Preventive and protective roles of dietary Nrf2 activators against central nervous system diseases

Abstract

Central nervous system diseases are major health issues and are often associated with disability or death. Most central nervous system disorders are characterized by high levels of oxidative stress. Nuclear factor erythroid 2 related factor (Nrf2) is known for its ability to regulate the expression of a series of enzymes with antioxidative, prosurvival, and detoxification effects. Under basal conditions, Nrf2 forms a complex with Kelch-like ECH associated protein 1, leading to Nrf2 inactivation via ubiquitination and degradation. However, following exposure of Keap1 to oxidative stress, Nrf2 is released from Keap1, activated, and translocated into the nucleus. Upon nuclear entry, Nrf2 binds to antioxidant response elements (ARE), thereby inducing the expression of genes such as glutathione s-transferase, heme oxygenase 1, and NADPH quinine oxidoreductase 1. Many dietary phytochemicals have been reported to activate the protective Nrf2/ARE pathway. Here, we review the preventive and protective effects of dietary Nrf2 activators against CNS diseases, including stroke, traumatic brain injury, Alzheimer’s disease, and Parkinson’s disease.

1. Introduction to the Nrf2 pathway

Nuclear factor erythroid 2 related factor (Nrf2) is a basic leucine zipper (bZIP) transcription factor with a characteristic Cap ‘n’ collar structure (1). It is one of the key regulator of phase II drug metabolizing enzymes, including glutathione S-transferase (GST), heme oxygenase 1 (HO-1) and NADPH quinine oxidoreductase 1 (NQO-1), all of which play important roles in antioxidant and pro-survival effects and the detoxification of xenobiotics (2–4). As with other nuclear transcription factors, Nrf2 translocates into the nuclear compartment upon activation and binds antioxidant response elements (ARE) that initiate expression of downstream genes. It is generally accepted that Kelch-like ECH associated protein 1 (Keap1) plays an essential role in the inactivation of Nrf2 (5), but there are also additional proteins that regulate the important Nrf2/ARE pathway (6).

1.1 Keap1-dependent pathway

1.1.1 Nrf2 under basal conditions

Nrf2 is composed of six functional domains known as Nrf2-ECH homologies (Neh) and designated as Neh1-6. Each Neh domain serves a unique function (7). The Neh1 domain is the CNC-bZIP domain, enabling Nrf2 to form a heterodimer with the ZIP domain of small musculoaponeurotic fibrosarcoma (Maf) proteins (8). The Neh2 domain mediates the binding of Nrf2 to Keap1, which acts as a cytosolic repressor of Nrf2 activation. Keap1 is composed of three functional domains: a bric-a-brac (BTB) domain, an intervening region (IVR), and a Kelch domain (also named DGR domain). Two Kelch domains can bind with high affinity to two motifs located in the Neh2 domain of Nrf2 (9). Under basal conditions, Nrf2 forms a complex with Keap1, which is in turn linked to a functional E3 ubiquitin ligase complex named Rbx1 via an adaptor protein, Cullin3 (10). The formation of this complex facilitates the ubiquitination and degradation of Nrf2 by the ubiquitin-proteasome system (Fig. 1).

Fig 1.

Mechanisms of select dietary Nrf2 activators. (A) Under physiological or baseline conditions, Nrf2 is sequestered by Keap1, leading to its ubiquitination and degradation by the proteasome. (B) Two mechanisms are involved in Nrf2 activation. The first is by modification of the cysteines in Keap1, which leads to conformational changes in this protein and the subsequent release of Nrf2. The second mechanism is by activation of kinases that phosphorylate Nrf2 and thereby free it from Keap1-mediated sequestration. After nuclear translocation, Nrf2 binds to the antioxidant response element (ARE) in the promoter regions of phase 2 enzyme genes. SFN, curcumin, and OSC all activate Nrf2 by the first mechanism, whereas RES and capsaicin function through the second mechanism and EGC and lycopene work through both mechanisms. Keap1: Kelch like ECH associated protein 1; SFN: sulforaphane; EGCG: epigallocatechin gallate; OSC: organosulfur compound; RES: resveratrol.

1.1.2 Dissociation of the Nrf2-Keap1 complex

The dissociation of the Nrf2-Keap1 complex is an essential prerequisite for Nrf2/ARE activation. Specifically, two cysteine residues (Cys273, Cys288) in the IVR domain and one cysteine residue (Cys151) in the BTB domain are essential for Keap1-mediated repression of Nrf2 activity under homeostatic, unstressed conditions (4). The oxidation of these cysteine residues affects the conformation of Keap1 and leads to dissociation of the Nrf2-Keap1 complex, thereby stabilizing and activating Nrf2, and upregulating the expression of phase II genes (10).

