Ameliorative Effect of N-Acetylcysteine Against 5-Fluorouracil-Induced Cardiotoxicity via Targeting TLR4/NF-κB and Nrf2/HO-1 Pathways

Figures

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  Figure 1. Effect of NAC on cardiac enzymes: (A) ALT, (B) AST, (C) LDH, and (D) CK. All data are presented as mean ± SD. *** p < 0.001 indicates significance compared to control and NAC rats, and ### p < 0.001 indicates significance compared to the 5-FU rats’ group, ns, non-significant.

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  Figure 2. Representative photomicrographs of heart sections: (A–D) control and NAC groups show a normal histological appearance of cardiac myocytes; (E,F) 5-FU group displays severe hemorrhage, with widely separated myocytes replaced by myocardial degeneration and necrosis; (G,H) 5-FU group demonstrates focal inflammation surrounding myocardial necrosis with shrunken, hypereosinophilic sarcoplasm; (I,J) 5-FU+NAC group shows focal areas of hemorrhage. (K) histopathological scoring. Thin arrow = myocardial necrosis, thick arrow = inflammation, star = hemorrhage. Image magnification = 100× and 400×. All data are presented as mean ± SD. *** p < 0.001 indicates significance compared to control and NAC rats, and ### p < 0.001 indicates significance compared to the 5-FU rats’ group.

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  Figure 3. Effect of NAC on (A) lipid peroxidation indicator MDA and antioxidant enzymes (B) SOD, (C) CAT, and (D) GPx in cardiac supernatant. All values are expressed as mean ± SD. *** p < 0.001 indicates significance compared to control and NAC rats, and ### p < 0.001 indicates significance compared to the 5-FU rats’ group. ns, non-significant.

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  Figure 4. Photomicrographs of Nrf2 immunoexpression in cardiac sections of all groups: (A,B) Normal group shows highly cytoplasmic-stained myocytes; (C,D) NAC group showing highly immunostained myocytes; (E,F) 5-FU group displays mild to moderate positive cytoplasmic staining in myocytes; (G,H) 5-FU group shows moderate cytoplasmic immunopositive staining in myocytes with mild staining of inflammatory cells; (I,J) 5-FU+NAC group demonstrates highly immunostained myocytes. Image magnification = 100× and 400×. The thin arrow indicates positive myocytes, while the thick arrow indicates positive inflammatory cells. (K) Image analysis of Nrf2 expression in different treatment groups. (L) Quantification of Nrf2 percentage expression in cardiac tissue was performed using ImageJ software (Version 1.52f). (M,N) Gene expression of Nrf2 and HO-1. All results are expressed as mean ± SD. *** p < 0.001 vs. control and NAC rats, and ### p < 0.001 vs. the 5-FU rats’ group. ns, non-significant.

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  Figure 5. Representative immunohistochemistry (IHC) of NF-κB expression in different cardiac sections. (A,B) The normal group shows negative nuclear staining in myocytes. (C,D) The NAC group demonstrates minimal nuclear immunostaining in myocytes. (E,F) 5-FU group exhibits strong nuclear staining in invading inflammatory cells. (G,H) 5-FU group demonstrates high nuclear positivity in both myocytes and inflammatory cells. (I,J) The 5-FU+NAC group displays mild faint nuclear staining in myocytes. Image magnification = 100× and 400×. The thin arrow indicates positive myocytes, while the thick arrow indicates positive inflammatory cells. (K) Image analysis of NF-κB expression in heart sections. (L) Histogram of NF-κB percentage immunolabeling. (M–O) Gene expression of NF-κB, TLR4, and MYD88. All values are presented as mean ± SD, *** p < 0.001 versus the control and NAC groups, ### p < 0.001 versus the 5-FU rats’ group. ns, non-significant.

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  Figure 6. Immunohistochemical staining of IL-1β in cardiac sections of all groups. (A,B) The normal group shows faint immunopositive staining in myocytes. (C,D) The NAC group demonstrates faint immunostaining in myocytes. (E,F) 5-FU group shows strong sarcoplasmic positivity with interstitially positive stained inflammatory cells. (G,H) 5-FU group exhibits extensive sarcoplasmic staining along with cytoplasmic and nuclear immunopositivity in inflammatory cells. (I,J) 5-FU+NAC group shows moderate cytoplasmic expression in myocytes with few positively stained inflammatory cells. Image magnification = 100× and 400×. The thin arrow indicates positively stained myocytes, while the thick arrow indicates positively stained inflammatory cells. (K) Images of IL-1β expression analysis in heart sections. (L) Histogram of IL-1β immunolabeling percentage. (M,N) Protein levels of TNF-α and IL-6 in different groups. All results are presented as mean ± SD, *** p < 0.001 indicates significance compared to control and NAC animals, ### p < 0.001 indicates significance compared to the 5-FU rats’ group. ns, non-significant.

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  Figure 7. Caspase-3 immunostaining in cardiac sections from different experimental groups. (A,B) Normal rat sections show negative to faint immunopositive staining in myocytes. (C,D) NAC group exhibits negative to faint immunostaining in myocytes. (E,F) 5-FU group demonstrates high positive staining in the sarcoplasm along with positively stained inflammatory cells. (G,H) 5-FU group shows significant immunopositive staining in perivascular inflammatory cells with sarcoplasmic positivity. (I,J) The 5-FU+NAC group illustrates weak cytoplasmic expression in myocytes and perivascular inflammatory cells. Image magnification = 100× and 400×. The thin arrow indicates positively stained myocytes, while the thick arrow indicates positively immunostained inflammatory cells. (K) Image analysis of caspase-3 expression in cardiac sections from different treatment groups. (L) Quantification of caspase-3 percentage expression in cardiac tissue. (M,N) Protein levels of TNF-α and IL-6 in different groups. All data are presented as mean ± SD, with *** p < 0.001 indicating significance compared to control and NAC rats and ### p < 0.001 indicating significance compared to the 5-FU rats’ group. ns,…