In addition to antioxidant effects, Nrf2/ARE activation can afford protection against the neuroinflammation evident following ischemia. In the ischemic brain, ROS, as with DAMPS, can activate the NLRP3 inflammasome, thereby converting prointerleukin (IL)-18 and pro-IL-1β into mature IL-18 and IL-1β forms, which are then released (Inoue et al., 2012). Glial cells are important drivers of neuroinflammation and NLRP3 activation (Frank et al., 2007; Gavillet et al., 2008). Xu et al. (2018) showed that the Nrf2/ARE pathway protects against the oxidative stress-induced NLRP3 activation in the BV2 microglial cell line exposed to OGDR. Moreover, neonatal rats treated with LPS 24 h prior to hypoxia-ischemia, were protected against neuropathological effects, whereas LPS administration 72 h before hypoxia-ischemia increased brain damage, which was prevented by treatment with N-acetylcysteine, a glutathione precursor (Wang et al., 2007). Such data support the idea that Nrf2 upregulation varies according to the time of ischemia exposure, as well as cell type (glial cells or neurons) and antioxidant reserve. Such data suggests complex effects, although it is clear that Nrf2-related pathways are important molecular targets for pharmacologic intervention in the management of ischemic stroke.

#### Multiple Sclerosis

Multiple sclerosis is the most frequent chronic neuroinflammatory disease of the CNS, characterized by demyelination, as well as focal inflammatory lesions in the brain and spinal cord, which culminates in axonal damage (Lassmann, 2011b). MS is also associated with gray matter synaptic loss and decreased neurogenesis, which contributes to cognitive impairment. MS clinical symptoms include sensory or motor impairment, fatigue, ataxia, and spasticity, as well as cognitive impairment (Chiaravalloti and DeLuca, 2008; Lublin et al., 2014). EAE is the most common experimental model of MS (Simmons et al., 2013). Activated macrophages and microglial cells can produce an array of neurotoxic factors, including proteases, nitric oxide, and ROS, with this being evident in both MS and the EAE model (Glass et al., 2010). Generation of ROS and other free radicals by macrophages is involved in demyelination and axonal damage in EAE. Evidence of lipid peroxidation, as a consequence of oxidative stress, can be detected in the exhaled breath samples of MS patients. Additional studies have shown decreased levels of antioxidant enzymes in blood and cerebrospinal fluid of MS patients (Lassmann, 2011a,b, 2014; Witte et al., 2014). Such data highlights the importance of heightened levels of immune-inflammatory activity and oxidative stress in MS.

Therapeutic strategies in the treatment of MS have primarily focused on dampening the heightened immune-inflammatory activity that is present, including by the use of: imunossupressors, such as synthetic glucocorticoids (e.g., dexamethasone, prednisone, and methylprednisone); monoclonal antibodies (Natalizumab); inhibitors of cell egress from lymphoid organs (Fingolimod), modulators of pro- and anti-inflammatory cytokines (interferon-beta); and microglial inhibitors (acetate glatiramer). However, all of these treatments have shown limited efficacy. Antioxidant therapies have proved to have some utility when used as adjuvants in MS treatment, with endogenous Nrf2 being an important treatment target for the induction of endogenous antioxidants (Kappos et al., 2010; Lim et al., 2014).

Classical treatment in MS has emphasized the role of heightened immune-inflammatory processes. As noted above, such processes are intimately linked to alterations in redox regulation. There is now a growing appreciation of the role the oxidant/antioxidant balance in MS. A growing body of data indicates an important role for ROS in MS, including: a central role in MS lesion development and progression and an initial contribution to the BBB breakdown and leukocyte infiltration that is crucial in early phases of lesion formation in MS (Van der Goes et al., 2001; Schreibelt et al., 2006). ROS can cause oxidative injury in CNS cells, including during demyelination, oligodendrocyte injury, and axonal degeneration. OPCs are highly vulnerable to oxidative injury due to their low levels of endogenous antioxidant enzymes and the relatively high levels of polyunsaturated fatty acids in the myelin sheets, which are more susceptible to lipid peroxidation (Juurlink et al., 1998; French et al., 2009). Furthermore, free radicals are able to impair the maturation of OPCs into myelin-forming mature cells by modulating the genes driving maturation and differentiation.

Antioxidant molecules and oxidative stress are upregulated in active brain lesions in MS patients, but not to a sufficient level to counteract the oxidative stress evident in MS brain tissue. NADPH oxidase subunits are strongly upregulated in the macrophages and microglia in active MS lesions, suggesting

an important role for heightened ROS, including as mediated by an oxidative burst, in the pathophysiology of active lesions.

Although a number of different cell types, such as macrophages, can have an antigen presenting capacity, DCs are the body's major antigen presenting cells. There is growing body of data indicating that Nrf2 may regulate the differentiation and function of macrophages and DCs, and therefore in antigen presentation and the regulation of adaptive immune response. In fact, Nrf2 deficiency modifies the phenotype and function of DCs by increasing the expression of co-stimulatory molecules and consequently the antigen-specific T cell reactivity (Al-Huseini et al., 2013). Some of these Nrf2 effects may be mediated via alterations in mitochondrial functioning, as indicated above, including in the mitochondrial regulation of immune cell responses.

Nrf2 is strongly upregulated in active MS lesions, with the expression of Nrf2-responsive genes being predominantly found in areas of initial myelin destruction (Licht-Mayer et al., 2015). Analyses of MS inflammatory lesions showed upregulation of Nrf2 and its downstream antioxidant enzymes, HO-1 and NQO1. In support of the importance of such Nrf2-mediated changes, experimental studies in Nrf2-deficient EAE mice show an increase in disease signals and peripheral cell infiltration (Lim et al., 2014). Accordingly, Johnson et al. (2010) showed that the absence of Nrf2 exacerbates the development of EAE. Part of the effects associated with Nrf2 deficiency may be related to the reduced levels of HO-1. Indeed, mice with a myeloid-specific HO-1 deficiency exhibit a higher incidence of lesions, accompanied by activation of antigen-presenting cells and the infiltration of the pro-inflammatory t helper (Th)17 cells and myelin-specific T cells (Tzima et al., 2009). Knockdown of KEAP1 (Kobayashi et al., 2016) or treatment with a wide range of small molecules that activate Nrf2 (Buendia et al., 2016) inhibits the development and severity of EAE.

#### ROLE OF Nrf2/ARE PATHWAY MODULATION BY DIETARY INTERVENTIONS IN NEUROLOGICAL DISEASES

A growing literature shows Nrf2 to regulate the expression of genes that reduce gluconeogenesis and lipogenesis, whilst also increasing fatty acids β-oxidation and mitochondria activity. Such data indicates that Nrf2 activity intimately interacts with cellular nutritional and energetic status (Holmstrom et al., 2013; Ludtmann et al., 2014; Tebay et al., 2015). The multiple molecular pathways that may underpin such interactions have been reviewed previously (Tebay et al., 2015). The present article reviews the current knowledge of dietary interventions, namely DER and HDF, on Nrf2 and the relevance of this to neurological disorders.

### Dietary Energy Restriction

Dietary energy restriction, the decrease of food intake without malnutrition, is the most powerful intervention known to delay aging processes and extend lifespan (Pearson et al., 2008; Hine and Mitchell, 2012). Several studies have comprehensively highlighted the beneficial effects of DER on cognitive function, metabolic health, and longevity, which have been especially associated with the two main DER protocols: caloric restriction (CR) and IF (Horne et al., 2015; Vasconcelos et al., 2018). In CR protocol, calorie consumption is chronically decreased in 20–40% of the ad libitum intake, whilst IF involves a restriction in the frequency of food intake, giving periods of free food intake coupled to fasting periods. These protocols were shown to extend life- and health-span and to counteract numerous age-related diseases. The vulnerability of the CNS to age progression is frequently expressed in neurological disorders, such as AD, PD, and stroke (Martin et al., 2006; Logsdon et al., 2017).

Dietary energy restriction, as a mild stress of energy restriction to the organism, is considered a hormetic stimulus, which is defined as a low dose stressor that induces adaptive responses able to improve resistance to more severe stressors and diseases. Within this context, the energetic challenge to the brain induced by DER triggers beneficial outcomes, including neurogenesis, synapses strengthening, and new synapse formation (Calabrese et al., 2010; Hine and Mitchell, 2012; Horne et al., 2015). Molecularly, DER induces the activation of protective transcription factors, such as Nrf2, that activate the expression of Phase II detoxifying enzymes, thereby increasing neuronal resistance to oxidative stress and death, and hence lowering the risk of neurodegenerative disorders. DER, especially IF, upregulates genes encoding the antioxidant enzymes that are modulated by Nrf2, including GPx, SOD2, and HO-1 (Hine and Mitchell, 2012; Mattson, 2012).

Some of the benefits of CR protocol are associated with Nrf2/ARE pathway activation (Pearson et al., 2008; Martin-Montalvo et al., 2011; Bruns et al., 2015). One of the proposed mechanisms of DER utility is via a transient ROS increase to hormetic levels that may activate Nrf2 (Hine and Mitchell, 2012). For instance, fasting, and its consequential effects on insulin levels, results in a small, transient increase in oxidative stress, triggering activation of the Nrf2/ARE pathway and the upregulation of its target genes (Kim and Novak, 2007).

Various age-related diseases, including AD and PD, are linked to decreased Nrf2 activity and display symptom improvement after Nrf2 activation by DER (Hine and Mitchell, 2012). CR can counteract the age-related loss of cellular antioxidant defenses, partly by promoting the up-regulation of Nrf2/ARE-driven genes, including GST and NQO1, in a variety of body tissues and organs, including the liver and brain (Chen et al., 1994; Hyun et al., 2006). Furthermore, a recent study showed that 30% CR for 12 weeks can prevent neurotoxicity, oxidative damage, and cognitive impairment induced by acrolein. Acrolein has been proposed to be involved in AD etiology, with the efficacy of CR

mediated, at least partly, through the amelioration of acroleininduced depletion of hippocampal SOD levels, indicating a positive effect of this protocol on Nrf2 signaling (Huang Y.J. et al., 2018).

The age-induced Nrf2 dysfunction in BBB endothelial cells is also prevented by CR, shedding light in the cerebrovascular protective effects of this DER protocol (Csiszar et al., 2014). Importantly, these age-dependent endothelial alterations are thought to play a role in both vascular cognitive impairment and AD (Gorelick et al., 2011; Zlokovic, 2011; Lin et al., 2013). Accordingly, numerous substances proposed as "DER mimetics" (i.e., compounds shown to promote the beneficial effects of DER in the absence of food intake restriction), such as curcumin, resveratrol, and quercetin (Ingram et al., 2006), have been shown to increase longevity and slow down the aging process, at least in part via Nrf2 activation (Balogun et al., 2003; Chen et al., 2005; Hsieh et al., 2006; Tanigawa et al., 2007; Calabrese et al., 2010).

Several studies in murine and primate models of PD have demonstrated that DER can protect dopaminergic neurons, decrease motor dysfunction, and alleviate PD symptoms (Duan and Mattson, 1999; Maswood et al., 2004; Qiu et al., 2012; Griffioen et al., 2013), even when the DER protocol is initiated after the induction of PD by MPTP (Holmer et al., 2005). Published data also indicate that energy consumption profoundly impacts the progression of AD (Mattson, 2012), with DER decreasing brain Aβ accumulation in the APP transgenic murine model of AD (Patel et al., 2005; Wang et al., 2005) and aged primates (Qin et al., 2006). Furthermore, in a triple transgenic AD mice, both IF and CR protocols, when starting at 3 months of age (before symptoms onset), can counteract age-related cognitive impairment (Halagappa et al., 2007). Interestingly, in this study, CR, but not IF, reduced Aβ deposition in the brain. The authors suggest that the IF mechanism of action may involve the prevention of Aβ-mediated negative effects on cognitive function.