1.1.3 Nuclear translocation of Nrf2

All transcription factors must enter the nucleus to be active and induce gene expression. In the case of Nrf2, this process can be completed within 15 min after tert-Butylhydroquinone (t-BHQ) treatment, the prototypical Nrf2 activator (11). The key mediators that regulate nuclear import and export of transcription factors are nuclear localization signals (NLS) and nuclear export sequences (NES). A number of such nuclear shuttling signals have been identified on Nrf2, including three NLS motifs (NLS1, NLS2 and NLS3) and two NES motifs (NES1 and NES2).

The direction of Nrf2 movement is determined by a homeostatic balance between import and export driving forces. Under basal conditions, NES1 is functional, and the import force is overshadowed by the export force, retaining Nrf2 in the cytosol and eliciting its degradation. Following exposure to oxidative or electrophilic stressors, Cys183 of NES1 becomes adducted, reducing its function. The import forces then overwhelm the export forces, culminating in Nrf2 nuclear translocation (12). In addition, Nrf2 nuclear translocation can be enhanced via phosphorylation by specific kinases. One such example is protein kinase C (PKC), which can phosphorylate Ser40 in the Neh2 domain (13), although its role in Nrf2 nuclear shuttling remains controversial. Another candidate kinase is Fyn, which phosphorylates Tyr568 in the Neh3 domain, thereby controlling nuclear export of Nrf2 (11, 14).

After translocation into the nucleus, Nrf2 forms a heterodimer with a group of nuclear bZIP proteins called small Maf proteins (5). This heterodimerization enhances the binding specificity of Nrf2 to the cis-acting enhancer ARE/EpRE (15) located in the promoter region of phase II genes (16, 17). As a consequence of this DNA binding event, Nrf2 is able to initiate the transcription of phase II genes (18).

1.2 Keap1-independent pathway

Recent studies have proposed a Keap1-independent ubiquitination model of Nrf2 degradation (Fig. 1) (19). In this model, glycogen synthase kinase-3β (GSK3β) phosphorylates Ser342 and Ser347 at Neh6 of Nrf2. Phosphorylated Neh6 can bind with the Skp, Cullin, F-box (SCF)-containing ubiquitin ligase adaptor β-TrCP. β-TrCP is a scaffolding protein that directly links Nrf2 to the Cullin1/Rbx1 ubiquitination complex, which is subsequently degraded. This GSK-3β and β-TrCP-dependent Nrf2 degradation model is supported by the finding that GSK-3β inhibitors stabilize Nrf2 in Keap1^−/− mouse embryo fibroblasts (MEFs) (14). Additionally, the cancer-chemopreventive agent nordihydroguaiaretic acid can activate Nrf2 and increase HO-1 protein levels by inhibiting GSK-3β phosphorylation in Keap1^−/− MEFs (20).

Several other kinases also regulate the Nrf2/ARE pathway. For example, PKC can phosphorylate Nrf2 at Ser40, which located in the Neh2 domain and is critical for its interaction with Keap1. This phosphorylation event results in the disassociation of the Keap1/Nrf2 complex (21). Phosphatidylinositol 3-kinase (PI3K) also positively regulates the transcriptional activity of Nrf2 (22). Finally, the mitogen-activated protein kinase (MAPK) family includes three members—extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38, all of which may modulate Nrf2 activation (1). However, such effects seem to depend on the cell type. For example, p38 is a negative regulator of Nrf2 in human hepatoma HepG2 and murine hepatoma Hepa1c1c7 cells (23), but it has the opposite role in MCF-7 mammary epithelial cells (24).

Some proteins can directly regulate the Nrf2/ARE pathway in the cytoplasm or in the nucleus. For example, p21 is a cytoplasmic mediator that binds the DLG motif of Nrf2 (the same site that binds to Keap1) through its C-terminal KRR motif. The competition between p21 and Keap1 for Nrf2 binding compromises Keap1-mediated ubiquitination of Nrf2 (25). Another protein, sequestosome-1 (also known as p62), binds and inactivates Keap1 and thus augments the expression of genes regulated by Nrf2 (6). In the nucleus, the BTB domain and CNC homolog 1 (BACH1) heterodimerizes with Maf and occupies ARE sequences to inhibit the expression of phase II genes. When challenged by oxidative insults, BACH1 is phosphorylated and exported to the cytoplasm. Maf proteins are thereby liberated and can form a heterodimer with Nrf2 to enhance the expression of phase II genes (18).

