Cancer is a prevalent systemic disease characterized by uncontrolled cell proliferation and distant invasion, resulting in multiple organ damage, and ultimately, leading to a high rate of morbidity and mortality worldwide. ^{1, 2}

One of the most important anticancer drugs in clinical practice is doxorubicin (DOX), also known as adriamycin, which belongs to the family of anthracycline antibiotics. ³ Since its approval, DOX has been utilized in treating different types of cancer, including solid tumors, like breast, ovarian, testicular, thyroid, lung, liver, stomach and bladder cancers, as well as soft tissue sarcomas. Moreover, the drug has been employed in the treatment of hematologic malignancies, such as multiple myeloma, acute lymphoblastic leukemia, Hodgkin’s and non-Hodgkin’s lymphomas, in addition to certain childhood cancers, including neuroblastoma, osteosarcoma, Ewing’s sarcoma and rhabdomyosarcoma. ^{1, 4}

Unfortunately, despite its broad antitumor spectrum, the clinical application of DOX is often limited by its irreversible and dose-dependent cardiotoxicity, which can result in left ventricular dysfunction (LVD) and heart failure (HF). ^{5, 6} On top of that, it has been estimated that the risk of LVD is approximately 7–26% when the cumulative dose of DOX reaches 550 mg/m ², and about 18–48% at a cumulative dose of 700 mg/m ². ⁷

Cardiotoxicity that is induced by DOX seems to be a multifactorial and complex process, and there has been a long list of proposed mechanisms in this regard, ⁸ one of which is the ability of DOX to generate excessive reactive oxygen species (ROS) and reactive nitrogen species (RNS) to the point that they would overpower the antioxidant defense system, causing disruption of cellular hemostasis and activation of the inflammatory response pathways, eventually leading to cell death, ^{9, 10} and from this point of view, developing a strategy that can hinder the development of the aforementioned events would be a possible approach to alleviate DOX-induced cardiotoxicity (DIC).

Ezetimibe (EZE) is a cholesterol-lowering drug that inhibits the absorption of dietary and biliary cholesterol from the small intestine by blocking the Niemann-Pick C1-Like protein (NPC1L1), which is a sterol transporter found on the brush border membrane of the intestinal epithelium. ¹¹

Apart from its beneficial impact on lipid profile, EZE has been shown to have pleiotropic properties, including antioxidant and anti-inflammatory effects that are independent of its ability to inhibit NPC1L1. ^{11, 12} Therefore, the current study was conducted to assess the hypothesis that EZE can provide protection against DIC in rats, which to our knowledge, has not been studied yet, and to elucidate the mechanisms underlying EZE’s possible cardioprotective effect.

One of the highly efficient antineoplastic agents with broad-spectrum application is the anthracycline antibiotic DOX. Nevertheless, the cardiotoxicity it causes represents a feared adverse effect and a potentially life-threatening response that can limit the clinical use of DOX and affect the quality of life of cancer patients, apart from their oncological prognosis. ^{2, 15}

Results of the current study revealed that treating rats with a single IP injection of 15 mg/kg DOX hydrochloride caused a significant elevation in the means of serum levels of cardiac injury biomarkers, including cTnI and NT-proBNP, which is consistent with the results obtained from previous studies. ^{16– 18}

Considering their high sensitivity, cardiac injury biomarkers are utilized for the early detection of cardiotoxicity, especially cardiac troponins, which are regulatory proteins that control calcium-mediated contractile function of the heart, and it has been demonstrated that newly elevated serum cTnI levels in patients receiving anthracyclines anticipate subsequent development of LVD. ^{7, 19, 20} Additionally, NT-proBNP, which is secreted by cardiomyocytes in increased amounts during conditions of volume or pressure overload of the heart, is widely utilized in the detection of HF, and in terms of chemotherapy, its use can be helpful, ^{7, 21– 23} based on studies that validated its role as a marker for the detection of DIC. ^{24, 25}