Glutathione is an important reducing agent of the phase II antioxidant response. Many genes involved in glutathione metabolism are modulated by Nrf2, including glutathione synthesis genes (GCLM, GCLC), GST, GPx, and GR. Several studies have reported an age-related disruption of the glutathione antioxidant system in rodents and humans, which may result in increased susceptibility to PD and AD (Rao et al., 1990; Samiec et al., 1998; Cho et al., 2003; Suh et al., 2004; Kennedy et al., 2005; Ballatori et al., 2009; Hine and Mitchell, 2012). Cho et al. (2003) showed that CR in rodents is able to prevent the age-related decrease in the levels of glutathione and glutathionerelated enzyme activities. Furthermore, many studies showed that GST and GPx levels and activities can be augmented by both fasting and CR (Leakey et al., 1989; Cho et al., 2003; Pearson et al., 2008; Mitchell et al., 2010; Vazquez-Medina et al., 2011). In one of these studies, these effects were evident in mice subjected to 30% CR for 2–4 weeks or short periods of fasting, following ischemia reperfusion injury (Mitchell et al., 2010). Results from the CALERIE Trial of Human Caloric Restriction also showed that GPx activity is increased by 10–30% CR over 6 months in overweight individuals (Meydani et al., 2011).

NQO1 is another important endogenous antioxidant defense enzyme modulated by Nrf2. Altered expression of NQO1 is correlated with many pathologies, including AD and PD (Lastres-Becker, 2017; Chhetri et al., 2018). Long term DR can increase NQO1 expression, resulting in enhanced antioxidant defenses in the brain and liver of aged rats (De Cabo et al., 2004; Hyun et al., 2006). This effect was also observed in studies using DER mimetics (Zhu et al., 2005; Higgins et al., 2009; Son et al., 2010).

DER and its mimetics can also counteract damage following ischemia reperfusion injury (Go et al., 1988; Khan et al., 2006; Saleh et al., 2010; Peng et al., 2012). After experimental stroke in rodents, 70 days of DER leads to a substantial attenuation of cognitive dysfunction as well as increasing hippocampal cell survival (Roberge et al., 2008). These effects seem, in part, to involve the Nrf2-triggering effect of DER (Mattson, 2012). Fasting for up to 4 days or 30% CR for 2–4 weeks results in augmented HO-1 expression and attenuates ischemic damage of the brain, liver, and kidney in rodents (Go et al., 1988; Mitchell et al., 2010; Verweij et al., 2011). Moreover, in vivo or in vitro data shows that DER mimetics, such as curcumin and plumbagin, also increase HO-1 expression and render rodents more resistant to acute stressors and oxidative damage (Farombi et al., 2008; Son et al., 2010). In one of these studies, plumbagin pre-treatment in a murine model of focal ischemic stroke led to the attenuation of brain injury and neurological deficits. These effects were attributed to Nrf2/ARE activation, given that Nrf2 knockdown prevents such neuroprotective effects (Son et al., 2010).

Current MS treatments have poor efficacy, both for symptom relief and disease progression (Lublin et al., 2014). Genetic risk factors do not fully explain the development of MS, with a number of environmental factors, including infections, smoking, low vitamin D levels and obesity, associated with increased incidence of MS (Ascherio, 2013). A number of studies show childhood/young adulthood obesity to be a risk factor for MS (Munger et al., 2009; Hedstrom et al., 2012, 2014; Langer-Gould et al., 2013). The chronic inflammatory state that is evident in obesity can promote autoimmunity though adipokine production (Calder et al., 2011). The effects of diet on the gut microbiome is thought to contribute to this, via the regulation of pro- and anti-inflammatory responses that regulate DC activation, MHC II presentation, and T cell differentiation in the gut (Goto et al., 2014; Furusawa et al., 2015). The gut microbiome in both RRMS patients and EAE models is altered compared to healthy controls (Chen et al., 2016a; Jangi et al., 2016). Several dietary habits such as high salt intake or long chain fatty acid intake, have been recently recognized as environmental contributors to the pathogenesis of MS and EAE, by expanding TH1 and TH17 cells (Berer et al., 2011; Kleinewietfeld et al., 2013).

Chronic CR has a potent anti-inflammatory effect, protecting against EAE symptoms (Piccio et al., 2008; Meydani et al., 2016). In these studies, CR reduced inflammation, demyelination, and neurodegeneration ameliorating relapsing-remitting EAE in SJL mice and progressive EAE in C57BL/6 mice. CR lowers plasma IL-6 concentration in the course of EAE accompanied by a decrease in leptin, suggesting CR-mediated alterations in the gut-brain axis and associated changes in gut-mediated inflammatory processes (Piccio et al., 2008). As leptin induces Th1 cell differentiation, such changes are likely to lower the levels of proinflammatory cytokine production (Matarese et al., 2005).

Accordingly, MS patients showed an increased concentration of leptin in the serum and cerebrospinal fluid, associated with reduced levels of CD4 (+) CD25+ regulatory T cells and augmented INF-γ release (Matarese et al., 2005).

Recent studies indicate that MS show characteristics of metabolic disease, with SPMS patients showing elevated peroxisomal metabolites (PlsEtn) and increased mitochondrial stress metabolites (VLCFA-PtdEtn), when compared to control patients (Senanayake et al., 2015). Furthermore, SPMS patients showed reduced seric anti-inflammatory hydroxylated longchain fatty acids called gastro-intestinal tract acids (GTAs), suggesting a diminished protection against MS-related inflammation. In addition, the oxidative stress-induced mitochondrial dysfunction in MS may provide biomarkers for the susceptibility to, and progression of, MS.

Importantly, although Nrf2/ARE signaling plays a critical role in cellular detoxification responses and prevention of age-related diseases, excessive Nrf2 activation has deleterious consequences. This is supported by studies showing KEAP1 knockout to be lethal in mice as a consequence of excessive constitutive Nrf2 activation (Wakabayashi et al., 2003), with Nrf2 overexpression (DeNicola et al., 2011; Lister et al., 2011) or mutation of KEAP2 (Zhang et al., 2010) promoting tumorigenesis. Consequently, pharmacological activation of Nrf2 may be dangerous, with the physiological activation of Nrf2 by DER likely to be a safer alternative.

#### High Energy Consumption

The Western lifestyle are frequently more sedate with greater levels of over-eating, characterizing a condition of chronic positive energy balance (Martin et al., 2009; Mattson, 2012). In contrast to DER, high-energy consumption is associated with many negative impacts on overall health and longevity, resulting in increased morbidity and mortality (Maffei et al., 1995; Caro et al., 1996; Herrmann et al., 2001). Consequently, being overweight or obesity has reached epidemic proportions, being now classed as the fifth largest cause of death worldwide (Razay et al., 2006; Procaccini et al., 2016). In fact, the World Health Organization estimated that the global burden of obesity and overweight was over 300 million and 1 billion of adults, respectively (World Health Organization [WHO], 2011). Obesity, in turn, is an important risk factor for diabetes, a metabolic disease associated to chronic hyperglycemia and an array of other complications (Tebay et al., 2015). A HFD (typically 40–60% of total calorie intake from fat) in humans and animals results in an augmented vulnerability to an array of medical conditions, including many psychiatric disorders where risk positively correlates with BMI (Lopes et al., 2001; Degirmenci et al., 2015). High BMI in human subjects is also linked to reduced blood flow in brain regions important for cognitive function (Willeumier et al., 2011) and reduced brain integrity (Gazdzinski et al., 2008; Stanek et al., 2011). Even a short-term HFD, for only 7 days, can cause cognitive impairment in humans (Edwards et al., 2011).

Accumulating data clearly shows that obesity is a risk factor for cognitive decline, dementia and neurodegenerative diseases, such as AD and PD (Mazon et al., 2017). One of the mechanisms by which diet-induced obesity can lead to neurological disorders is through increased neuroinflammation and ROS production and the downregulation of endogenous antioxidant enzymes, resulting in increased oxidative damage to the CNS (Edwards et al., 2011; Matsuda and Shimomura, 2013; Guillemot-Legris and Muccioli, 2017; Mazon et al., 2017). Diet-induced obesity can also lead to neurological disorders through microglia activation and BBB disruption, which can trigger neuroinflammation and synaptic impairment, thereby resulting in cognitive decline and neurodegeneration (Pistell et al., 2010; Zlokovic, 2011; Knight et al., 2014; Tucsek et al., 2014) (**Figure 3**).

Nrf2 activation can improve energy expenditure and prevent weight gain in HFD mice (Shin et al., 2009). Moreover, Nrf2/HO-1 pathway activation can ameliorate long-term HFD-induced cognitive impairment, inflammation, as well as the accumulation of Aβ and hyperphosphorilated tau in the hippocampus (FangFang et al., 2017). In young subjects, Nrf2 can counteract the vascular oxidative damage and augmented ROS production associated with obesity (Ungvari et al., 2011a; Csiszar et al., 2015). However, aging is associated with Nrf2/ARE signaling pathway dysfunction, increasing the susceptibility of the elderly to obesity-driven oxidative stress. As oxidative stress is already exacerbated in these individuals by various other factors, the dysfunctioning of Nrf2 signaling will further aggravate this pro-oxidative scenario and contribute to the development of neuropsychiatric disorders in the aging brain (Morrison et al., 2010; Ungvari et al., 2011b,c).

Morrison et al. (2010) showed that 20-month old male mice fed a HFD composed of 60% fat, but not 41% fat, presented increased hippocampal oxidative stress and cognitive decline after 4 months, when compared to controls. These changes were associated with a decrease in Nrf2 levels and activity, suggesting that Nrf2 signaling impairment may be a mechanism of HFDinduced cognitive dysfunction in the aging brain.

Interestingly, numerous studies have demonstrated that DER can counteract the negative effects of HFD and promote a wide array of beneficial effects on the health of overweight individuals (Mattson, 2012).

A HFD can result in neuronal insulin resistance, a marker of diabetes, contributing to cognitive impairment. Moreover, AD risk is positively correlated to brain insulin resistance and diabetes (Pipatpiboon et al., 2012; Tucsek et al., 2014; FangFang et al., 2017). Studies in an AD murine model show that, unlike DER, HFD and T2D symptoms can aggravate Aβ deposition in the brain and contribute to cognitive impairment (Ho et al., 2004; Takeda et al., 2010). In an AD post-mortem study, insulin resistance positively correlated with Aβ plaques and negatively correlated with last recorded cognitive function (Talbot et al., 2012).

A recent study in triple transgenic AD mice showed that HFD (60% fat) for 4 months resulted in cognitive impairment, coupled to increased oxidative stress and neuronal cell death. These changes were attributed to suppressed Nrf2/ARE pathway activation along with reduced expression of the Nrf2 target genes

HO-1 and manganese SOD (MnSOD) (Sah et al., 2017). Tarantini et al. (2018) fed Nrf2 knockout mice a HFD (60% fat) for 5 months, with Nrf2 deficiency significantly increasing HFDinduced brain inflammation, oxidative stress, synaptic disruption and BBB permeability, as well as raising levels of amyloid precursor protein (APP) gene expression, the proteolysis of which produces Aβ. The authors concluded that Nrf2 dysfunction exacerbates the obesity-induced adverse effects in the brain and plays a role in vascular cognitive impairment and AD (Tarantini et al., 2018).

As with AD, calorie intake throughout life can influence the incidence of PD in some individuals (Mattson, 2012). Several lines of evidence have shown that diet, adiposity and T2D are linked to PD (Logroscino et al., 1996; Anderson et al., 1999; Johnson et al., 1999; Abbott et al., 2002; Hu et al., 2006, 2007; Morris et al., 2011; Schernhammer et al., 2011; Xu et al., 2011). All these factors are correlated to a HFD, given that over 80% of T2D patients are overweight or obese, whilst a HFD is frequently used as a model of diabetes (Centers for Disease Control and Prevention (CDC), 2004; Morris et al., 2011).

Many studies have shown that a HFD renders SN neurons more vulnerable to PD neurotoxins and increased ROS levels (Choi et al., 2005; Morris et al., 2010, 2011; Bousquet et al., 2012). In one of these works, mice fed a HFD or control diet for 5 weeks were treated with the PD-related neurotoxin 6-OHDA, with the HFD mice presenting with higher oxidative stress and neurodegeneration (dopamine depletion in the striatum and SN). These poorer outcomes were all correlated with heightened levels of adiposity and insulin resistance (Morris et al., 2010).