1.3 Self-limitation of the Nrf2 pathway

After exposure to oxidative stress, the activated Nrf2/ARE pathway boosts the expression of phase II genes by Keap1-dependent and independent means. However, there are endogenous regulatory mechanisms to prevent excessive activation of this pathway. First, AREs are located in the promoter region of Cul3, Rbx1, and Keap1 genes. Thus, when the Nrf2/ARE pathway is activated, the transcription of these inhibitory proteins is simultaneously promoted. This negative feedback loop is defined as an autoregulatory arm of the Nrf2/ARE pathway (22). Second, the Keap1-Cul3-Rbx1 complex can translocate into the nucleus, an event that is mediated by prothymosina (ProTα), a Keap1-binding protein with a nuclear localization signal. As a result, 10–15% of the Keap1-Cul3-Rbx1 complex is located in the nucleus. Upon nuclear entry, the Keap1-Cul3-Rbx1 complex releases ProTα and binds Nrf2, leading to the ubiquitination and degradation of nuclear Nrf2 (26). Third, oxidative stressors such as hydrogen peroxide promote the activation of GSK-3β by phosphorylating its tyrosine 216 residue. Activated GSK-3β subsequently phosphorylates Fyn (p-Fyn, a member of Src family) at a threonine residue(s), leading to the accumulation of p-Fyn in the nucleus and subsequent phosphorylation of Nrf2 at tyrosine 568. Subsequently, phosphorylated Nrf2 interacts with Crm1 or exportin 1 and is exported out of nucleus (27), thereby leading to its inactivation and loss of phase II enzyme expression. These mechanisms would all serve to prevent neoplastic transformation of tissue from excessive activation of the prosurvival Nrf2/ARE pathway.

2. The neuroprotective effects of the Nrf2/ARE pathway against CNS diseases

There are more than 200 detoxification and antioxidant genes driven by the Nrf2/ARE pathway. Thus, Nrf2 can elicit neuroprotection against CNS disorders in manifold ways. In this review, we will focus on the protective roles of Nrf2 against acute neurological diseases.

2.1 Acute neurological diseases

2.1.1 Traumatic brain injury (TBI)

TBI is a major cause of death and disability worldwide, especially in children and young adults. Oxidative stress is a major component of the pathogenesis of TBI. Many studies have demonstrated that Nrf2 is capable of reducing the brain damage caused by TBI (28).

Following TBI, the brain exhibits a significant increase in Nrf2 expression and phase II enzymes, such as HO-1 and NQO1 (29). HO-1 catalyzes the first and rate-limiting step of heme catabolism (30). The neuroprotective effects of HO1 can be attributed to two mechanisms: the breakdown of heme and the generation of antioxidants. Hemoglobin, oxidase, and peroxidase are the three main classes of proteins containing heme (31, 32). Breakdown of these proteins causes a net reduction in superoxide and other reactive oxygen species (ROS). The breakdown products of heme also exhibit neuroprotective effects. For example, biliverdin and bilirubin are both potent antioxidants (32, 33). NQO1 itself also has anti-oxidative properties, indicating an important role in neuroprotection (34). The increased expression of these enzymes after traumatic insults suggests that the Nrf2/ARE pathway is an endogenous compensatory adaptation against the damage elicited by TBI.

(−)-Epicatechin, a type of natural flavonol, has also been demonstrated to exert neuroprotective effects via activation of Nrf2/ARE. EC has been shown to decrease TBI lesion volume and neuronal degeneration in mice, and to increase the expression of Nrf2 and HO-1 following TBI. However, the neuroprotective effects of Epicatechin are abolished in Nrf2^−/− mice (35). Similar results have also been reported by other groups (36). Taken together, these findings demonstrate that endogenous activation of Nrf2 protects the brain from TBI.

Nrf2 activation can ameliorate blood-brain barrier (BBB) dysfunction. The BBB barrier loses integrity following direct exposure to mechanical or shearing forces and secondary oxidative and inflammatory damage after TBI. Secondary damage causes a loss of endothelial cells and tight junction proteins, further exacerbating BBB dysfunction (37). While direct lesions after trauma cannot be reversed, the secondary damage can perhaps be ameliorated. One approach to ameliorating the secondary oxidative damage is Nrf2 activation. For example, Zhao and colleagues reported that the BBB retains its integrity in wildtype mice treated with an Nrf2 activator, in contrast to Nrf2 knockout mice (37). These findings demonstrate that Nrf2 activation exerts robust BBB protective effects.

2.1.2 Ischemic stroke

Stroke is the leading cause of disability and the third-leading cause of mortality across the world. Ischemic stroke is the most common cause of stroke. The pathological processes of stroke are complex, ranging from excitotoxicity, oxidative stress, and inflammation to mitochondrial dysfunction (38, 39). Nrf2 serves as a natural brake on ischemic injury. Following middle cerebral artery occlusion (MCAO) in rodents, Keap1 levels are naturally decreased, and this loss is paralleled by an increase in Nrf2 and its downstream proteins, such as thioredoxins, glutathione (GSH) synthases, and HO-1 (40). Thioredoxins are 12-kDa enzymes containing a conserved Cys-X-X-Cys motif in their active center. This motif plays an important role in eliminating oxidized proteins via the exchange between cysteine thiols and protein disulfides (41). GSH is the most abundant antioxidant peptide in the CNS and scavenges various reactive oxygen species, including superoxide, nitric oxide, the highly reactive hydroxyl radical, and peroxynitrite (42). GSH can also mitigate toxicity secondary to high cysteine concentrations (43).

