRF-2, nuclear factor erythroid 2-related factor 2; DAB, 3,3′-diaminobenzidine; S.E.M., standard error of the mean.

• 

  Figure 6. Protective effect of MB on hepatic immunoreactivity of Caspase-3 and NF-κB in DOX-treated rats evoked hepatic damage in rats. Photomicrographs of hepatic tissues of all groups. Caspase-3 and NF-κB reactivity were detected in tissues using DAB chromogen, resulting in a brown color (DAB, ×400). The control group exhibited weak cytoplasmic Caspase-3 and NF-κB staining. The MB-alone group displayed minimal immunoreactivity similar to the control. In contrast, the DOX group showed strong Caspase-3 and NF-κB staining. The DOX+MB group demonstrated markedly reduced immunostaining intensity for both markers. Quantitative analysis of Caspase-3 and NF-κB immunoreactivity is expressed as mean ± S.E.M. (n = 10). a: significant difference compared with the control group; b: significant difference compared with the DOX-treated group at p < 0.05. Abbreviations: NF-κB, nuclear factor kappa B; DAB, 3,3′-diaminobenzidine; S.E.M., standard error of the mean.

• 

  Figure 7. Proposed mechanism of DIHT.

• 

  Figure 8. The proposed mechanism of MB’s hepatoprotective effect.