The present study also showed that DIC was associated with notable changes in oxidative stress biomarkers in cardiac tissue, where the means of the antioxidant enzymes SOD and GPx were significantly depleted, while there was a significant increment in the mean of MDA contents, provided that MDA is a highly reactive dialdehyde derived from peroxidation of polyunsaturated fatty acids by means of reactive species. ^{26, 27}

The abovementioned outcomes are compatible with those acquired from prior studies, ^{17, 28, 29} which reinforces the theory that one of the major mechanisms underlying the development of DIC is oxidative stress, and the latter can be defined as a state of imbalance in which the production of ROS, such as superoxide anion (O ₂ ^{• –}), hydrogen peroxide (H ₂O ₂) and hydroxyl radical (HO ^•), as well as RNS, such as peroxynitrite anion (ONOO ⁻), exceeds the capacity of the endogenous antioxidant system, resulting in cellular damage. ^{10, 30, 31}

One of the reasons that makes the heart particularly susceptible to injury by DOX-induced oxidative stress is that antioxidant enzymes, including SOD, catalase (CAT) and GPx are less abundantly expressed in cardiomyocytes as compared with other cell types. Another reason is that DOX has a tendency to accumulate in the mitochondria, and since the heart is the body’s most metabolically active organ, it tends to have the highest mitochondrial content, rendering the cardiac cell the most vulnerable to oxidative damage. ^{5, 32}

Studies elucidated that DOX induces oxidative stress as the quinone moiety in its structure undergoes one-electron reduction through the action of NADH- or NADPH-dependent enzymes to form a semiquinone radical, which tends to react rapidly with an oxygen molecule (O ₂), where it donates an electron to O ₂ to form O ₂ ^{• –}, while recycling itself back to the quinone form, and this cycle is referred to as the redox cycling of DOX. Subsequently, the generated O ₂ ^{• –} can be transformed by SOD into H ₂O ₂ that can be converted either into O ₂ and water (H ₂O) by means of CAT or GPx, or into a highly reactive and toxic HO ^• in the presence of ferrous ion (Fe ²⁺). One more point to consider is that O ₂ ^{• –} can also react with nitric oxide (NO) to form the RNS ONOO ⁻. Eventually, overproduced HO ^• and ONOO ⁻ interact with the biomolecules nearby, causing lipid peroxidation, protein modification, DNA damage as well as disruption of mitochondrial function, ^{33– 35} as shown in Figure 4.

Our results also manifested that DOX administration significantly increased the means of cardiac NF-κB levels and its downstream proinflammatory cytokines, including TNF-α and IL-1β relative mRNA expression levels, and these observations are in agreement with those reported in other studies, ^{36– 38} thereby supporting another theory through which DOX induces cardiotoxicity, and that is inflammation.

Researchers pointed out that inflammation in DIC occurs as a result of NF-κB activation in response to oxidative stress, ^{10, 39} provided that NF-κB is a family of DNA binding proteins that function as homo or heterodimers to mediate the expression of numerous genes involved in cell proliferation, inflammation, immunity and apoptosis. ^{40, 41} In its resting state, NF-κB presents in the cytoplasm as a complex with a member of inhibitory proteins known as inhibitors of NF-κB (IκΒs) that mask nuclear localization signals to prevent NF-κB from being imported into the nucleus. However, in response to stimulation, an IκB kinase (IKK) phosphorylates IκB to promote its ubiquitination and proteasomal degradation, after which NF-κB becomes freed from IκB and translocates into the nucleus, where it upregulates the expression of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6 and others, with consequent development of cardiac inflammation that can lead to cardiac remodeling and dysfunction. ^{39, 42– 44}

In our research, we aimed to investigate whether EZE can safeguard against DIC in rats. Interestingly, treating rats with either 10 or 20 mg/kg EZE before DOX administration significantly mitigated the cardiotoxic potential of the anticancer drug, and that was evidenced by a significant decline in the means of serum cTnI and NT-proBNP levels compared with those of DOX group, and to our knowledge, the current research is the first to estimate serum levels of the two earlier mentioned biomarkers in rats after receiving EZE for the prevention of a cardiac pathological condition.

