SCD is a complicated pathophysiologic disorder caused in part by several pro-oxidant mechanisms, resulting in chronic and systemic oxidative stress. Erythrocytes are continuously exposed to a free radical environment in healthy biological systems [8]. However, potential indicators of the severity of SCD include increased ROS generation and the byproducts of their oxidative reactions [13]. In SCD patients, the main source of pro-oxidants is the sickle erythrocyte, where unstable autoxidative HbS and higher metabolic turnover due to recurrent HbS polymerization and depolymerization produce enhanced ROS formation. In addition, sickle RBCs are subjected to continuous exogenous oxidative onslaughts, contributing to the evolution of SCD vasculopathy. Notably, nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases), reported in sickle RBCs, may act as an incubator for oxidized Hb redox forms [14,15,16]. Further, ROS accumulation damages key sickle cell components. Increased ROS generation in sickle RBCs could contribute to cell membrane damage and premature hemolysis [17], and trigger sickle cell adhesion [15,18], consequently leading to severe anemia, vaso-occlusion, increased susceptibility to infections, chronic inflammatory diseases, and microvascular damage in organs [19]. These multifactorial events create a cyclic cascade, resulting in higher levels of ROS and oxidative damage, lower quality of life and life expectancy [20,21]. Many investigations have found that sickle erythrocytes produce twice as many superoxide (O₂^•−), hydrogen peroxide (H₂O₂), hydroxyl radical (HO^•), and lipid oxidation products as HbA-containing erythrocytes [22]. The two primary antioxidant enzymes, glutathione peroxidase (GPx) and catalase (CAT), remove H₂O₂ that is generated either by a two-electron transfer or because of sickling. CAT is usually more significant than GPx because it can convert H₂O₂ into H₂O, without burning cellular-reducing equivalents (glutathione (GSH) or nicotinamide adenine dinucleotide phosphate (NADPH)), which is a more energy-efficient way of eliminating H₂O₂. Interestingly, in transgenic sickle mouse models and SCD patients, certain investigations have found reduced CAT activity, but some contradicting reports have indicated instead increased CAT activity in SCD patients [21,23]. However, an increase in CAT activity may represent a defensive mechanism to scavenge H₂O₂ [21,23]. Yet, others have found that the activity of CAT in human sickle RBCs is not affected [24]. In disorders such as SCD where vascular oxidative stress is ensued by accumulations of ROS (superoxide anion, hydrogen peroxide, and the hydroxyl radical), vascular oxidative damage has long been linked to exposure to phosphatidylserine on RBCs [21]. Furthermore, in SCD patients, the normal antioxidant capacity of the RBC is impaired due to defects in the availability of antioxidants—lower levels of GSH, and enzymatic antioxidants such as peroxiredoxin 2 and non-enzymatic antioxidants such as vitamins C and E [24]. These findings, when considered collectively, point to oxidative stress playing a significant role in the pathophysiology of SCD [8,21,25].

Nrf2 is a cap’n’collar (CNC) transcription factor family member, including the founding member NF-E2p45. The transcription factor NF-E2p45 was initially discovered to bind to the erythroid gene regulatory element NF-E2 that is situated in the promoter region of the heme biosynthetic porphobilinogen deaminase gene (PBGD) [26]. Additionally, it was discovered that the transcription factor Nrf2 from humans binds to the gamma-globin gene regulatory region [27]. Along with NF-E2p45, the CNC family also consists of Nrf1, Nrf2, Nrf3, Bach1 (tBTB domain and CNC homolog 1), and Bach2. In contrast to the other proteins in the CNC family, Bach1 and Bach2 act as transcriptional repressors [26]. The basic leucine zipper (bZip) transcription factor is translocated to the nucleus and forms heterodimers with small musculoaponeurotic fibrosarcoma proteins (sMaf) K, G, and F [28]. This heterodimer then recognizes the antioxidant response element (an enhancer sequence) that is present in the regulatory regions of over 250 genes also known as AU-rich elements (AREs) [29]. There are 589 amino acids in Nrf2, containing seven Neh1-7 (N-terminal Nrf2-ECH homology) evolutionarily highly conserved domains. The Neh1 domain is a basic leucine zipper (bZip) motif. This motif recognizes DNA and facilitates its binding, and it is critical for Nrf2 dimerization with sMaf proteins [28]. The Neh2 domain has the ETGE motif located in the hydrophilic loop of the β-loop-β-structure at the C-terminal region and has a strong affinity for the β-propeller of Keap1-DC domain [30]. Similarly, the conserved DLG motif of Neh2 is located in a flexible region upstream of the core β-helix, and also binds Keap1-DC; however, with a noticeably reduced affinity [31]. Neh6, a serine-rich region containing the DSGIS and DSAPGS motifs, works as a degron to mediate Nrf2’s nuclear degradation [32]. The transactivation domains for Nrf2 include Neh4 and Neh5 and serve as the site for HRD1 binding [31,33]. These two domains regulate coordinately the transactivation of different cytoprotective genes [34]. Another domain, Neh7 (209-316 amino acid), was also found in Nrf2 which serves as a binding site for retinoid X receptor α (RXRα). The direct binding of RXRα to this domain inhibits the functions of Nrf2, and thereby inhibits expression of Nrf2 target genes [35,36].