Activation of the Nrf2 pathway is critical for scavenging ROS, and this scavenging function contributes to robust neuroprotection against ischemic brain injury. Wu et al. reported that the Nrf2/ARE pathway activator myricetin lessened the production of ROS, decreased infarct volume, and reduced neuronal loss after ischemia (44, 45). This conclusion is supported by another study employing a second Nrf2/ARE pathway activator, Panax notoginseng saponins (46). Finally, Nrf2^−/− mice subjected to stroke exhibit higher levels of ROS than wildtype mice (47), supporting the aforementioned notion that Nrf2 is a natural compensatory mechanism.

2.1.3 Hemorrhagic strokes

Hemorrhagic strokes include intracerebral hemorrhages (ICH) and subarachnoid hemorrhages (SAH), which are classified based on the location of bleeding and blood accumulation. That is, ICH involves bleeding into brain parenchyma whereas SAH involves bleeding into the subarachnoid space.

In addition to the intracranial hypertension and brain herniation caused by hematoma and edema, ischemia and oxidative stress also contribute to brain injury after ICH (48). HO-1 is upregulated within 24 hr after ICH, peaking at day 5, and subsiding on day 8, indicating a temporal window of activation of the Nrf2/ARE pathway (49). However, HO-1 elicits detrimental effects in ICH, as shown by smaller injuries in HO-1 knockout mice (50). Post-injury administration of the selective Nrf2 activator tBHQ has been shown to attenuate neurodegeneration and improve neurological outcomes after ICH (51, 52). Consistent with these latter observations, Nrf2^−/− mice showed more severe neurological deficits and cellular damage after ICH (53). Although HO-1 induction by tBHQW would be expected to elicit detrimental effects after ICH (see above), the parallel activation of many other antioxidant response genes by Nrf2 may ameliorate the negative consequences of greater heme breakdown by HO-1.

Although SAH per se does not directly lead to parenchymal cerebral damage, it is accompanied by vasospasms that indirectly cause ischemia and acute brain injury (54). In rodent SAH models, there is activation of Nrf2 in cerebral vessels, as shown by increased Nrf2 nuclear translocation and DNA binding in both endothelial and smooth muscle cells of the basilar artery (55). tBHQ treatment also markedly upregulates the expression of Keap1, Nrf2, HO-1, NQO1, and GSTα1 after SAH. Furthermore, activation of the Keap1/Nrf2/ARE pathway is also associated with attenuation of cognitive dysfunction after SAH (56).

2.2 Neurodegenerative Diseases

2.2.1 Alzheimer’s disease (AD)

AD is a progressive neurodegenerative disorder characterized by memory loss and cognitive dysfunction. Pathological hallmarks of AD include the cerebral deposition of amyloid-beta (Aβ) peptides in senile plaques and neurofibrillary tangles of hyper-phosphorylated tau aggregates. Aβ, generated from the cleavage of amyloid precursor protein, is thought to be critical for neuronal cell death in AD (57). Aβ itself has been shown to induce the production of H₂O₂ in cultured cells (58), revealing that oxidative damage may play a role in AD progression. In turn, mitochondrial ROS production increases Aβ production in a self-amplifying cascade and leads to neurodegeneration in vivo (59). As will be argued below, natural Nrf2/ARE activation may break the progression of AD by reducing oxidative stress-mediated cell death.

Postmortem tissue from AD victims exhibits increased levels of proteins that are downstream of Nrf2. For example, NQO1 is increased in AD brains (60). Similar patterns have been reported for HO-1, p62, and glutathione redox system genes in the hippocampus and cerebellum in AD (61). However, some studies have found a decrease of Nrf2 in AD brains (62). One explanation for these discrepancies may be the stage of disease at the time of tissue collection. There may be upregulation of endogenous defense systems at early stages of the disease but loss of natural protective mechanisms at late stages.

Many studies have demonstrated positive effects of Nrf2 activation on AD phenotypes. An activator of the Nrf2/ARE pathway, 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic acid-Methyl Amide (CDDO-MA) has been shown to improve memory and decrease plaque formation, Aβ, and markers of oxidative stress in Tg19959 transgenic AD mice (63). Similar effects have been reported with other Nrf2 activators, such as tBHQ and curcumin. Administration of Nrf2 activators ameliorates long-term memory loss in addition to the accumulation of ROS or Aβ peptides (62, 64). In hypobaric hypoxia-induced dementia, tropomyosin receptor kinase A expression and ERK phosphorylation were increased by acetyl-L-carnitine, resulting in Nrf2 nuclear translocation. The activated Nrf2/ARE pathway in turn ameliorated memory impairments in this model (65).

2.2.2 Parkinson’s disease (PD)

Mitochondrial dysfunction is believed to be critical in the pathogenesis of PD (66, 67). Parkin and PINK1 genes play important roles in mitochondrial quality control and are mutated in some familial PD cases (68). The mitochondrial inhibitors 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its metabolite 1-methyl-4-phenylpyridine, 6-hydroxydopamine, and rotenone are commonly used to induce a PD-like phenotype in animals (58). Mitochondrial function can also be inhibited by dopamine (DA) quinone, a by-product of DA oxidation (69). DA quinone itself can induce oxidative stress due to its ability to bind protein cysteine residues and GSH, leading to GSH depletion and changes in cellular function. Furthermore, GSH depletion results in mitochondrial complex I inhibition, which further exacerbates mitochondrial dysfunction (58). Taken together, these findings suggest that oxidative stress is an important factor in the pathogenesis of PD. In addition, there is evidence of oxidative damage to proteins, lipids, and DNA in postmortem tissue from PD victims (70).