Moreover, results of the present study demonstrated that pretreatment with EZE at a dose of 10 or 20 mg/kg caused a significant increment in the means of cardiac SOD and GPx levels, along with a significant reduction in the mean of cardiac MDA levels compared with those of DOX group, and these outcomes emphasize that the drug possesses antioxidant effect, as it was reported in an in vitro study, where EZE protected endothelial cells against oxidative stress induced by oxidized low-density lipoproteins, in which a reduction in ROS formation, as well as an enhancement in SOD and CAT activities were observed. ⁴⁵

Our study also showed that in comparison with DOX-challenged rats, pretreatment with either 10 or 20 mg/kg EZE significantly decreased the means of cardiac NF-κB levels and TNF-α relative mRNA expression levels, while the 20 mg/kg dose was also capable of significantly reducing the mean of cardiac IL-1β relative mRNA expression levels, and these findings can be attributed to the anti-inflammatory effect of the drug that was mentioned in previous studies, including one that utilized THP-1 human monocytic cell line treated with water-soluble cholesterol to induce inflammation, where the results manifested that EZE treatment dramatically decreased the proinflammatory cytokine TNF-α production by silencing NF-κB signaling. ⁴⁶

The antioxidant and anti-inflammatory actions of EZE were also evidenced in other studies, and in this regard, a recent one pointed out that EZE alleviated acetic acid-induced ulcerative colitis in rats by hampering oxidative stress and inflammation. ¹¹ Also, in a transient middle cerebral artery occlusion model in rats, EZE attenuated oxidative stress and prevented neuroinflammation, resulting in an improvement in neurological outcome. ¹²

The rationale behind EZE attenuation of oxidative stress and inflammation can be related to its ability to activate nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that modulates the expression of genes involved in cellular defense against oxidative stress insults, including the ones encoding SOD, CAT, GPx and other antioxidants. ^{47, 48} Under basal conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2 that mediates its ubiquitination and proteasomal degradation to prevent unwarranted expression of Nrf2-target genes. However, under stressed conditions, Nrf2 dissociates from Keap1 and translocates into the nucleus, where it upregulates the expression of antioxidant genes through binding to their antioxidant response element (ARE), which is an enhancer sequence placed at the promoter region of several cytoprotective genes. Consequently, the activity of antioxidants would be boosted, leading to suppression of oxidative stress-mediated activation of the NF-κB signaling pathway, ⁴⁹ as shown in Figure 5.

The possible mechanism through which EZE induces Nrf2 activation has been elucidated in one study, where the drug was found to disrupt the interaction between Nrf2 and Keap1 through adenosine monophosphate-activated protein kinase (AMPK)-mediated phosphorylation of p62, an adaptor protein that functions as a central scaffold in various cellular processes, including autophagy. Accordingly, upon its phosphorylation, p62 binds to Keap1, resulting in autophagic degradation of the latter, after which the liberated Nrf2 translocates into the nucleus to upregulate the expression of antioxidant genes, ^{48, 50} and this can explain, at least in part, why EZE exerted a protective effect against DIC in rats ( Figure 5).

The current research demonstrates, for the first time, that pretreatment with the cholesterol-lowering drug EZE can attenuate DIC in rats, as evidenced by a notable reduction in the serum levels of cardiac injury biomarkers. Also, we believe that the cardioprotective effect of EZE was mediated by suppression of DOX-induced oxidative stress and NF-κB-mediated inflammation, where EZE raised the levels of antioxidant enzymes, diminished lipid peroxidation and downregulated the gene expression of proinflammatory cytokines in cardiac tissues, thereby, it has the potential to serve as a promising candidate for alleviating DOX-induced cardiac injury.

The current study followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, ¹³ and was commenced after receiving approval from the Research Ethics Committee of the College of Medicine, University of Baghdad (approval date: December 31, 2023; approval number: 03-31).