Uncovering the Cardioprotective Potential of Diacerein in Doxorubicin Cardiotoxicity: Mitigating Ferritinophagy-Mediated Ferroptosis via Upregulating NRF2/SLC7A11/GPX4 Axis

Abstract

Doxorubicin (DOX)-induced cardiotoxicity (DIC) is a life-threatening clinical issue with limited preventive approaches, posing a substantial challenge to cancer survivors. The anthraquinone diacerein (DCN) exhibits significant anti-inflammatory, anti-proliferative, and antioxidant actions. Its beneficial effects on DIC have yet to be clarified. Therefore, this study investigated DCN's cardioprotective potency and its conceivable molecular targets against DIC. Twenty-eight Wister rats were assigned to CON, DOX, DCN-L/DOX, and DCN-H/DOX groups. Serum cardiac damage indices, iron assay, oxidative stress, inflammation, endoplasmic reticulum (ER) stress, apoptosis, ferritinophagy, and ferroptosis-related biomarkers were estimated. Nuclear factor E2-related factor 2 (NRF2) DNA-binding activity and phospho-p53 immunoreactivity were assessed. DCN administration effectively ameliorated DOX-induced cardiac cytomorphological abnormalities. Additionally, DCN profoundly combated the DOX-induced labile iron pool expansion alongside its consequent lethal lipid peroxide overproduction, whereas it counteracted ferritinophagy and enhanced iron storage. Indeed, DCN valuably reinforced the cardiomyocytes' resistance to ferroptosis, mainly by restoring the NRF2/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling axis. Furthermore, DCN abrogated the cardiac oxidative damage, inflammatory response, ER stress, and cardiomyocyte apoptosis elicited by DOX. In conclusion, for the first time, our findings validated DCN's cardioprotective potency against DIC based on its antioxidant, anti-inflammatory, anti-ferroptotic, and anti-apoptotic imprint, chiefly mediated by the NRF2/SLC7A11/GPX4 axis. Accordingly, DCN could represent a promising therapeutic avenue for patients under DOX-dependent chemotherapy.

Keywords: diacerein; doxorubicin cardiotoxicity; ferritin heavy chain polypeptide 1; ferroptosis; iron; nuclear factor E2-related factor 2/solute carrier family 7 member 11/ glutathione peroxidase 4 axis; nuclear receptor coactivator 4-dependent ferritinophagy; oxidative stress.

Figure 1

Schematic representation of the study protocol.

Figure 2

HW/BW ratio and cardiac toxicity indices among different studied groups. (A) HW/BW ratio (mg/g). (B) Serum cTn-I levels (pg/mL). (C) Serum CK-MB activity (U/L). (D) Serum LDH activity (U/L). CON: control; DOX: doxorubicin; DCN-L: diacerein low dose; DCN-H: diacerein high dose; HW: heart weight; BW: body weight; cTn-I: cardiac troponin-I; CK-MB: creatine kinase isoenzyme-MB; LDH: lactate dehydrogenase. p < 0.05, values expressed as mean ± SD (n = 7), * vs. CON group, # vs. DOX group, & vs. DCN-L+DOX group.

Figure 3

Modulatory effect of DCN on histopathological analysis of heart tissue. (A) In light microscopic images of all research groups, the CON group demonstrates normal cardiac muscle in the longitudinal section with a syncytium of branching and anastomosing myocardial fibers (black arrows). On higher magnification, the central nuclei (arrowhead), blood capillaries (V), and some lipofuscin granules are seen near the nuclei (curved arrow), with flat fibroblasts forming the interstitial space (green arrow). The (DOX) group demonstrates disorganized myocardial fibers with degenerative changes (blue arrow) in the form of focal disturbance of some myocardial fibers (dark blue arrow), interstitial edema in between the disrupted cardiomyocytes, and pyknotic and karyolitic nuclei (black and yellow arrowheads, respectively). Areas of sarcoplasmic vacuolation (star) are observed. The DCN-L+DOX group demonstrates some degree of enhancement as organized myocardial fibers (black arrow) with central vesicular nuclei (black arrowhead). However, the area of hemorrhage (hg), distribution of some fibers (bifid arrow), and karyolitic nuclei (yellow arrowhead) of some cardiomyocytes are still observed. The DCN-H+DOX group demonstrates apparent enhancement where they display normal acidophilic sarcoplasm (black arrow) and central vesicular nuclei (black arrowhead), but pyknotic nuclei in a few cells (yellow arrowhead) and congested blood capillaries (V) are still observed. (B) Histopathological score of heart injury, significant at p-value < 0.05. Values are expressed as mean ± SD, with 7 rats in each group. * Significant vs. CON group, # significant vs. DOX group, & significant vs. DCN-L+DOX group (H&E ×200, scale bar = 100 μm, H&E ×400, scale bar = 50 μm). CON: control; DOX: doxorubicin; DCN-L: diacerein low dose; DCN-H: diacerein high dose.