Several studies suggest that Nrf2 plays a naturally protective role in PD (71) . The substantia nigra of PD patients exhibits higher levels of Nrf2 downstream genes, such as NQO1 and HO-1 (72). Furthermore, Nrf2^−/− mice exhibit increased sensitivity to the dopaminergic toxins MPTP and 6-OHDA (73). Administration of the Nrf2 activators tBHQ and 1,2-dithiole-3-thione ameliorated the PD phenotype in experimental mouse models (25). Chen and colleagues have demonstrated that astrocytes with Nrf2 activation, but not neurons, can reduce the neurotoxicity of MPTP (25), indicating that it is astrocytic Nrf2 that is critical for neuroprotection against PD models.

3. Phytochemicals with Nrf2-ARE activating ability

As discussed above, Nrf2 plays a protective role against acute and chronic brain disorders, making it a promising target for clinical intervention (74). There are several categories of Nrf2 activators. Below we will focus on dietary Nrf2 activators, as they are widely available and safe.

3.1 Sulforaphane

Sulforaphane (1-isothiocyanate-4-methylsulfinyl butane, SFN)—a naturally occurring isothiocyanate compound—is present in cruciferous vegetables such as broccoli, brussel sprouts, cabbage, and cauliflower. SFN has indirect antioxidant properties by virtue of Nrf2 pathway activation and upregulation of genes downstream of ARE (75, 76).

Oral bioavailability is one of key limiting factors for phytochemicals. SFN passively diffuses into cells because of its lipophilicity and small molecular size (77, 78). Within the cell, SFN is conjugate to glutathione (SFN-GSH) in the presence of glutathione-S-transferase (GST), which maintains a concentration gradient across the cell membrane and ensures continued passive diffusion of SFN into the cell (79). SFN is an electrophile that can react with protein thiols to form thionoacyl adducts. Hong and colleagues reported several sensor cysteines in human Keap1 that can be modified by SFN, including Cys-77, −226, −249, −257, −489, −513, −518, and −583 (80). Another sensor cysteine for SFN is Cys151 in mouse Keap1, which is essential for the association of Cul3 ubiquitin ligase (81). By modifying these cysteines, SFN can prevent ubiquitination of Nrf2, thereby leading to Nrf2 stabilization. Stabilized Nrf2 is then free to translocate into the nucleus and initiate the Nrf2/ARE pathway. In addition to targeting Keap1, SFN can also enhance Nrf2 mRNA and protein expression by reducing methylation of the first 15 CpGs of Nrf2 promoters (82). Other targets of SFN, such as DNA methytransferases and histone deacetylase (83) are out of the scope of this review.

Many studies have demonstrated protective effects of SFN against CNS diseases via activation of the Nrf2/ARE pathway. For example, Ping et al. reported that administration of SFN could significantly reduce infarct volume by increasing Nrf2 and HO-1 expression in experimental ischemia models (84). In addition, SFN reduced the numbers of apoptotic neurons, activated microglia, and oxidative parameters after ischemia. In TBI models, SFN is able to preserve BBB function by reducing loss of endothelial cell markers and tight junction proteins. Nrf2 is a key mediator in this process, as SFN administration increases the expression of Nrf2-driven genes, including GSTα3, GPX, and HO-1 in the parietal cortex and brain microvessels (47). SFN also has protective effects against chronic neurodegeneration, such as experimental AD and PD. The accumulation of Aβ peptides in AD is accelerated by oxidative stress, impaired protein-folding in the endoplasmic reticulum, and deficient proteasome- and autophagic-mediated clearance of damaged proteins (85). Kwak et al demonstrated that SFN can increase proteasome function in vivo, which offers a viable strategy to protect neuronal cells from oxidative damage in AD (86). Finally, SFN can inhibit 6-OHDA-induced DA neuron loss by increasing Nrf2 nuclear translocation and HO-1 expression in experimental PD (87, 88). These latter findings have also been confirmed in rat organotypic nigro-striatal co-cultures (89).

3.2 Curcumin

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a diferuloylmethane derived from the rhizomes of turmeric. Curcumin shows a wide range of beneficial properties, including antioxidant and anti-inflammatory effects. Unlike SFN, the bioavailability of curcumin is poor. Many studies have found that limited tissue distribution, rapid metabolism, and short half-life severely curtail its bioavailability (90–93). Because of its poor bioavailability, the effect of curcumin on Nrf2/ARE pathway is highly dose-dependent. Curcumin treatment at a concentration of 5 µM does not have a significant effect on Nrf2 activation, whereas 15 and 30 µM curcumin does activate Nrf2 (94). Curcumin exerts its protective effects by both Nrf2-dependent and independent means. Curcumin has two electrophilic α, β-unsaturated carbonyl groups, which can modify the thiol groups of Keap1 and release Nrf2, thereby activating the Nrf2/ARE pathway and inducing the expression of phase II enzymes (95, 96). Furthermore, the ability of curcumin to increase the expression of HO-1 has been demonstrated in vascular endothelial cells, astrocytes, and cultured hippocampal neurons (97–99). Finally, curcumin can upregulate NQO1 expression (100).