Innate and adaptive immune systems have a pivotal role in MS pathogenesis. Nrf2 agonists are promising candidates in the treatment of MS, since CDDO-Im 1-[2-cyano-3-,12 dioxooleana-1,9(11)-dien-28-oyl] imidazole, a Nrf2 activator, promoting the differentiation of the less inflammatory Th2 t cell phenotype in stimulated splenocytes of C57BL/6 mice, thereby lowering INFγ and TNFα production as well as NF-κB DNA binding (Zagorski et al., 2018). However, it has more recently been proposed that inflammatory processes are secondary to primary cytodegenerative processes in some neural cells, such as oligodendrocytes and neurons, with some studies strongly suggesting that alterations in mitochondrial functioning, and consequent increase ROS, can drive the initial neurodegeneration in MS (Witte et al., 2014). Furthermore, increased oxidative stress is correlated with decreased Complex IV electron transport chain gene expression and Nrf2 activity in the non-lesioned gray matter of the frontal and parietal cortex, from post-mortem MS and control groups, suggesting that mitochondrial function is correlated with, if not regulated by, Nrf2 in MS (Pandit et al., 2009).

Epidemiological studies also indicate a positive correlation between MS severity and fatty acids intake, with a long-term, higher level saturated fat consumption being associated with an increased frequency of MS as well as augmented EAE symptomatology (Schwarz and Leweling, 2005; Thompson, 2008; Winer et al., 2009). Moreover, overweight and obese 20-year olds

have a higher risk of developing MS compared to those of normal weight (Hedstrom et al., 2014).  Timmermans and colleagues showed an increased clinical score and proinflammatory genes (IL6, INFγ, and IL1β), as well as higher levels of infiltrating CD3+ T cells in the CNS of female animals subjected to a HFD. Similarly, Fernandez-Real and Pickup (Fernandez-Real and Pickup, 2008) showed an increase in inflammatory cytokines, soluble adhesion molecules, and chemokines in the blood of obese people. Accordingly, the Nrf2 activator, CDDO-IM, prevented the body weight gain in animals submitted to HFD, partly by regulating expression of fatty acid synthesis and oxidation enzymes in the liver (Shin et al., 2009).

As in MS, a HFD also is a risk factor for cardiovascular diseases and cerebral ischemia. Recent research has revealed an increase in the prevalence of acute ischemic stroke in children and young adults, correlated with such risk factors, as obesity and lipid disorders (George et al., 2011). Impairment in vascular function is evident in male Wistar rats submitted to 8-week HFD before transient middle cerebral artery occlusion (MCAO), with HFD significantly increasing not only body weight and adiposity as well as associated processes, but also augmenting the infarct size in rats (Li et al., 2013). Such data highlights how alterations in the regulation of energy intake are intimately linked to levels of Nrf2, in the regulation of susceptibility to, and severity of, a wide array of seemingly distinct medical conditions.

Interestingly, both HFD and lack of Nrf2 can increase infarct area after cerebral ischemia. Deutsch et al. (2009) demonstrated that HFD-rats submitted to cerebral ischemia by MCAO showed smaller lumens and thicker MCA walls, when compared to normal diet controls. This is attributed to increased expression of metalloproteinase-2 expression and collagen-1 deposition, suggesting impairment on neurovascular functions (Deutsch et al., 2009; Osmond et al., 2010; Li et al., 2013). In another study, the volume infarct of Nrf2-deficient mice subjected to MCAO for 90 min and 24 h of reperfusion was increased when compared to the control group (Shah et al., 2007). Intracerebral ventricular pre-administration of tert-butylhydroquinone reduced the infarct area in the MCAO mouse brain (Shih et al., 2005). Corroborating these findings, pharmacological agonism of Nrf2, by dimethyl fumarate, reduced NF-κB activation and protected the calcium-activated potassium (BK) channel-mediated coronary vasodilatation in HFD mice (Lu et al., 2017). These findings suggest that both Nrf2 and HFD have important roles in neurovascular and cerebral ischemia modulation.

It is also of note that dietary impacts on the composition of the gut microbiome may be of some relevance to the data presented above. There is considerable interest in the role of gut microbiome changes in the etiology, course and treatment of AD, PD, and MS (Anderson and Maes, 2017), mediated via changes in the gut-brain and gut-liver axes. By increasing gut permeability, gut bacteria and tiny fragments of partially digested food can trigger an immune reaction, with a wide array of consequences, including the possible production of α-synuclein in the gut, and its transport via neurons to the brain in the etiology of PD (Chen et al., 2018). Generally, an increase in the gut bacteria producing the small chain fatty acid, butyrate, is beneficial across a wide array of medical conditions. It is of note that butyrate increases levels of Nrf2 (Anderson et al., 2016), suggesting that some of the benefits of butyrate may be mediated not only by its histone deacetylase inhibitor capacity and its induction of melatonin, but also by its induction of Nrf2 (Dong W. et al., 2017).

## CONCLUSION

It is widely accepted that oxidative stress plays a central role in neurological disorders. This underpins the importance of targeting Nrf2 to counteract such oxidative stress and associated brain diseases (Patel, 2016). Dietary interventions such as DER protocols, in contrast to a HFD, can promote small energetic challenges to the brain that enhance Nrf2/ARE pathway activation (Mattson, 2012). Nrf2 up-regulates the expression of several pro-survival genes and counteracts oxidative damage to the CNS, thereby preventing neurodegeneration and obesityrelated brain disorders (Calkins et al., 2009). Hence, DER may be a valuable treatment option for brain disorders, including as adjunct therapy with other known Nrf2 activators. However, controversial reports showed that NRF2 activation can result in drug resistance and oncogenic effects (Sporn and Liby, 2012). Thus, additional large-scale studies are warranted to further explore the effects of dietary interventions in Nrf2/ARE signaling and to establish the best dietary protocols for humans to optimize the beneficial effects of Nrf2 for the prevention and/or early intervention in the etiology and course of neurological disorders.

## AUTHOR CONTRIBUTIONS

AV, NdS, CS, and CM contributed to the design of the manuscript, literature review, writing of the manuscript, and creation of the figures.

### FUNDING

This publication was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2012/24727-4 and #2016/07427-8), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq #160570/2012- 3), and University of São Paulo (USP).

## ACKNOWLEDGMENTS

The authors would like to thank George Anderson of CRC Scotland & London for English editing services.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Vasconcelos, dos Santos, Scavone and Munhoz. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Critical Role of Nrf2 in Experimental Ischemic Stroke

Lei Liu<sup>1</sup> , Logan M. Locascio<sup>1</sup> and Sylvain Doré1,2 \*

<sup>1</sup> Department of Anesthesiology, Center for Translational Research in Neurodegenerative Disease and McKnight Brain Institute, University of Florida, Gainesville, FL, United States, <sup>2</sup> Departments of Neurology, Psychiatry, Pharmaceutics, and Neuroscience, University of Florida, Gainesville, FL, United States

Ischemic stroke is one of the leading causes of death and long-term disability worldwide; however, effective clinical approaches are still limited. The transcriptional factor Nrf2 is a master regulator in cellular and organismal defense against endogenous and exogenous stressors by coordinating basal and stress-inducible activation of multiple cytoprotective genes. The Nrf2 network not only tightly controls redox homeostasis but also regulates multiple intermediary metabolic processes. Therefore, targeting Nrf2 has emerged as an attractive therapeutic strategy for the prevention and treatment of CNS diseases including stroke. Here, the current understanding of the Nrf2 regulatory network is critically examined to present evidence for the contribution of Nrf2 pathway in rodent ischemic stroke models. This review outlines the literature for Nrf2 studies in preclinical stroke and focuses on the in vivo evidence for the role of Nrf2 in primary and secondary brain injuries. The dynamic change and functional importance of Nrf2 signaling, as well as Nrf2 targeted intervention, are revealed in permanent, transient, and global cerebral ischemia models. In addition, key considerations, pitfalls, and future potentials for Nrf2 studies in preclinical stroke investigation are discussed.

#### Edited by:

Javier Egea, Institute of Health Research of the University Hospital of La Princesa, Spain

#### Reviewed by:

Alexander A. Mongin, Albany Medical College, United States Siew Hua Gan, Monash University Malaysia, Malaysia

\*Correspondence:

Sylvain Doré<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Inflammation Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 26 October 2018 Accepted: 08 February 2019 Published: 05 March 2019

#### Citation:

Liu L, Locascio LM and Doré S (2019) Critical Role of Nrf2 in Experimental Ischemic Stroke. Front. Pharmacol. 10:153. doi: 10.3389/fphar.2019.00153 Keywords: antioxidant response element, global cerebral ischemia, middle cerebral artery occlusion, permanent MCAO, preclinical models, reperfusion, stroke, transient MCAO

### INTRODUCTION

Ischemic stroke is one of the leading causes of death and long-term disability worldwide (Benjamin et al., 2018), an event which is more disabling than it is fatal. In the U.S. alone, ∼795,000 people each year suffer a new or recurrent stroke, of which 87% are ischemic (Dirnagl et al., 2003; Feigin et al., 2017; Benjamin et al., 2018). However, effective clinical approaches are still limited. A successful therapeutic strategy for salvaging ischemic brain tissue and promoting functional outcomes is to quickly restore blood flow during acute ischemic stroke by introducing intravenous recombinant tissue plasminogen activator (rtPA), which has been available since 1996 (Prabhakaran et al., 2015; Romano and Sacco, 2015), in combination with thrombectomy. However, more than 95% of patients do not benefit from rtPA due to the narrow therapeutic window (4.5 h) and limited indications (Hacke et al., 2008; Fonarow et al., 2011; Sandercock et al., 2012; Emberson et al., 2014). In addition, there is no efficacious treatment that exhibits long-term recovery improvement. Therefore, development of new therapeutic strategies by targeting vital cellular components of the ischemic cascade is urgently needed.

The transcriptional factor Nrf2 is a major regulator of cellular and organismal defense mechanism against endogenous and exogenous stresses by coordinating basal and stress-inducible activation of multiple cytoprotective genes (Leonardo and Doré, 2011; Suzuki et al., 2013; Hayes and Dinkova-Kostova, 2014; Tonelli et al., 2017; Cuadrado et al., 2018; Yamamoto et al., 2018). Since its discovery in 1994 (Moi et al., 1994), Nrf2 biology has been extensively studied in understanding the structure, molecular mechanism, function, regulation of Nrf2 activity, downstream pathways and implications as a therapeutic target of diseases (Ma, 2013; Suzuki et al., 2013; Kumar et al., 2014; Cuadrado et al., 2018; Yamamoto et al., 2018). It is now recognized as a master regulator of redox homeostasis through the control of a wide array of target genes that share a common DNA sequence called the antioxidant response element (ARE) in the promoter region ensuring that its activity increases in response to redox perturbation, energy or nutrient fluxes, inflammation, toxicity, and disease conditions (Ma, 2013; Cuadrado et al., 2018; Yamamoto et al., 2018). The equivalent response element in mice and rats is called the electrophile response element (EpRE) (Friling et al., 1990; Wasserman and Fahl, 1997; Itoh et al., 2010; Yamamoto et al., 2018). Besides mediating antioxidant responses, Nrf2 contributes to the regulation of many primary and secondary metabolic processes (Hayes and Dinkova-Kostova, 2014; Tonelli et al., 2017). Consequently, targeting Nrf2 has emerged as an attractive therapeutic strategy for stroke prevention and reversal (Calkins et al., 2009; Leonardo and Doré, 2011; Wang Y. C. et al., 2011; Ma, 2013; Kumar et al., 2014; Tonelli et al., 2017).

This paper outlines the current understanding of the Nrf2 regulatory network and critically examined the recent evidence for the contribution of Nrf2 pathway in experimental ischemic stroke models. It includes extensive literature review for Nrf2 studies in experimental ischemic stroke mouse and rat models published by June 30, 2018 with a special focus on in vivo evidence for the role of Nrf2 in ischemic brain injury. We summarized the dynamic regulation of Nrf2 signaling, functional importance, and its targeted intervention in permanent, transient, and global cerebral ischemia preclinical models. Finally, we also assessed key considerations, pitfalls, and the potential for future Nrf2 studies in stroke investigation.