Figure 4

Modulatory effect of DCN on Masson’s trichrome staining heart tissue. (A) The CON group demonstrates sparse collagen fibers in between the cardiomyocytes. (B) The DOX group displays plentiful collagen fiber (arrows) deposition in between the cardiomyocytes. (C) The DCN-L+DOX group shows moderate collagen fibers (arrows) between cardiomyocytes and around the blood capillaries. (D) The DCN-H+DOX group demonstrates minimal collagen fibers (arrows) between the cardiomyocytes. (E) Area percentage of collagen fibers in all groups. (Masson’s trichrome stain ×400, scale bar = 50 μm). p < 0.05, values expressed as mean ± SD (n = 7), * significant vs. CON group, # significant vs. DOX group, & significant vs. DCN-L+DOX group. CON: control; DOX: doxorubicin; DCN-L: diacerein low dose; DCN-H: diacerein high dose.

Figure 5

Cardiac ATF3, NCOA4-mediated ferritinophagy, and ferrous iron content among different studied groups. (A) Cardiac ATF3 (ng/mg tissue protein). (B) NCOA4 relative gene expression. (C) Cardiac Fe²⁺ (mg/mg tissue protein). CON, control; DOX, doxorubicin; DCN-L, diacerein low dose; DCN-H, diacerein high dose; ATF3, activating transcription factor 3; NCOA4, nuclear receptor co-activator 4. p < 0.05, values expressed as mean ± SD (n = 7), * significant vs. CON group, # significant vs. DOX group, & significant vs. DCN-L+DOX group.

Figure 6

Modulatory effect of DCN on phospho-p53 immunohistochemical expression in heart tissue. (A) The CON group demonstrates a negative phospho-p53 immunoreaction. (B) The DOX group displays a strong positive phospho-p53 reaction in most cardiomyocytes’ nuclei (yellow arrows). (C) The DCN-L+DOX group demonstrates a moderate phospho-p53-positive reaction as a faint brown color in some cardiomyocytes. (D) The DCN-H+DOX group exhibits a weak phospho-p53-positive reaction in a few cardiomyocytes’ nuclei (arrows). (E) Area percentage of phosphor-p53 in all groups. (phospho-p53 immunostaining ×400, scale bar = 50 m). p < 0.05, values are expressed as mean ± SD (n = 7), * significant vs. CON group, # significant vs. DOX group, & significant vs. DCN-L+DOX group. CON, control; DOX, doxorubicin; DCN-L, diacerein low dose; DCN-H, diacerein high dose.

Figure 7

DCN enhanced NRF2 signaling in rats’ cardiac tissue. (A) Cardiac NRF2 DNA-binding activity (µg/mg tissue protein). (B) Relative SLC7A11 mRNA expression. (C) Cardiac GPX4 activity (U/mg tissue protein). (D) Relative FTH1 mRNA expression. CON, control; DOX, doxorubicin; DCN-L, diacerein low dose; DCN-H, diacerein high dose; NRF2, nuclear factor erythroid 2–related factor 2; SLC7A11, solute carrier family 7 member 11; GPX4, glutathione peroxidase 4; FTH1, ferritin heavy chain 1. p < 0.05, values are expressed as mean ± SD (n = 7), * significant vs. CON group, # significant vs. DOX group, & significant vs. DCN-L+DOX group.

Figure 8

Modulatory effect of DCN on cardiac ER stress and apoptotic markers. (A) Relative PERK mRNA expression. (B) Cardiac ATF4 (pg/mg tissue protein). (C) Cardiac GRP78 levels (μg/mg tissue protein). (D) Cardiac CHOP levels (ng/mg tissue protein). (E) Cardiac cleaved caspase-3 levels (ng/mg tissue protein). CON, control; DOX, doxorubicin; DCN-L, diacerein low dose; DCN-H, diacerein high dose; PERK, protein kinase R-like ER kinase; ATF4, activating transcription factor 4; GRP78, glucose-regulated protein 78; CHOP, C/EBP homologous protein. p < 0.05, values are expressed as mean ± SD (n = 7), * significant vs. CON group, # significant vs. DOX group, & significant vs. DCN-L+DOX group.

Figure 9

Correlation between cardiac NRF2 and other measured parameters in the DCN high-dose-treated group. (A) CK-MB activity (U/L). (B) cTn-I levels (pg/mL). (C) LDH activity (U/L). (D) ATF3 level (ng/mg protein). (E) NCOA4 mRNA level. (F) Fe²⁺ (mg/mg protein). (G) 4-HNE (ng/g tissue). (H) GSH content (mg/g tissue). (I) HMGB1 (ng/mL) in the high-dose DCN-treated group.

Figure 10

The proposed mechanisms implicated in DCN’s cardioprotective potency against DIC in rats.