Although curcumin has poor bioavailability, it is highly lipophilic and may cross the BBB, accumulating in the brain at a sufficient concentration to activate the Nrf2 pathway. Indeed, several studies have shown neuroprotective effects of curcumin. Yang et al. reported that curcumin could protect neurons from brain focal ischemia in vivo through the induction of Nrf2/HO-1 (101). This finding is in agreement with studies on ethanol-induced brain damage (102). Similarly, Li et al. also reported that curcumin can protect the brain against ischemia/reperfusion injury by activating the Nrf2/ARE pathway (103). Furthermore, pre-incubation of cultured neurons with low concentrations of curcumin for 12 hours improves cellular resistance to glucose oxidase-mediated oxidative damage (104). The protective effects of curcumin have also been reported with post-treatment after the onset of the injury (105). Thus, curcumin has both neuroprotective and neuro-preventive properties.

Epidemiological studies suggest that oral curcumin exposure through the curry-rich Indian diet is associated with a 4.4 reduction in the incidence of AD in India compared to the USA (106). However, one limitation of these findings is that they are correlative and that a causal link between curcumin and lower AD risk has not yet been established. It is also unclear if elderly patients with diseases such as AD are diagnosed as readily in underdeveloped countries as they are in developed nations. Nevertheless, Scapagnini et al. suggested that curcumin can protect cortical neurons against apoptotic cell death induced by β-amyloid peptide (99). Similarly, Cole and colleagues reported that 6 months of dietary supplementation of transgenic AD mice (Tg2576) with curcumin reduces inflammation, oxidative cerebral damage, and Aβ-induced cognitive deficits (107, 108). These authors also discovered that curcumin could easily enter the brain and inhibit the formation and toxicity of Aβ oligomers (109). These studies support the view that curcumin exposure via ingestion of the turmeric spice exerts similar beneficial effects.

3.3 Epigallocatechin gallate

Epigallocatechin gallate (EGCG) is the most abundant catechin compound in green tea. It is well established that EGCG is a potent antioxidant and anti-inflammatory agent (110). Epidemiological studies show that consumption 200–300 mg of EGCG per day is beneficial, as it is the most potent Nrf2 activator among all green tea catechins (111).

EGCG has a low absorption rate (probably <5%) and an average Tmax of 2 hours after oral administration (112, 113), due to its instability in the air and inactivation in the gastrointestinal tract and liver (114). Thus, EGCG also has poor bioavailability, similar to curcumin. Despite these pharmacokinetic properties, EGCG exhibits robust diffusion through bodily tissues, including the endothelium of the BBB (115).

EGCG has the capacity to activate Nrf2/ARE and induce HO-1 expression (116). Several studies have shown that EGCG can also interact with more upstream kinases, such as ERK, PI3K, PKC and JNK, causing the disassociation of Nrf2/Keap1 complex. In endothelial cells, EGCG induced HO-1 expression by activating Akt and ERK1/2, whereas in B lymphoblasts, EGCG induced Nrf2 translocation and HO-1 expression by activation of p38 MAPK and Akt (117). As one might expect, the effect of EGCG on MAPKs is dose and time-dependent. Low concentrations of EGCG result in the activation of ERK and induction of ARE-mediated gene expression, whereas higher concentrations cause activation of MAPKs such as JNK, thereby leading to apoptosis (111, 118).

Han et al. have reported protective effects of EGCG against ischemia/reperfusion injury in a rat model of MCAO (119). Administration of EGCG improved neurologic scores, reduced infarct volume, and ameliorated neuronal apoptosis due to increased GSH biosynthesis (via Nrf2 activation) and decreased ROS content. EGCG also can induce the expression of HO-1 in cultured neurons through the Nrf2/ARE pathway, protecting neurons against oxidative stress-induced cell death (120). Furthermore, EGCG is a potent neuroprotective agent in AD models. By inducing the expression of Nrf2 and HO-1, EGCG increases important endogenous antioxidants in microglial cells. EGCG suppresses Aβ-induced cytotoxicity by reducing the activation of the MAPK signal transduction cascade (121).

3.4 Allyl sulfides

Allyl sulfides are organosulfur compounds (OSCs) found abundantly in garlic, and include diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). OCSs exert health benefits, and have been used as a food and medicinal herb for thousands of years.