### OVERVIEW OF THE NRF2 REGULATORY NETWORK

### A Brief History

Nrf2 is widely expressed in mammalian cells. In 1991, a study in the field of toxicology revealed that oxidative stress activates antioxidant genes through the antioxidant-response element (ARE), a cis-acting regulatory element that contributes to cellular defense in eukaryotes (Rushmore et al., 1991; Hayes and Dinkova-Kostova, 2014). In 1994, Nrf2 was firstly reported in the molecular biology field as an NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region (Moi et al., 1994; Raghunath et al., 2018). However, its biological function was unclear. In 1996, the first study on Nrf2 knockout mice showed that no overt abnormal phenotype was detected although the mice were susceptible to stresses (Chan et al., 1996). In 1997, a landmark study illustrated that the induction of two ARE-driven genes, glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase-1 (NQO1), was abolished in the Nrf2 knockout mice by the phenolic antioxidant butylated hydroxyanisole (BHA), revealing that Nrf2 controls drug-metabolizing enzymes in vivo (Itoh et al., 1997). Recently, series of breakthrough studies have revealed that Nrf2 coordinately regulates a wide array of antioxidant response element/electrophile responsive element (ARE/EpRE)-driven genes that play critical roles in controlling endogenous resistance to various intrinsic and extrinsic stressors (Ma, 2013; Suzuki et al., 2013; Hayes and Dinkova-Kostova, 2014; Tonelli et al., 2017; Yamamoto et al., 2018).

### Structure Domain of Nrf2

Nrf2 belongs to the cap'n'collar subfamily of the basicregion leucine zipper (CNC-bZIP) transcription factors. It is a modular protein composed of seven functional domains, Nrf2-ECH homology (Neh) domains 1–7, which have distinct functions (**Figure 1A**) (Chan et al., 1993; Katoh et al., 2005; Hayes and Dinkova-Kostova, 2014; Tonelli et al., 2017). The Neh1 domain contains the well-conserved CNC-bZIP region that heterodimerizes with small musculoaponeurotic fibrosarcoma oncogene (sMaf) proteins and binds ARE/EpRE sequence in DNA. This CNC-bZIP region, which can be found in several species, is vital for Nrf2 function. The N-terminal Neh2 is the domain by which Nrf2 binds to its primary negative regulator Keap1 through its low-affinity DLG and the high-affinity ETGE motifs. The C-terminal Neh3 region represents a transactivation domain that binds to chromo-ATPase/ helicase DNA-binding protein (CHD) 6 and activates transcription of Nrf2 target genes in concert with the Neh4 and Neh5 domains. The Neh4 and Neh5 regions of Nrf2 are transactivation domains that recruit the cAMP response element-binding protein (CREB)-binding protein (CBP) and/or receptor-associated coactivator (RAC) 3. The Neh6 domain, independent of Keap1, mediates the repression of Nrf2 stability with another negative regulator the dimeric β-transducin repeat-containing protein (β-TrCP), a substrate adaptor for the S-phase kinase-associated protein 1 (Skp1)–Cul1–Rbx1 core E3 complex, through DSGIS and DSAPGS motifs. The Neh7 domain is involved in the negative control of Nrf2 by physically binding to the retinoid X receptor (RXR).

### Overview of the Nrf2 Pathway

Organisms are equipped with a defense system to maintain homeostasis against constant intrinsic and extrinsic insults that result in the damage of nucleic acids, proteins, and membrane lipids. Nrf2 is a master regulator of the inducible cell defense system by controlling a broad range of cytoprotective genes (Hayes and Dinkova-Kostova, 2014; Suzuki and Yamamoto, 2015). Under basal homeostatic conditions, Nrf2 in the cytoplasm is predominately bound to the Kelch-like ECHassociating protein 1 (Keap1) through the Keap1–Cullin3 (Cul3)-Rbx1 ubiquitin E3 ligase complex and is constitutively degraded by the proteasome. Cul3 serves as a scaffolding protein that is bound to both Keap1 and Rbx1. As a result, Nrf2 abundance and activity are maintained at low levels (**Figure 1B**). Under stress conditions, Nrf2 protein is liberated from Keap1 mediated repression. The stabilized and accumulated Nrf2

translocates into the nucleus and, as a heterodimer with one of the small Maf proteins, binds to the ARE/EpRE in the promoter region of Nrf2 target genes, thus activating a wide array of cytoprotective genes (Ma, 2013; Kumar et al., 2014; Suzuki and Yamamoto, 2015; Taguchi and Yamamoto, 2017; Bellezza et al., 2018; Yamamoto et al., 2018).

It is becoming increasingly clear that Keap1 plays a central role in the regulatory mechanism of Nrf2. (1) Keap1 acts as a sensor of oxidative and electrophilic stresses for Nrf2 with a subcellular localization in the perinuclear cytoplasm (Suzuki and Yamamoto, 2017; Yamamoto et al., 2018). (2) Keap1 is a major repressor of Nrf2 through the activity of the Keap1–Cul3 complex. It is an adaptor protein for Cul3-dependent ubiquitination and specifically targets Nrf2 (Itoh et al., 1999, 2003; Kobayashi et al., 2004), thereby marking Nrf2 protein for rapid degradation by the ubiquitin– proteasome system. Under normal conditions, Nrf2 undergoes constitutive ubiquitination by the Keap1-Cul3 complex and resultant proteasomal degradation, leading to its low level of activity. Exposure to cellular insults like electrophiles or reactive oxygen species (ROS) brings activity of the E3 ubiquitin ligase Keap1-Cul3 complex to a halt, inhibiting Nrf2 ubiquitination and enabling Nrf2 protein stabilization and transcriptional activation. (3) Keap1 serves as a "floodgate" for Nrf2: Normally it functions to suppress Nrf2 nuclear translocation in response to cellular insults, the gene promotes translocation of Nrf2 into the nucleus to activate cytoprotective target gene expression. Keap1 has a relatively long half-life of 12 h compared to Nrf2's half-life of 20 min, contributing to its importance in the Nrf2 regulatory network.

## Target Genes and Functions of Nrf2

The Nrf2-Maf complex binds to the specific promoter of its target genes. In the past decade, more than 200 target genes of Nrf2 have been identified through the analyses of gene expression profiling and chromatin immunoprecipitation (ChIP) (Suzuki et al., 2013; Kumar et al., 2014; Suzuki and Yamamoto, 2017; Rojo de la Vega et al., 2018). The Nrf2 target ARE-driven genes have been defined in encoding proteins for functional regulation of a wide range of biological processes including detoxification, antioxidation, anti-inflammation, NADPH regeneration, and intermediary metabolisms (**Figure 1B**). Nrf2 exerts multiple defense processes counteracting various stresses by the induction of these genes. Consequently, Nrf2 plays a fundamental role in maintaining the redox homeostasis of the cell.

### Target Genes and Functions of Nrf2—Detoxification of Xenobiotics

Xenobiotics, including toxic chemicals and drugs, are detoxified by various drug-metabolizing enzymes and transporters. This detoxification mechanism is divided into three phases (Croom, 2012; Raghunath et al., 2018). Phase I enzymes mainly consist of cytochrome P450 oxidases that modify the xenobiotics through oxidation, reduction, or hydrolysis. Phase II conjugating enzymes include glutathione S-transferases, which catalyze the conjugation of reactive electrophile species with glutathione (GSH), attenuating the toxic potential of xenobiotics. Phase III transporters eliminate the GSH conjugates and offer protection against deleterious chemicals. It has been demonstrated that Nrf2 controls the expression of many drug-metabolizing enzymes [like distinct GST subunits and [NAD(P)H:quinone oxidoreductase 1) NQO1] and transporters [(drug-resistanceassociated proteins (MRP)].

### Target Genes and Functions of Nrf2—Antioxidant

A major function of Nrf2 is to resist oxidative stress, thereby maintaining redox homeostasis between reactive oxidants and endogenous antioxidant systems. Groups of Nrf2 targeted genes that regulate antioxidant defense and oxidant signaling have been defined. (1) Glutathione (GSH)-based: glutamate–cysteine ligase catalytic (GCLC) subunit and glutamate-cysteine ligase modifier (GCLM) control the entry of cystine into cells; glutathione peroxidase (GPX) 2 produces oxidized glutathione (GSSG) during the reduction of peroxides; and glutathione reductase (GSR) 1 reduces GSSG for maintenance of reduced intracellular GSH levels. (2) Thioredoxin (TXN)-based: Thioredoxin (TXN) 1, thioredoxin reductase (TXNRD) 1, and sulfiredoxin (SRXN) 1 reduce oxidized protein thiols. (3) Others: G6PDH and 6PGD reduce synthesis of NADPH, antioxidant protein Trx, stress response protein heme oxygenase 1 (HO1), and other proteins. Nrf2 coordinately regulates key components in the antioxidant system that precisely controls antioxidant defense at multiple levels, thus ensuring an adequate response to oxidants in time and space (Lin and Beal, 2006; Ma, 2013).

#### Target Genes and Functions of Nrf2—Anti-inflammation

Besides protecting against xenobiotic and oxidative insults, Nrf2 plays a role in anti-inflammation, which is supported by several recent findings (Kobayashi et al., 2016; Suzuki and Yamamoto, 2017). Several findings support the anti-inflammatory effect. The absence of Nrf2 leads to exacerbated inflammation in different murine models. Attenuated inflammation by Nrf2 is related to the inhibition of the nuclear factor-κB (NF-κB) pathway and proinflammatory cytokine level (Wardyn et al., 2015). Most recently, it was demonstrated that Nrf2 suppressed lipopolysaccharide (LPS)-induced inflammation by blocking the transcriptional upregulation of proinflammatory cytokines, including IL-6 and IL-1β. ChIP-seq and ChIP-qPCR analyses revealed that Nrf2 binds near the promoter region of these genes, and this Nrf2-mediated inhibition is independent of the Nrf2-binding motif and ROS level (Ahmed et al., 2017).

### Target Genes and Functions of Nrf2—Heme and Iron Metabolism

Nrf2 also modulates the key components of heme and iron metabolism. HO1 catalyzes the cleavage of heme (which cannot be recycled) to form iron, carbon monoxide (CO), and biliverdin, which is immediately reduced to bilirubin (Doré and Snyder, 1999; Doré et al., 1999). HO1 is considered to be a stress protein that can be highly induced in a cell-specific manner by numberous conditions (Ewing et al., 1992). Iron is intimately linked to oxygen by acting as an essential nutrient cofactor in enzymes for oxygen transport, oxidative phosphorylation, and metabolite oxidation. However, excess labile iron—not bound to ferritin—would facilitate the formation of oxygenderived free radicals capable of damaging biomolecules (Rouault, 2013; Kerins and Ooi, 2017). The biological utilization of iron is a tightly regulated process. Constitutive Nrf2 activation and subsequent deregulation of iron metabolism have been implicated in cancer development. Nrf2-mediated upregulation of HO1 or the iron storage protein ferritin can lead to enhanced proliferation and therapy resistance.

### Target Genes and Functions of Nrf2—NADPH Regeneration and Other Metabolic Processes

NADPH is an essential cofactor for many drug-metabolizing enzymes and antioxidants. Nrf2 controls NADPH production by regulating key NADPH-generating enzymes, such as glucose-6-phosphate dehydrogenase (G6pd), 6-phosphogluconate dehydrogenase (Pgd), isocitrate dehydrogenase 1 (Idh1), and malic enzyme 1 (Leonardo and Doré, 2011; Dinkova-Kostova and Abramov, 2015). In addition, Nrf2 controls the expression of multiple metabolic enzymes, indicating its unique role between redox and intermediary metabolism. Nrf2 also regulates fatty acid oxidation and lipases that influence lipid metabolism and transcription factors (Hayes and Dinkova-Kostova, 2014).

### Regulatory Mechanisms of Nrf2

Besides the predominant view that Nrf2 activity is mainly regulated at the protein stability level by Keap-mediated repression, accumulated evidence show that Nrf2 activity is tightly controlled at the transcriptional and post-translational levels during both basal and stressful conditions (Hayes and Dinkova-Kostova, 2014; Tonelli et al., 2017; Yamamoto et al., 2018). Given the central role of Nrf2 in cellular defense, the regulatory mechanisms of Nrf2 activity are intricate and multifaceted.

### Regulatory Mechanisms of Nrf2–Transcriptional Regulation

Nrf2 mRNA is broadly expressed across different species (Ma, 2013; Tonelli et al., 2017; Yamamoto et al., 2018), and its basal levels vary within different organs. Nrf2 activity is at least partially regulated at the transcriptional level**.** (1) The rodent Nrf2 gene comprises two ARE-like sequences in its promoter region and, in response to electrophilic stimuli, appropriately autoregulates its own expression level in concert with sustaining the induction of ARE-driven genes, offering a positive feedback mechanism to augment the Nrf2 signal. In contrast, the ARElike sequences in the human Nrf2 gene promoter region are reportedly associated with reduced expression of Nrf2, which enhances vulnerability to lung cancer. (2) The Nrf2 gene promoter comprises a binding site for NF-κB, which can be induced by environmental stress, injury or inflammation. Indeed, Nfe2l2 transcription level in human monocytes is indeed activated by LPS-induced inflammation. (3) It was reported that Nfe2l2 transcription in tumor cells can be amplified by the Notch signaling pathway and the phosphoinositide 3-kinase (PI3K)- Akt pathway, ultimately contributing to the increased expression of Nrf2.