Regulation of NRF2 expression is mediated via three signaling pathways involving Keap1 (Kelch-like ECH-associated protein 1), HRD1, an E3 ubiquitin ligase involved in protein degradation [37], and E3 ligase adapter β-TrCP (β-transducin repeat-containing protein). These three proteins facilitate NRF2 proteasomal degradation by different mechanisms. As discussed above, Nrf2 has seven highly conserved domains (Neh1–Neh7) that make interactions possible with various different proteins, especially Keap1 [35]. The hydrophilic area of lysine residues (7K) in Neh2 is essential for Keap1-dependent polyubiquitination and Nrf2 degradation, and it also contains the ETGE and DLG motifs that are necessary for the interaction with Keap1 [31,38], whereas, the Keap1 has five domains and 624 amino acid residues. The intervening region (IVR) lies between the BTB and the Kelch domain, two protein–protein interaction motifs. The BTB domain and IVR’s N-terminal region work together to homodimerize Keap1 and connect to Cullin3 (Cul3) [39]. Cul3 is the core protein of the E3 ubiquitin–protein ligase complex, which facilitates target protein ubiquitination and proteasomal breakdown [39]. The interaction with Neh2 is mediated by the C-terminal region and the Kelch domain. With 27 cysteines in human protein, Keap1 has a high concentration of cysteine residues [40,41,42,43]. According to the “hinge and latch” model, the dimer of two keap1 molecules interacts with Nrf2 [44]. Thus, under basal conditions, the Nrf2 protein is tightly controlled by the Keap1–Cul3–E3 ubiquitin ligase complex to keep Nrf2 at its low level. Moreover, another recent research has revealed that the deubiquitinating enzyme USP15 also plays a significant part in controlling the ubiquitination and degradation of Nrf2 [45]. Keap1 is deubiquitinated by USP15, which also stabilizes and improves the E3 ligase activity of the Keap1–Cul3–E3 complex [39,45]. As a result, Nrf2 is eventually degraded. However, under induced conditions or oxidative stress, the controlling activity of the Keap1–Cul3–E3 ubiquitin ligase complex is impaired, and Nrf2 level increased [45]. Keap1 has been hypothesized to release Nrf2 by covalent modifications of the crucial cysteine residues (Cys-151, Cys-273, and Cys-288), since it is a thiol-rich protein and is therefore sensitive to an electrophile [46]. In other words, post-translational modifications in cysteine residues lead to the dissociation of the Cul3-based E3 ligase complex from Keap1. This dissociation helps prevent the proteasomal degradation of Nrf2 and leads to Nrf2 stabilization [47]. After dissociation, Nrf2 accumulates in the cytosol, then translocate to the nucleus, although the de novo generated Nrf2 was also found to accumulate in the cytoplasm and migrate into the nucleus rather than detaching from Keap1 [48]. In the nucleus, sMaf binds to Nrf2 and forms a heterodimerized form that can recognize and bind to the ARE sequences of various antioxidant enzymes (such as NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferases (GST), superoxide dismutase (SOD), and heme oxygenase 1 (HO-1), etc.) and promote their expression to cope with oxidative stress [49].