OSCs have neuroprotective effects by induction of the expression of phase II enzymes. Due to their lipid solubility, they are easily absorbed, but are nevertheless barely detectable in the blood or urine, even after consumption of large amounts of garlic (122). This disappearance is probably caused by rapid hepatic metabolism as they pass through the liver (123). Within the liver, allicin from garlic shows a remarkable first-pass effect and is very efficiently metabolized into DADS and allyl mercaptan (124). This may prevent OSCs from reaching high concentrations in extrahepatic tissues after dietary delivery. Further studies are needed to determine the bioavailability and pharmacokinetics of OSCs in humans.

DATS is thought to react with Keap1 at Cys288, which would cause the release of Nrf2, and activation of genes downstream of ARE (125). Allyl sulfides also activate Nrf2/ARE in a similar manner as EGCG. Allyl sulfides interact with upstream kinases and lead to the dissociation of the Nrf2-Keap1 complex. Xu et al. reported that DATS protected B35 neural cells against oxygen glucose deprivation-induced cell damage via upregulation of the PI3K/Akt-mediated Nrf2 pathway and the expression HO-1 (126). DAS has also been shown to upregulate antioxidant genes and enzymes by activating Nrf2 through ERK/p38 pathways in lung MRC-5 cells (127) and in renal cells (128).

3.5 Resveratrol

The polyphenol Resveratrol (trans-3,5,4’-trihydroxystilbene, RES) is abundant in grapes and red wine. It is thought to be beneficial against cancer, diabetes, neurodegenerative, and cardiovascular diseases (129–131). As with allyl sulfides, the bioavailability of RES is very poor, approximating <1% when delivered orally (130). Such limited bioavailability can be explained by its low water solubility, limited stability, and rapid metabolism (131).

RES activates the Nrf2/ARE pathway activation by interacting with upstream kinases. Cheng et al. reported that RES activated the Nrf2 pathway and induced HO-1 and glyoxalase via ERK phosphorylation, resulting in suppression of methylglyoxal-induced insulin resistance in HepG2 cells (132). RES pretreatment also has neuroprotective effects against cerebral ischemia/reperfusion injury by activation of Nrf2 and upregulation of HO-1 in vivo (133, 134). In addition, Gaballah and colleagues recently observed that RES increased Nrf2 DNA binding activity in the rotenone model of neuronal oxidative stress in experimental PD (135).

3.6 Lycopene

Lycopene is a polyunsaturated hydrocarbon phytochemical present in tomatoes and carrots. It belongs to the tetraterpene carotenoid family (136) and exhibits health-benefits through its antioxidant activity. There are few studies about the bioavailability of lycopene, though the structural localization of lycopene in chloroplasts of fruit and vegetables is an important limitation in terms of bioavailability (137). The distribution of lycopene among tissues is tissue-specific, with high concentrations in testes, adrenals, liver, and prostate, but low concentrations in others tissues (138).

Several studies have demonstrated that lycopene can stimulate the expression of phase II enzymes by activation of the Nrf2/ARE pathway (139). Lycopene has been shown to elicit Nrf2 translocation and upregulate the ARE system in HepG2 and MCF-7 cells (140). Lycopene pretreatment increases the expression of Nrf2/HO-1 and reduces ischemic injury following global cerebral ischemia, indicating an important role of the Nrf2/ARE pathway in lycopene-medicated neuroprotection. Finally, lycopene also exerts protective effects against Aβ-induced mitochondrial oxidative stress and dysfunction in cultured rat cortical neurons, mitigating neuronal apoptosis and revealing a potential therapeutic approach against Aβ-induced neurotoxicity (141).

3.7 Capsaicin

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major active pungent molecule in hot chili pepper, has been shown to have immunomodulatory functions and cytotoxicity towards cancer cells (142). Capsaicin is easily absorbed following percutaneous, oral, gastrointestinal and systemic routes of administration. Due to its low half-life in plasma and rapid metabolism in the liver, capsaicin also exhibits poor bioavailability. The mechanism of Nrf2 regulation by capsaicin has been described by Joung and colleagues (143), who showed that capsaicin induced ROS production by inhibiting NQO1, which in turn caused oxidative stress, activated the PI3K/Akt pathway, and induced dissociation of the Nrf2-Keap1 complex in HepG2 cells. Notably, a capsaicin-rich diet exhibits favorable effects on Aβ levels and cognitive function (144). However, the role of Nrf2 in this process remains unclear.

The activation of the Nrf2/ARE pathway by so many dietary compounds is not coincidental. Plants often produce substances that elicit a robust stress response, as they need to defend themselves from attack by microorganisms, insects, reptiles, birds, and mammals. Many phytochemicals are therefore toxic in high doses. This feature partly explains why cells respond to many phytochemicals with robust activation of the Nrf2/ARE pathway, as this pathway is so intimately involved in detoxification and redox balance. In effect, many phytochemicals may actually be eliciting via Nrf2 a primordial form of preconditioning, which is defined as an adaptive cellular response to mild stress that prepares cells to survive subsequent challenges.

There are numerous other dietary phytochemicals not discussed above that can regulate the Nrf2/ARE pathway and exert neuroprotective effects. A description of every single ARE activator is beyond the scope of this review. However, Table 1 lists additional phytochemicals that exhibit neuroprotective properties against CNS diseases.