### Regulatory Mechanisms of Nrf2—Post-transcriptional Regulation

A microRNA (miRNA) is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. At least eight miRNAs have been identified as direct modulators of Nrf2 expression at the transcriptomic level (Ayers et al., 2015). The miR-144 was identified to negatively regulate Nrf2 level in reticulocytes of homozygous sickle cell disease (HbSS) patients (Bryan et al., 2013; Kurinna and Werner, 2015) (140). Increased miR-144 is associated with the reduction of both Nrf2 level and GSH regeneration as well as impeded oxidative stress defense.

#### Regulatory Mechanisms of Nrf2—Post-translational Regulation in Nrf2 Protein Stability

#### Keap1-Dependent Repression Mechanism

(1) The prevailing view is that Keap1 is a primary repressor of Nrf2. Under basal conditions, Keap1 constantly targets Nrf2 for ubiquitination and proteasomal degradation, resulting in the disruption of Nrf2 protein stability and maintenance of Nrf2 signaling capacity at a very low level. In response to electrophiles or stressors, Nrf2 is liberated from the Keap1 repression (i.e., derepression) by inhibiting ubiquitylation and proteasomal degradation that allows newly-synthesized Nrf2 to be rapidly stabilized and activates transcriptional activation of cytoprotective genes. This view is supported by many reports of Keap1 knockout mice and Keap1 knockdown human cells that exhibited sufficient Nrf2 activity (Kensler et al., 2007; Yates et al., 2009; Bellezza et al., 2018; Yamamoto et al., 2018). Blockage of Keap1 expression by miR-141 or miR-200a triggers increased Nrf2 activity at the protein stabilization level. Transgenic complementation rescue assay is a comprehensive and powerful approach to delineate the in vivo functions of proteins (Motohashi et al., 2011; Katsuoka and Yamamoto, 2016). The esophagus lesions and resultant mortality in Keap1 knockout mice were fully retrieved by concurrent Nrf2 disruption, and aberrant phenotypes caused by Keap1 deficiency can also be rescued by transgene-derived Keap1. Epigenetic silencing of the Keap1 gene by hypermethylation of its promoter results in upregulation of Nrf2 in patients with gliomas and different cancers of breast, lung, prostate and colorectal (Tonelli et al., 2017). (2) The function of Keap1 within various electrophiles is related primarily to three critical cysteine residues: Cys151, Cys273, and Cys288 (Yamamoto et al., 2018). Keap1 is a thiolrich protein with many cysteine residues and thus is sensitive to electrophiles (McMahon et al., 2010). Therefore, covalent modifications of the cysteine residues had been proposed to influence the function of Keap1, which releases Nrf2 (McMahon et al., 2010). Affirmatively, covalent binding of electrophiles to critical cysteine residues has been observed. Meanwhile, various lines of studies in cultured cells, zebrafish, and mice demonstrate that Keap1 functions as a sensor due to the prominent contribution of the cysteine residues. Multiple studies involving substitution of Cys151 strongly support its role as a main cysteine sensor by modulating the activity of the ubiquitin E3 ligase Keap1-Cul3 complex to respond to different electrophiles. (3) To activate Nrf2 by inhibiting Keap1 activity: Many ARE activity inducing agents are soft electrophiles that inhibit Keap1 by modifying Cys151, Cys226/Cys613, Cys-273/Cys-288, or Cys-434. Thus, the presence of multiple Cys-based sensors in Keap1 allows Nrf2 to be de-repressed in response to many xenobiotic stressors. (4) To activate Nrf2 by competitive inhibition of Keap1: p62, the first mammalian selective autophagy cargo receptor (Chu, 2018), is able to directly interact with the Nrf2- binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2 (Komatsu et al., 2010; Lau et al., 2010). Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in Nrf2 stabilization and transcriptional activation of Nrf2 target genes. Additionally, p62 is a target gene of Nrf2 and creates a positive feedback loop by inducing ARE-driven gene transcription (Jain et al., 2010). Oxidative stress-triggered Nrf2 results in the induction and accumulation of p62 that in turn activates Nrf2 activation (Komatsu et al., 2010).

#### Keap1-Nondependent Repression Mechanism

(1) The Nrf2 is has been described to be suppressed by b-TrCP and GSK-3; in addition to the well-studied Keap1, E3 ubiquitin ligase adaptor β-TrCP is another negative repressor of Nrf2 stabilization (Rada et al., 2012). Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity (Chowdhry et al., 2013). Intraperitoneal injection of the GSK-3 inhibitor through the GSK-3/β-TrCP axis led to increased Nrf2 and HO1 levels in liver and hippocampus (Rada et al., 2012). Activation of the GSK3β/β-TrCP axis by gene knockdown of PHLPP1 in NRK52E cells enhanced Nrf2-responsive antioxidant enzymes HO1 and NQO1 (Mathur et al., 2018). (2) The Nrf2 is suppressed by CRIF1 and RNF4: CR6-interacting factor 1(CRIF1) physically interacts with both N- and C-terminal regions of Nrf2 and promotes Nrf2 ubiquitination and subsequent proteasomemediated Nrf2 protein degradation (Kang et al., 2010). Crif1 knockdown BMMSCs caused increased oxidative stress and apoptosis after irradiation injury, partially due to a suppressed antioxidant response mediated by decreased Nrf2 nuclear translocation (Chen L. et al., 2017). RING finger protein 4 (Rnf4) mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in promyelocytic leukemia-nuclear bodies (Malloy et al., 2013). Respiratory syncytial virus-induced Nrf2 degradation occurs in a SUMO-specific E3 ubiquitin ligase—RING finger protein 4 (Rnf4)-dependent manner.

#### CEREBRAL ISCHEMIA AND RELEVANT PRECLINICAL ANIMAL MODELS

Ischemic stroke is caused by cerebral arterial occlusion that leads to a critical reduction or loss of regional cerebral blood flow, and it occurs as a consequence of multiple vascular diseases, including cardioembolism, atherosclerosis, small vessel disease, and cryptogenic diseases (Adams and Biller, 2015; Mehndiratta et al., 2015). Interruption of the blood supply initiates complex spatial and temporal events involving hemodynamic, biochemical, and neurophysiologic alterations that ultimately lead to a pathological disturbance and a wide range of clinical symptoms (Lo et al., 2003; Iadecola and Anrather, 2011). The severity and temporal evolution of ischemic injury depend on the extent of cerebral blood flow (CBF), localization, duration of ischemia and coexisting systemic diseases among individual patients (Martini and Kent, 2007; Bang et al., 2008; Shen and Duong, 2008; Liebeskind, 2010).

Experimental ischemic stroke models are indispensable to our understanding of the events occurring in the ischemic and reperfused brains, enabling us to elucidate the pathophysiological mechanisms of ischemic brain injury and develop novel therapeutic treatments (Bosetti et al., 2017; Quinn et al., 2018). The vast majority of stroke models are carried in rats or mice because they present clear advantages of lower cost, resemblance to human cerebrovascular anatomy and physiology, and reproducibility of studies (Durukan and Tatlisumak, 2007; Durukan et al., 2008). In addition, genetic modification is usually applied in mice to illuminate molecular pathophysiological conditions like stroke. Cerebral ischemia in humans is divided into two categories: focal and global. Focal cerebral ischemia occurs when CBF is disrupted within a specific brain region, whereas global cerebral ischemia occurs when CBF is blocked throughout most or all of the brain. Given that ischemic stroke in humans occurs mostly in the territory of the middle cerebral artery (MCA), experimental focal cerebral ischemia models, including permanent and transient types, have served as the most widely used tool in the stroke research field (Dorr et al., 2007; Mehta et al., 2007). In focal cerebral ischemia, there is no blood flow at the infarct core, but there is usually a gradient of blood flow from the inner core to the neighboring ischemic area due to the collateral circulation. In global cerebral ischemia, global blood flow is completely stopped or remarkably reduced, depleting the energy supply and hindering cerebral metabolism and function. The reperfusion of blood flow effectively treats acute stroke; however, it can also exacerbate tissue damage and limit the recovery of function. Oxidative stress plays a key role in the pathogenesis of cerebral ischemia-reperfusion injury.

(1) Permanent cerebral ischemia (pdMCAO and pMCAO): Technically, the MCA can be selectively occluded at a distal or proximal site, referred to as permanent distal MCA occlusion (pdMCAO) or permanent (proximal) MCA occlusion (pMCAO) cerebral ischemia model, respectively. The pdMCAO model produces highly reproducible ischemic cortical lesions that are predominantly restricted to the barrel regions of cortex, inducing definable sensorimotor deficits that closely mimics ischemic stroke in humans. Thus, it is believed to be one of the most predictable and useful stroke models, allowing researchers to look at long-term recovery with high survival rates (Doyle and Buckwalter, 2014). Permanent cerebral ischemia can also be generated by the intraluminal suture method used in MCAO (pMCAO).

(2) Transient focal cerebral ischemia (tMCAO): This model is easy to perform in a controlled manner. The intraluminal suture MCAO model in rats and mice is the most frequently used model. This model exhibits reproducible MCA region infarctions that depend on the shape, size, and insertion length of the thread, allowing reperfusion by retracting the suture. MCAO generates ischemic cell death in the striatum and overlying the frontal, parietal, temporal, and portions of the occipital cortex. MCAO also precipitates variable damage in the thalamus, cervicomedullary junction, substantia nigra, and hypothalamus. Ischemic brain injury widely affects diverse brain regions and leads to complex motor, sensory, autonomic, and cognitive deficits (Carmichael, 2005). Usually ischemia must be induced for 60–120 min to obtain reproducible infarct volumes in transient focal ischemia models. In contrast, focal ischemia for more than 3 h precludes reversibility (Kasner and Grotta, 1998; Sicard and Fisher, 2009).

(3) Global cerebral ischemia (GCI): global cerebral ischemia (GCI) during cardiac arrest results in selective and delayed neuronal death of pyramidal neurons in the hippocampal CA1 region, similar to the situation in humans, and consequent cognitive decline (Traystman, 2003; Ostrowski et al., 2016).

### ROLE PLAYED BY NRF2 DURING CEREBRAL ISCHEMIA

In recent years, studies reported findings concerning the dynamic change of Nrf2 signaling, its functional importance, and its targeted intervention in cerebral ischemia. These findings provide insights into whether, when and how Nrf2 functions during brain injury. Accordingly, we mainly focused on the following questions. (1) What is the dynamic regulation of the Nrf2 signaling following cerebral ischemia? (2) Does the evidence from Nrf2−/<sup>−</sup> mice support the functional importance of Nrf2 during ischemic injury? (3) Whether Nrf2 induction is protective against ischemic injury and is facilitative for recovery? Specific focus is given to the in vivo evidence in different rodent cerebral ischemia models. Then, the pitfalls and concerns of current Nrf2 experimental ischemic stroke studies are discussed, which would be valuable for future studies.

### THE DYNAMIC REGULATION OF THE NRF2 SIGNALING DURING CEREBRAL ISCHEMIA

Normally, Nrf2 is largely localized in the cytoplasm and is maintained at a low basal level due to its binding affinity to Keap1. However, when cells are exposed to excessive oxidative stimuli during cerebral ischemia, Nrf2 is liberated from Keap1, translocates into the nucleus, and binds to the ARE sequence, thereby upregulating the expression of its target genes, which code cytoprotective proteins like anti-oxidative enzymes. In recent years, studies have provided substantial in vivo evidence of dynamic alternation of Nrf2 expression, as well as its target genes, and cellular and subcellular distribution of Nrf2 during different stages of cerebral ischemia. These findings, utilizing focal ischemia models with or without reperfusion in addition to global ischemia models, help us to identify the role of the Nrf2 regulatory network in the context of cerebral ischemia.

