Multifaceted Cardioprotective Potential of Reduced Glutathione Against Doxorubicin-Induced Cardiotoxicity via Modulating Inflammation-Oxidative Stress Axis

Abstract

Doxorubicin (DOX) is a potent chemotherapeutic agent used to treat many types of cancer. Its use is limited because of the reported accompanied cardiotoxicity, which is driven by oxidative stress and inflammation. Herin, we explored the cardioprotective impact of reduced glutathione (GSH) against DOX-induced cardiac damage in a mice model and highlighted the dynamic interplay between pro-inflammatory and antioxidant mechanisms, with tissue damage markers and oxidative byproducts. Mice were divided into four groups and administered DOX, GSH, or a combination, and the outcomes were compared to untreated controls. DOX administration caused significant mortality, weight loss, elevated serum markers of cardiac injury (CK-MB and LDH), oxidative stress (MDA and iron), pro-inflammatory cytokines (IL-6, IL-17, and IL-23), and upregulated pro-inflammatory gene expression of STAT-3 and NFκB as well as downregulated gene expression of NRF-2 and HO-1. Histological analysis showed myocardial fibrosis, vacuolization, and apoptosis, as confirmed by a TUNEL assay. Meanwhile, treatment with GSH improved survival rate, attenuated weight loss, and restored cardiac function markers. Furthermore, GSH suppressed oxidative stress and inflammation, modulated gene expression, and declined histopathological damage. These findings demonstrated the multifaceted cardioprotection of GSH through the restoration of redox homeostasis and modulation of the pro- and anti-inflammatory responses. GSH supplementation emerges as a promising adjunct therapy to mitigate DOX-induced cardiotoxicity, offering a strategy to improve cardiac health in cancer patients undergoing doxorubicin chemotherapy.

Keywords: cardioprotective mechanisms; doxorubicin-induced cardiotoxicity; glutathione (GSH); inflammation; oxidative stress.

Figure 1

Effect of DOX, GSH, and DOX + GSH treatments on survival rate, body weight, and heart weight in experimental groups. Data represents means ± SEM in each group.** p < 0.05 represents significant difference vs. normal control group. ## p < 0.05 represents significant difference vs. DOX group. a represents non-significant difference vs. normal control group.

Figure 2

Analysis of cardiac biomarkers in response to different treatments. The upper left panel shows serum levels of CK-MB (U/L) for the control, DOX, GSH, and DOX + GSH groups. The upper right panel presents CK-MB levels (ng/mg tissue) in cardiac tissue for the same groups. The lower panel displays LDH levels (U/L) in serum across all treatment groups. Data are presented as the mean ± SEM. ** p < 0.05 represents a significant difference vs. the normal control group. ## p < 0.05 represents a significant difference vs. the normal control group. a represents a non-significant difference vs. the DOX group.

Figure 3

Assessment of oxidative stress markers and iron content in tissue. The left panel displays malondialdehyde (MDA) levels (μmol/g tissue) for the control, DOX, GSH, and DOX + GSH groups. The right panel shows iron content (μmol/g tissue) in the same treatment groups. Data are presented as the mean ± SEM. ** p < 0.05 represents a significant difference vs. the normal control group. ## p < 0.05 represents a significant difference vs. the normal control group. a represents a non-significant difference vs. the DOX group.

Figure 4

Cytokine levels in tissue following different treatments. The upper left panel shows interleukin-6 (IL-6) concentrations (ng/100 mg of tissue). The upper right panel presents interleukin-17 (IL-17) levels (ng/100 mg of tissue). The lower panel displays interleukin-23 (IL-23) concentrations (ng/100 mg of tissue). Data are presented as the mean ± SEM. ** p < 0.05 represents a significant difference vs. the normal control group. ## p < 0.05 represents a significant difference vs. the normal control group. a represents a non-significant difference vs. the DOX group.

Figure 5

Expression levels of key mRNA markers in tissue following various treatments. The upper left panel shows STAT3 mRNA expression. The upper right panel presents NRF2 mRNA expression. The lower left panel displays NF-κB mRNA expression. The lower right panel shows HO-1 mRNA expression in the DOX, GSH, and DOX + GSH groups, normalized to β-actin. Data are presented as the mean ± SEM. ** p < 0.05 represents a significant difference vs. the normal control group. ## p < 0.05 represents a significant difference vs. the normal control group. a represents a non-significant difference vs. the DOX group.

Figure 6

Representative H&E staining of mice heart. (a,b) refers to the control and reduced-glutathione-treated groups, respectively, that showed a standard architecture in the heart samples. (c) The Dox-treated group displayed a small cluster of myocardial fibers with small and large cytoplasmic vacuoles (black arrow), hemorrhage (white arrowheads), and inflammatory infiltration (black arrowheads). (d) Dox + reduced-glutathione-treated animals revealed recovery of the myocardial pattern with minor hemorrhage spots. (Magnification ×400).

Figure 7

Tunnel assay displayed apoptotic cell death (blue color) with a minor-to-moderate count in the case of both the control and reduced-glutathione-treated groups (a,b), while DOX-treated samples revealed an intense count of blue apoptotic cell deaths (c); in contrast, DOX + reduced-glutathione-treated animals displayed a reduced count of apoptotic cell deaths (d). The Tunnel assay analysis chart showed elevated apoptotic cell death within the DOX-treated group when compared with the control and/or GSH-treated group, while this count was decreased in the DOX + GSH-treated group (Magnification ×200). Representative chart for fluorescent staining quantitative analysis expression (n = 5 per group). All data are represented as the mean ± SEM. ** p < 0.05 represents a significant difference vs. the normal control group. ## p < 0.05 represents a significant difference vs. the normal control group. a represents a non-significant difference vs. the DOX group.