Table 1.

Other dietary Nrf2 activators

Compound	Origin	Molecular Mechanism	Ref
3H-1,2-dithiole-3- thione (d3t)	Brussels sprouts, cabbage	↑ Phosphorylation of Akt	(144)
		↑ Nrf2 nuclear translocation
		↑ HO-1
3-O-caffeoyl-1- methylquinic acid	Phyllostachys edulis	↑ Phosphorylation of EKR and JNK	(145)
		↑ Nrf2 nuclear translocation
		↑ HO-1
Brazilin	Caesalpinia echinata	↑ Phosphorylation of ERKs and PI3K/Akt	(146)
		↑ HO-1
Cafestol	Coffee	↑ Binding of Nrf2 to ARE	(147)
		↑ Expression of NQO1
Carnosol	Herb rosemary	↑ ERK, Akt, p38, MAPK	(148)
		↑ HO-1
Chaocone	Cardamom	↑ Expression of HO-1, GPX2 and TrxR1	(149)
		↑ Nrf2 nuclear translocation
Chlorophyllin	Green vegetables	↑ Phosphorylation of PI3K/Akt	(150)
		↑ HO-1, NQO1
Cinnamaldehyde	Cortex Cinnamomi	↑ Expression of Nrf2	(151)
		↑ Nrf2 nuclear translocation
		↑ HO-1 NQO1
Eupatilin	Artemisia asiaitica	↑ Phosphorylation of ERKs and PI3K/Akt	(152)
		↑ Nrf2 nuclear translocation
		↑ HO-1
Fisetin	Strawberries, apples, Japanese wax tree	↑ Phosphorylation of PI3K/Akt, PKC and p38	(153)
		↑ HO-1
		↓ ROS
Isoorientin	Gentiana olivieri	↑ Phosphorylation of PI3K/Akt	(154)
		↑ HO-1, NQO1
Kahweol	Coffee	↑ Binding of Nrf2 to ARE	(155)
		↑ Activation of pI3K and p38
		↑ HO-1
Omega-3 fatty acids	Cold water fish, fish oil, flaxseed	↑ Nrf2 nuclear translocation	(156)
		↑ HO-1
Piperine	Black pepper	↑ Phosphorylation of MAPK,	(157)
		↑ HO-1,
		↑ Nrf2 nuclear translocation
Plumbagin	Plumbago	↑ HO-1, Phosphorylation of Akt and ERK	(158)
Quercetin	Red kidney bean, caper, radish, onion	↑ Phosphorylation of p38	(159)
		↑ Nrf2 nuclear translocation
		↓ Nrf2 ubiquitination,
		↑ HO-1, NQO1
Xanthohumol	Hops	↑ Expression of phase II enzymes	(152)
		↑ activation of Nrf2 via covalent
		modification of Keap-1 at cysteine 27 residue
Zerumbone	Tropical ginger	↑ HO-1,	(160)
		↑ Nrf2 nuclear translocation

4. Conclusion

In summary, Nrf2 is sequestered under normal homeostatic conditions when redox balance is properly maintained by other cellular components. However, upon loss of redox equilibrium and other types of injury, the Nrf2 pathway is naturally activated as an endogenous compensatory mechanism. As with a preconditioning stimulus, various dietary phytochemicals can activate the Nrf2/ARE pathway and induce expression of many pro-survival genes, thereby exerting powerful neuroprotective effects against CNS diseases. Therefore, targeting the Nrf2 pathway with rational drug design is a promising therapeutic approach. However, further studies are warranted to identify the mechanism underlying Nrf2 activation by dietary compounds and to establish their protective effects in models of CNS disorders.

Abbreviations

AD

Alzheimer’s disease

ARE

antioxidant response elements

BBB

blood-brain barrier

BTB

bric-a-brac domain

bZIP

basic leucine zipper

CDDO

2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic acid

CNS

central nervous system

EGCG

epigallocatechin gallate

ERK

extracellular signal-regulated protein kinase

GSH

glutathione

GPX

GSH peroxidase

GSK3β

glycogen synthase kinase-3β

GST

glutathione s-transferase

HO-1

heme oxygenase 1

ICH

intracerebral hemorrhages

IVR

intervening region

JNK

c-jun N-terminal kinase

Keap1

Kelch-like ECH associated protein 1

Maf

musculoaponeurotic fibrosarcoma

MAPK

mitogen-activated protein kinase

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Neh

Nrf2-ECH homologies

NES

nuclear export sequences

NLS

nuclear localization signals

NQO-1

NADPH quinine oxidoreductase 1

Nrf2

nuclear factor erythroid 2 related factor

PD

Parkinson’s disease

PKC

protein kinase C

PI3K

phosphatidylinositol 3-kinase

ROS

reactive oxygen species

SAH

subarachnoid hemorrhages

SFN

sulforaphane

TBI

traumatic brain injury

tBHQ

tert-butylhydroquinone