A number of studies with permanent cerebral ischemia models (pdMCAO and pMCAO) investigated the Nrf2/ARE pathway in response to ischemic insults (**Table 1**). Several permanent cerebral ischemia studies showed that, at 24 h after pMCAO, the protein expression levels of Nrf2 (total and nuclear) and its target antioxidant genes HO1 and SOD were upregulated in the ischemic cortex of mice or rats (Chang et al., 2013; Zhang J. et al., 2014; Zhao et al., 2014). The obvious increase of Nrf2 downstream antioxidant proteins can be sustained for at least 3 days following ischemia, revealed by the 1.8- to 3.6-fold increase in HO1, NQO1, SOD2, and GPx proteins after pdMCAO (Liu et al., 2018). The inflammatory factors IL-1β and IL-6 were dramatically increased in mouse brains for at least 48 h after pdMCAO (Clausen et al., 2017).  Minimal Nrf2 and HO1 positive cells were detected in the sham group, indicating low baseline levels of Nrf2 and HO1 in the nonischemic cortex areas, levels which increased significantly after pMCAO. Such signals were detected in neurons, astrocytes, and microglia, indicating concurrent expression of Nrf2 and HO1 in most cortical cells. On the contrary to above data, Nrf2 was reported to be significantly decreased at the mRNA level 6 h after pdMCAO, whereas the overall protein level was comparable to that under basal conditions (Clausen et al., 2017). A study reported a significantly decreased level of Nrf2 protein along with an increased level of antioxidant protein HO1 in rat cortex after pMCAO (Wang et al., 2018).

It appears that the majority of tMCAO studies support the activation of the Nrf2/ARE pathway in response to focal cerebral ischemia and perfusion (**Tables 2**, **3**). Because the ischemia-reperfusion injury measured by infarct volume is widely accepted to be most severe at 24 h following tMCAO, most studies investigated the Nrf2 pathway by Western blot (WB) or immunohistological analyses at that time point, with some extending to earlier (2–8 h) or later stages (3–14 d). In these studies, the Nrf2 pathway was examined in different ischemic brain tissues including the cortex, hemisphere, hippocampus, striatum, and cerebellum. The researchers selected 1–2 h ischemia when designing these tMCAO ischemia-reperfusion animal models. We summarized these studies together in order to provide an overview of this field. Most studies measured total Nrf2 level in the ischemic cortex or brain at 24 h after tMCAO (1–2 h); studies subjects showed Nrf2 upregulation at the mRNA level (Li et al., 2013; Guo et al., 2014) and protein level (up to 2– 3 fold higher) (Han et al., 2014; Li et al., 2015; Peng et al., 2015; Shi et al., 2015; Cai et al., 2017; Miao et al., 2018; Shang et al., 2018). Such an increase in Nrf2 protein might begin at 8 h (Cai et al., 2017) and be sustained over 3–14 days (Ding et al., 2015; Lin et al., 2016; Bai et al., 2017; Shang et al., 2018). Following nuclear translocation, Nrf2 protein accumulated in the nucleus (Li et al., 2013; Lv et al., 2017) and increased the Nrf2-binding activity to the ARE. Studies presented that the high nuclear Nrf2 protein level (Ding et al., 2015; An et al., 2018) and increased DNA binding activity of Nrf2 with the ARE can be observed, at 7 days after tMCAO (Li et al., 2014).  Several reports showed that the higher expression level of HO1 can continue over 2–14 days (Zhang M. et al., 2014; Ding et al., 2015; Lin et al., 2016). At 24 h after tMCAO, the total and nuclear Nrf2 proteins levels in the ischemic hippocampus also implied significantly higher expression levels (Shi et al., 2015; Lou et al., 2016), which may continue for at least 2 days. In addition, it was reported by immunohistochemistry that, Nrf2 levels in the peri-infarct regions began to show a significant increase at 2 h with a peak at 8 h of reperfusion after 1 h tMCAO, and its target antioxidative proteins such as thioredoxin, glutathione, and HO1 showed significant increases at 24–72 h (Tanaka et al., 2011). Another study presented that Nrf2 immunoreactivity was detectable in the neurons, endothelial cells, astrocytes, and microglia in the peri-infarct area at 7 days after 1 h tMCAO (Shang et al., 2018). A luciferase mouse model, a Keap1-dependent oxidative stress detector, was employed to visualize the time-dependent Nrf2 expression from brain ischemia onset through 7 days after tMCAO (Takagi et al., 2014; Nakano et al., 2017). The in vivo optical signals of Nrf2 expression were not detected in the earliest stages but peaked at 24 h after ischemia. Such Nrf2 expression was mainly detected in in the penumbra area, largely localizing inside neurons and astrocytes (Srivastava et al., 2013; Takagi et al., 2014). By quantifying Nrf2 labeled with green fluorescent protein (GFP), researchers have found that Nrf2 and HO1 exhibited higher protein levels in the ischemic cortex at 4– 48 h and striatum at 24 h after ischemia, which are consistent with the findings above. In contrast, some studies present the results that indicate suppression of the Nrf2/ARE pathway during tMCAO.  On the contrary to above findings, several reports found that no change was detected in the markers above (Wu L. et al., 2013; Zhao et al., 2016; Wu G. et al., 2017; Zhang W. et al., 2018).

The activation of the Nrf2/ARE pathway following global ischemia damage remains controversial (**Table 4**). This might be because of the various experimental factors like animal background, age, model, observation time points after ischemia, presence or absence of reperfusion, and site of the sample. At 24 h after GCI (2 vessel occlusion, 2VO), hippocampal Nrf2 content increased nearly 2-fold as determined by biochemical assay (Atef et al., 2018); striatal nuclear Nrf2 protein level and DNA binding activity of Nrf2 increased about 1.5-fold following reperfusion compared with the sham group; and there was notable upregulation in the detoxification and antioxidant proteins HO1, NQO1, GCLC, and GCLM (Ya et al., 2017). At 3 days after GCI (2VO), the total, cytoplasmic, and nuclear

TABLE 1 | Preclinical studies of Nrf2 in permanent

 cerebral ischemia models of mice and rats.

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1

Continued

protein levels of Nrf2 were upregulated in the hippocampal CA1 region of rats although the increase in nuclear Nrf2 was not significant (Chen B. et al., 2016), a finding which is supported by another report (Lei et al., 2016). No change in HO1 protein level was detected at 3 days following brain injury (Chen B. et al., 2016). An immunofluorescence-based study showed that the Nrf2 level was slightly (but not significant) higher in the rats hippocampal CA1 area along with a decline of HO1 expression at 7 days after GCI (2VO) (Tulsulkar and Shah, 2013). In a rat GCI (4VO) model, activation of Nrf2 in the hippocampal CA1 region was examined over 3 days by four metrics including nuclear translocation of Nrf2, total Nrf2 protein level, DNAbinding of Nrf2, and induction of Nrf2 regulated proteins (Tu et al., 2015). The cytosolic but not nuclear Nrf2 protein was slightly upregulated from 12 to 72 h as evidenced by WB and immunostaining analyses, whereas the total Nrf2 protein level remained unchanged at 24 h. No change in the Nrf2 DNA binding activity was detected at 24 h and 72 h. The Nrf2-regulated antioxidant proteins HO1, NQO1, SOD2, and GPx1 were slightly altered within 72 h. These findings are supported by another report of GCI with less time of ischemia (Wang et al., 2013) that presented the upregulation of HO1 protein at 3 days. Lastly, it should be pointed out that the reports are inconclusive concerning the protein expression levels of Nrf2 with its target genes and subcellular distribution of Nrf2 following GCI. For example, hippocampal Nrf2 protein was reported to have not changed (Yang Y. et al., 2015), significantly decreased (Liu H. et al., 2015), or increased (Ashabi et al., 2015; Lee et al., 2015) following ischemia.

#### FUNCTIONAL BENEFIT OF NRF2 IN CEREBRAL ISCHEMIA—IN VIVO EVIDENCE FROM NRF2−/<sup>−</sup> MICE

Multiple lines of evidence demonstrate the beneficial contribution of Nrf2 in various pathological conditions, and most come from studies using Nrf2 knockout (Nrf2−/−) or knockdown animals. Reports showed that Nrf2−/<sup>−</sup> mice do not exhibit any overt abnormal phenotype regarding size, body weight, food intake, mobility, fertility, or other characteristics at baseline (Itoh et al., 1997; Wang et al., 2007; Wang Y. C. et al., 2011; Wang B. et al., 2012; Leonardo et al., 2013, 2015; Doré, 2015; Liu et al., 2018). In addition, Nrf2−/<sup>−</sup> mice exhibit similar cerebrovascular architecture in anastomosis, vascular physiology, and blood pH compared to their WT counterparts (Leonardo et al., 2015). Basal GST and NQO1 activities, but not GSH content, modestly but significantly decreased in multiple brain regions of Nrf2−/<sup>−</sup> mice compared to their Nrf2+/<sup>+</sup> littermates, suggesting that the constitutive synthesis of brain GSH does not rely on Nrf2 function, at least in young adult mice (Shih et al., 2005).

However, in response to the various ischemic insults, it is becoming clear that Nrf2 plays a critical role in protection against ischemic brain damage (**Tables 1**–**3**). Indeed, older and recent studies with different cerebral ischemia models have revealed the functional benefit of Nrf2 on infarct volume, brain edema, and

Liu et al. Nrf2 in Experimental Ischemic Stroke

TABLE

2

Continued

(Continued)

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TABLE

3

Preclinical

studies

of

Nrf2

in

transient

cerebral

ischemia

models

of

mice

and

rats

(MCAO,

2

h).

(Continued)

TABLE

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3

Continued

**202**

leveledema

 and

mRNA/protein

 expression level (<sup>↑</sup> or ↓, increase or decrease at indicated time point; no label at indicated time point, no significant difference); h, hour; d, day; wk, week.

neurobehavioral

 deficitseatmest,

posttreatmeatme;

 IF,

Immunofluorescence;

 KDa, kilodalton that indicates Nrf2 protein molecular weight by Western blot; the changes in brain lesion, edema, neurological

 deficits, and

TABLE

4

Preclinical

studies

of

Nrf2

in

global

cerebral

ischemia

models

of

mice

and

rats.

Frontiers in Pharmacology | www.frontiersin.org

leveledema

 and

mRNA/protein

 expression level (<sup>↑</sup> or ↓, increase or decrease at indicated time point; no label at indicated time point, no significant difference); h, hour; d, day; wk, week.

neurobehavioral

 deficitsntt is generated by the; IF,

Immunofluorescence;

 KDa, kilodalton that indicates Nrf2 protein molecular weight by Western blot; the changes in brain lesion, edema, neurological

 deficits, and

neurobehavioral deficits after ischemia. Following permanent ischemia injury without reperfusion, Nrf2 deficiency exacerbated the acute development of brain lesions, revealed by severe infarct volume at 3 days but not at 1 day after pdMCAO, and these mice exhibited relatively poor sensorimotor function over 28 days (Liu et al., 2018). At 2 days after pdMCAO, Nrf2−/<sup>−</sup> mice had significantly larger cortical infarct volume and more severe neurological deficits than WT controls (Wang B. et al., 2012). Another group showed that Nrf2 deficiency slightly (but not significantly) enlarged the cortical infarct size at 1 day and sustained considerably more damage at 7 day after pdMCAO (P < 0.01), which further supports the findings above (Shih et al., 2005). Moreover, it is well-known that reperfusion after a long period of ischemia can exacerbate brain damage. Following cerebral ischemia injury with reperfusion, several studies showed that Nrf2−/<sup>−</sup> mice exhibited aggravated acute brain damage in infarct volume and neurological deficits scores at 1 day after tMCAO with 1 h (Li et al., 2013; Yao et al., 2016; Zhang et al., 2017), 1.5 h (Shah et al., 2007), and 2 h (Shi et al., 2015) of ischemia. In these studies, no significant difference in blood pH, PaO2, PaCO2, or cerebral blood fluid (CBF) was observed before, during, or after ischemia-reperfusion between WT and Nrf2−/<sup>−</sup> at indicated time points as monitored by laser-Doppler flowmetry (Shah et al., 2010). In contrast, another contradictory report showed that no significant difference in infarct volume was observed between Nrf2−/<sup>−</sup> and WT mice 2 days after 1 h tMCAO (Narayanan et al., 2015). This discrepancy might be related to the different genetic background or age of mice or the experimental setting.

Nrf2 disruption depresses the capacity of the cellular antioxidant system and diminishes upregulation of its target cytoprotective proteins in response to ischemia (**Tables 1**–**3**). A study showed that, along with severe ischemic brain damage 3 days after pdMCAO, Nrf2 deficiency destroyed the ischemiainduced increase of antioxidant/detoxification proteins NQO1, HO1, SOD2, and Gpx1 (Liu et al., 2018). Oxidative stress and inflammation plays a key role in the pathological process of ischemia. Excessive production of ROS and reactive nitrogen species (RNS) contributes to the activation of inflammatory processes, further aggravating brain injury and functional deficits during or after stroke. Toll-like receptor 4 (TLR 4) initiates inflammatory processes and induces the expression of inflammatory elements IL-6, TNFα, and IL-1β through activation and nuclear translocation of NF-κB in cerebral ischemia/reperfusion injury (Arslan et al., 2010; Wang Y. C. et al., 2011). At 24 h after 1 h tMCAO, Nrf2 knockout upregulated the TLR4 and NF-κB proteins mediated by the inflammatory response in the ischemic cortex (Li et al., 2013).

Astrocytes interact closely with neurons to provide structural and functional support at multiple levels, including ion and water homeostasis, chemical signal transmission, blood flow regulation, immune and oxidative stress defense, and supply of metabolites (Liu et al., 2017). Given that the ARE-regulated genes are preferentially activated in glial cells, which have more effective detoxification and antioxidant capacities than neurons (Vargas and Johnson, 2009; Gan et al., 2012; Haskew-Layton et al., 2013), Nrf2 in glia cells can protect neurons from a wide array of insults like stroke (Vargas and Johnson, 2009).  Indeed, in the ischemic cortex, Nrf2 deficiency exacerbated reactive astrogliosis, dysfunction in glutamate metabolism, and water permeability within 3 days after pdMCAO. The spatiotemporal pattern of reactive astrogliosis correlated well with acute ischemic damage progression. Interestingly, the microglia activation was affected considerably under Nrf2 absence.

### NRF2 TARGETED INTERVENTION IN ISCHEMIC STROKE—ADVANCEMENTS FROM EXPERIMENTAL ISCHEMIC STROKE MODEL

Given the fundamental role of Nrf2 in redox homeostasis, many studies have presented the unique contribution of Nrf2 in neuroprotection against various diseases (Ma, 2013; Kumar et al., 2014; Sun et al., 2017; Cuadrado et al., 2018). For ischemic stroke, Nrf2/ARE activation is expected to trigger a cytoprotective response counteracting deleterious ischemic events. The beneficial effects of some candidate Nrf2 inducers were directly proved by the findings in Nrf2−/<sup>−</sup> mice. The typical findings were listed below (just to name a few), and more are also depicted in **Tables 1**–**4**.

### Dimethyl Fumarate and Monomethyl Fumarate

Dimethyl fumarate (DMF) and its primary metabolite monomethyl fumarate (MMF) are two typical Nrf2 inducers. Several studies support their neuroprotective efficacy against ischemic brain injury through the activation of the Nrf2/HO1 pathway. DMF and MMF dramatically reduced infarct volume, brain edema, and neurological deficits over 7 days after tMCAO, along with the suppressed acute glial activation (Yao et al., 2016). Importantly, such evident protection was abolished in Nrf2−/<sup>−</sup> mice, indicating that the Nrf2 pathway is required for the benefit of DMF and MMF. During the late phase (7–14 d), DMF also acted as a potent immunomodulator, reducing the infiltration of neutrophils and T cells and the number of activated microglia and macrophages in the infarct region (Lin et al., 2016). Interestingly, DMF also attenuated brain edema formation during ischemia and stabilized the blood brain barrier (BBB) by preventing disruption of interendothelial tight junctions (Kunze et al., 2015). DMF protected against acute brain damage in edema volume and sensorimotor deficits after pdMCAO through anti-inflammatory cytokines (IL-10 and IL-12p70), further supporting the neuroprotective role of MMF (Clausen et al., 2017).

#### Sulforaphane

Sulforaphane (SFN), a typical Nrf2 activator, is a naturally occurring isothiocyanate found in cruciferous vegetables. Upregulation of Nrf2 by sulforaphane pretreatment was associated with increased HO1 expression in perivascular astrocytes in the peri-infarct regions and cerebral endothelium of the infarct core. BBB disruption, lesion progression, and neurological deficits were reduced after tMCAO. This indicates that the Nrf2 defense pathway in the cerebral microvasculature provides a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke victims (Alfieri et al., 2013). There is a contradictory finding that sulforaphane treatment increased the overall mRNA levels of transcription of Nrf2, Hmox1, GCLC and GSTA4 but failed to reduce the infarct volume and motor deficits in a photothrombotic mouse model (Porritt et al., 2012).

#### Tert-Butylhydroquinone

Tert-butylhydroquinone (tBHQ) is a Nrf2 inducer widely used as a food additive. Nrf2 activation by tBHQ pretreatment reduced cortical damage and sensorimotor deficits from 24 h up to even 1 month after pdMCAO. Interestingly, larger infarcts were observed in Nrf2−/<sup>−</sup> mice 7 d after stroke, but not 24 h after stroke (Shih et al., 2005). This Nrf2-specific action of tBHQ in vivo was also validated in another study with tMCAO (Shah et al., 2007). A recent study showed that Nrf2 upregulation by tBHQ significantly reduced the expression of cytoplasmic thioredoxin interacting protein, nod-like receptor protein 3 (NLRP3) inflammasome, and downstream factors caspase-1, IL-1β, and IL-18, all of which contribute to the Nrf2-mediated neuroprotection against ischemic outcomes after tMCAO (Hou et al., 2018). Interestingly, there is a contradictory report that tBHQ-exacerbated stroke damage (Sun et al., 2016), revealed by increased post-stroke mortality and worsened outcomes after pMCAO.

#### MiR-93 Antagomir

MicroRNAs (miRNAs) play vital roles in regulating neuronal survival during cerebral ischemia/reperfusion injury. MiR-93, a direct negative modulator of Nrf2 expression at the transcriptomic level, serves as a potential therapeutic target for acute ischemic stroke. MiR-93 levels in the ischemic cortex of mice increased at 24 h and 48 h after tMCAO. MiR-93 antagomir treatment reduced infarction volume, neural apoptosis and neurological deficits through the Nrf2/HO1 antioxidant pathway (Wang et al., 2016).

#### Korean Red Ginseng

Korean Red Ginseng (Ginseng) is one of the most widely used herbal medicines with reported antioxidant and antiinflammatory properties, displaying promising potential in neuroprotection. Ginseng pretreatment ameliorated short- and long-term sensorimotor deficits over 28 days, prevented the acute enlargement of lesion volume, attenuated reactive astroglial progression but not microglial activation, and enhanced the induction of Nrf2 target antioxidant proteins after pdMCAO in WT mice, an effect that was abolished in Nrf2−/<sup>−</sup> mice (Liu et al., 2018). Nrf2-dependent spatiotemporal reactive astrogliosis correlated well with acute ischemic damage progression. In contrast, Nrf2 deficiency exacerbated the ischemic conditions. This supported the conclusion that Ginseng pretreatment protects against acute sensorimotor deficits and promotes its long-term recovery after pdMCAO, at least partly, through Nrf2 activation. Attenuated reactive astrogliosis contributes to the Nrf2-depedent neuroprotection.

## (–)-Epicatechin

The (**–**)-Epicatechin (EC) is especially abundant in cocoa, dark chocolate, and green tea, and it boosts antioxidant activity while supporting vascular function. EC-treated ischemic WT mice displayed a reduction of forelimb motor coordination impairments associated with reduced anatomical injury and microglia/macrophage activation in pMCAO and tMCAO models of adult WT mice, impairments which were abolished in tissues and neurons from Nrf2−/<sup>−</sup> mice (Shah et al., 2010). Interestingly, this neuroprotection was observed in 12 monthold WT and Nrf2−/<sup>−</sup> mice, indicating that age influences Nrf2 function (Leonardo et al., 2013, 2015).

#### Resveratrol

Resveratrol is a natural polyphenolic compound that is found in some dietary sources such as grapes, plums, and red wine. Transresveratrol pretreatment had neuroprotective effects on ischemic brain damage after tMCAO. This neuroprotective effect is likely exerted by upregulated expression of Nrf2, HO1 and SOD that ameliorates oxidative damage (Ren et al., 2011). Meanwhile, resveratrol preconditioning-mediated neuroprotection is reduced after tMCAO (Narayanan et al., 2015). WT and Nrf2−/<sup>−</sup> cortical mitochondria exhibited increased uncoupling and ROS production after resveratrol pre-treatments. Nrf2−/<sup>−</sup> astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular antioxidants.

### Carbon Monoxide

Carbon monoxide (CO) is a gaseous second messenger produced when heme oxygenase enzymes catabolize heme. At low doses (Otterbein et al., 1999; Koehler and Traystman, 2002; Leffler et al., 2011), despite being traditionally viewed as a toxic agent (Cheng et al., 2012), CO displayed sustained neuroprotective efficacy against brain damage and progressive functional deficits via the Nrf2 pathway in tMCAO and pdMCAO models (Meffert et al., 1994; Maines, 1996; Alkadhi et al., 2001). Brain lesions in mice exposed to CO were 29.6 ± 12.6% smaller at 7 days after pdMCAO. Additionally, 18-h CO treatment led to Nrf2 dissociation from Keap1, nuclear translocation, increased activity of Nrf2 binding to the ARE sequence of the HO1 gene, and elevated HO1 expression from 6 to 48 h after CO exposure, the neuroprotection of which was abolished in Nrf2−/<sup>−</sup> mice (Wang B. et al., 2011).

#### Tetramethylpyrazine

Tetramethylpyrazine (TMP) is one of the main components of Ligusticum wallichii Franchat (Chuan Xiong) that has been used to treat neurovascular and cardiovascular diseases including stroke in traditional Chinese medicine. TMP has been found to protect against pdMCAO injury and reduce inflammation and ischemia-induced neutrophils through Nrf2/HO1 activation (Kao et al., 2013; Chang et al., 2015).

#### Curcumin

Curcumin is a phenolic pigment extracted from the rhizome of Curuma longa Linn, which has been widely used as medicine in many Asian countries. It has strong neuro-protective effects on neurological injuries, with virtually no toxicity, even at a high dose; though, its absorption-bioavailability is the main issue, and few groups are actively looking to test innovative formulation to circumvent this serious limitation. Curcumin significantly reduced infarct size, brain edema, neurological dysfunction, and oxidative stress levels in tMCAO rats, and this neuroprotective effect involves the Akt/Nrf2 and NF-κB pathways (Wu J. et al., 2013; Li et al., 2016).

### PITFALLS AND CONSIDERATIONS OF CURRENT NRF2 EXPERIMENTAL ISCHEMIC STROKE STUDIES

Although most reports support the beneficial effect of Nrf2 activation by a multitude of variety of inducers against ischemic brain damage, many findings regarding the role of Nrf2 during ischemia or ischemic-reperfusion injury are contradictory. This could be attributed to a number of inconsistencies across studies, as briefly overview here. (1) Nrf2 antibody: Getting potent, selective and highly specific antibodies against the various species of Nrf2 has been an ongoing serious issue. Furthermore, what is the correct molecular weight of Nrf2 protein? The biologically relevant molecular weight of Nrf2 protein has been proven to be ∼95–110 KDa, but not ∼55–65 KDa, based on its 2-kb open reading frame or its tertiary structure (Lau et al., 2013; Kemmerer et al., 2015). Many reports presented the expression level of Nrf2 protein by Western blots at apparent different (or unspecified) molecular weights, which might be mainly responsible for the contradictory results among research groups, the lack of good negative controls to be run in parallel should be documented. Additionally, this issue may also contribute to the discrepancy regarding findings of Nrf2 in subcellular distribution and cell-type expression under normal and inducible conditions. (2) Questions remain as to which cell types would be most responsible for the neuroprotection associate with Nrf2. (3) Some reports also questioned the potential issues in the Nrf2 knockout lines and strains. (4) Furthermore, while many of these reported cytoprotective enzymes that are induced by Nrf2 activation, there are activate debates as to whether traditional freely available substrates are sufficiently high to explain the resulting beneficial properties of these metabolites/end-products. (5) While many of these compounds are assumed to be the bioactive entity, there are well-recognized issues on such drugs to be bioavailable and cross the blood brain barrier to then reach the target at a concentration that would be biologically relevant; thus, there is the possibility that indirect pathways are stimulated that would then indirectly activate the Nrf2 pathway. (6) Several of the studies have tested preventive medicine when give before ischemia while others have tested the therapeutic potential of the given drug concoction; though, one should not extrapolate conclusion of the universal protection until it is rigorously tested and independently validated. (7) Nrf2/ARE pathway: Is the Nrf2/ARE pathway activated or not? To validate that Nrf2 is activated, many reports based their conclusions on selected protein level measurements of total Nrf2 with or without its target markers like HO1 by WB. Based on the current understanding of Nrf2 activation, nucleus translocation of Nrf2 is essential to its activation, which is revealed by nuclear Nrf2 protein levels. Evidence that targets distinct components of the Nrf2/ARE pathway cascade will be expected. In addition, the application of pharmacological or genetic blockage of Nrf2 will be helpful to demonstrate whether Nrf2/ARE pathway activation is specific to the neuroprotection of the candidate Nrf2 inducer. (8) Ischemic brain tissue: Which part of the ischemic cerebral hemisphere was used for measurement? Despite variable ischemic damage to different brain regions (or together with reperfusion), various ischemic brain tissue like the cortex, ischemic region, and cerebral hemisphere was used for measurement, possibly leading to inconsistent results. These tissue samples such as the infarct core, peri-infarct, and healthy brain regions, could "dilute" and even mask the changes of markers interest during cerebral ischemia. The apparent alternation of Nrf2 expression happens inside the peri-infarct area. Accordingly, tissue from the same site of the peri-infarct area appears to be appropriate for the subsequent measurement that leads to the final conclusion. (9) Histological and neurobiological outcomes: Which marker and which time points are expected to have the functional benefit of a potent Nrf2 inducer? Most reports only selected infarct volume or neurological deficit score in the acute ischemic stage (1–3 days) without tracking long-term efficacy. As we know, ischemic insult induces severe motor, sensory, emotional, and cognitive deficits (Ferro et al., 2016), and long-term functional recovery is considered to be the goal of stroke intervention. More accurate and long-term histological and functional evaluations are expected for future studies. (10) Nrf2 activators: As mentioned above, a number of Nrf2 activators have been identified that appears to upregulate Nrf2/ARE pathway. However, thus far, only limited compounds including sulforaphane, CDDO-methyl ester, and DMF have consistently indicated sufficient pharmacokinetic and pharmacodynamic properties to act in brain (Lastres-Becker et al., 2016; Yamamoto et al., 2018). Future rigorous studies are needed to provide the necessary information concerning the clinical application prospect of Nrf2 targeted intervention.

This review focusses on Nrf2 activity in the context of ischemic stroke, though, over- or prolonged Nrf2 activation could potentially be problematic. For example, sustained activation of Nrf2 in drosophila would shorten its life expectancy (Tsakiri et al., 2013), and it would also promote the malignant transformation of human cells (Yang X. et al., 2015). It has also been reported that mutations in Nrf2 and Keap1 would promote cancer cell survival (Shibata et al., 2008); furthermore, many cancer cells would overexpress Nrf2 and this would be associated with cell resistance to cancer therapies (Taguchi and Yamamoto, 2017).

#### CONCLUSIVE REMARKS AND PERSPECTIVE

Emerging evidence demonstrates that the Nrf2 network plays a crucial role in cellular adaption by controlling a wide range of cytoprotective proteins, counteracting distinct endogenous and exogenous insults while providing a promising optimal therapeutic target against various diseases from cancer to brain disorders. By using various ischemic stroke rodent models, recent preclinical studies provide direct in vivo evidence revealing the contribution of the Nrf2 pathway in ischemic stroke pathogenesis and neuroprotection. This review highlighted the promising potential of interventions targeting Nrf2, implying that the

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moderate activation of Nrf2 favors the attenuation of brain damage and long-term recovery from cerebral ischemia. Because the investigations of Nrf2 in stroke are still in the initial stages, future research is expected to elucidate the natural properties of Nrf2 in stroke leading to the development of novel drugs that target Nrf2.

#### AUTHOR CONTRIBUTIONS

LL and SD conceived the study and designed the databases analysis. LL prepared the manuscript with input from SD. LL and LML searched databases, collected data, performed analyses, and prepared the tables and figure. All authors reviewed, discussed and approved the final manuscript.

#### FUNDING

This work was supported in part by the American Heart and Stroke Association Postdoctoral Fellowship 16POST31220032 (LL). Publication of this article was funded in part by the University of Florida Open Access Publishing Fund.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Liu, Locascio and Doré. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nrf2 as a Potential Mediator of Cardiovascular Risk in Metabolic Diseases

Rafael M. da Costa1,2 \*, Daniel Rodrigues<sup>1</sup> , Camila A. Pereira<sup>1</sup> , Josiane F. Silva<sup>1</sup> , Juliano V. Alves<sup>1</sup> , Núbia S. Lobato<sup>2</sup> and Rita C. Tostes<sup>1</sup>

<sup>1</sup> Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil, <sup>2</sup> Special Academic Unit of Health Sciences, Federal University of Goiás, Jataí, Brazil

#### Edited by:

Javier Egea, Institute of Health Research of the University Hospital of La Princesa, Spain

#### Reviewed by:

Soon Yew Tang, University of Pennsylvania, United States Carole L. Wilson, Medical University of South Carolina, United States

> \*Correspondence: Rafael M. da Costa<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Inflammation Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 07 November 2018 Accepted: 26 March 2019 Published: 12 April 2019

#### Citation:

da Costa RM, Rodrigues D, Pereira CA, Silva JF, Alves JV, Lobato NS and Tostes RC (2019) Nrf2 as a Potential Mediator of Cardiovascular Risk in Metabolic Diseases. Front. Pharmacol. 10:382. doi: 10.3389/fphar.2019.00382 Free radicals act as secondary messengers, modulating a number of important biological processes, including gene expression, ion mobilization in transport systems, protein interactions and enzymatic functions, cell growth, cell cycle, redox homeostasis, among others. In the cardiovascular system, the physiological generation of free radicals ensures the integrity and function of cardiomyocytes, endothelial cells, and adjacent smooth muscle cells. In physiological conditions, there is a balance between free radicals generation and the activity of enzymatic and non-enzymatic antioxidant systems. Redox imbalance, caused by increased free radical's production and/or reduced antioxidant defense, plays an important role in the development of cardiovascular diseases, contributing to cardiac hypertrophy and heart failure, endothelial dysfunction, hypertrophy and hypercontractility of vascular smooth muscle. Excessive production of oxidizing agents in detriment of antioxidant defenses in the cardiovascular system has been described in obesity, diabetes mellitus, hypertension, and atherosclerosis. The transcription factor Nrf2 (nuclear factor erythroid 2–related factor 2), a major regulator of antioxidant and cellular protective genes, is primarily activated in response to oxidative stress. Under physiological conditions, Nrf2 is constitutively expressed in the cytoplasm of cells and is usually associated with Keap-1, a repressor protein. This association maintains low levels of free Nrf2. Stressors, such as free radicals, favor the translocation of Nrf2 to the cell nucleus. The accumulation of nuclear Nrf2 allows the binding of this protein to the antioxidant response element of genes that code antioxidant proteins. Although little information on the role of Nrf2 in the cardiovascular system is available, growing evidence indicates that decreased Nrf2 activity contributes to oxidative stress, favoring the pathophysiology of cardiovascular disorders found in obesity, diabetes mellitus, and atherosclerosis. The present minireview will provide a comprehensive overview of the role of Nrf2 as a contributing factor to cardiovascular risk in metabolic diseases.

Keywords: Nrf2, oxidative stress, cardiovascular risk, metabolic diseases, therapeutic target

#### OVERVIEW OF REACTIVE OXYGEN SPECIES AND CARDIOVASCULAR FUNCTION

Although reactive oxygen species (ROS) were initially presumed to cause cell damage, they are now recognized as important molecules that regulate many cell signaling and biological processes, such as induction of defense genes, activation of transcription factors, phosphorylation of kinases, and mobilization of ions in transport systems (reviewed in Droge, 2002; Touyz and Briones, 2011; Brown and Griendling, 2015). In the cardiovascular system, ROS generation is important to maintain endothelial and vascular smooth muscle cells (VSMCs) function, including vascular tone control, inflammationrelated responses, cell growth and proliferation, modulation of extracellular matrix production, apoptosis and angiogenesis (Siti et al., 2015; reviewed in Galley and Straub, 2017). Alterations of the balance between cellular ROS production and the capacity to rapidly detoxify reactive intermediates play an important role in the development of risk factors for cardiovascular diseases (reviewed in Harrison et al., 2003 and Touyz et al., 2018). Such events are observed in patients with essential hypertension (Redón et al., 2003) and various experimental models of arterial hypertension, such as spontaneously hypertensive rats (SHR) that exhibit increased levels of vascular superoxide anion and vascular smooth muscle hypercontractility (Paravicini et al., 2004). The same occurs in obesity and experimental models of diabetes, in which insulin resistance and hyperglycemia culminate in increased NAD(P)H oxidase enzyme activity and endothelial nitric oxide synthase (eNOS) uncoupling, thereby contributing to increased ROS generation and impaired vascular function (Youn et al., 2012; da Costa et al., 2017; Neves et al., 2018).

Vascular cells generate ROS in response to several stimuli, including cytokines, angiotensin II, endothelin-1, aldosterone and platelet-derived growth factor (PDGF) (reviewed in Thannickal and Fanburg, 2000). ROS signaling in endothelial cells and VSMCs involves alterations in the intracellular redox state and oxidative modification of regulatory and contractile proteins (da Costa et al., 2017). The oxidative modification of these redox-sensitive proteins alters their conformation, stability, activity and/or ability to interact with other proteins, resulting in modulation of vascular function. Redox-sensitive proteins include proteins involved in calcium handling as well as contractile proteins, proteins involved in various signaling and transcriptional activities. Redox modulation of calcium handling proteins directly affects cardiac and vascular contraction by altering intracellular calcium levels. Examples of redox-sensitive calcium handling proteins are calcium-calmodulin kinase II (CaMKII), the ryanodine receptor on the sarcoplasmic reticulum, sarcoplasmic reticulum ATPase (SERCA), and phospholamban (reviewed in Burgoyne et al., 2012 and Steinberg, 2013). Protein kinases and phosphatases are also affected by direct redox modification, resulting in modulation of various signal transduction pathways in cardiac and vascular cells, including altered modulation of calcium sensitivity, phosphorylation of the myofilaments and receptor tyrosine kinase signaling (reviewed in Knock and Ward, 2011 and Steinberg, 2013).

The long-term effects of ROS in cardiovascular function depend on the balance between signals promoting proliferation or growth inhibition and/or cell death. This dual role is evidenced by the findings that endothelial apoptosis initiated by tumor necrosis factor-alpha (TNF-α) is attenuated by ROS scavenging (Xia et al., 2006) and that, during ischemia-reperfusion injury, ROS trigger apoptosis, whereas ROS generated during ischemic preconditioning prevent apoptosis (Maulik et al., 1999; Hattori et al., 2001; reviewed in Becker, 2004). In vascular cells, ROS can alter this balance leading to either excessive angiogenesis or loss of endothelial cells (reviewed in Papaharalambus and Griendling, 2007).

To contain ROS generation, there are antioxidant defenses that prevent or neutralize the generation of highly reactive compounds in biological systems. Among the major intracellular antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase are major players. In cooperative action, these enzymes catalyze ROS in molecular oxygen and water (reviewed in Rodriguez and Redman, 2005). Other agents, such as ascorbic acid (reviewed in May and Harrison, 2013) and phenolic acids (reviewed in Santhakumar et al., 2014), which do not present enlightened mechanisms of action, also contribute to redox balance.

Although the exact mechanisms underlying cardiovascular oxidative stress during obesity, diabetes, and atherosclerosis still remain unclear, the discovery of mechanisms and subsequent new pharmacological interventions related to the disruption of the antioxidant system has become of great interest to identify key mediators that will potentially instigate and exacerbate these cardiovascular complications.

## Nrf2 ACTIVITY AND REGULATION

Several cell types throughout evolution have developed adaptive programs to counteract oxidative stress, and the role of the nuclear factor erythroid 2-related factor-2 (Nrf2) in these programs has been a subject of recent interest. Nrf2 is a basic leucine zipper region (bZip) transcription factor of the Cap n'Collar family, with approximately 589 amino acid residues and seven conserved domains (Neh1 – Neh7) (reviewed in Kansanen et al., 2013). When the levels of ROS and electrophiles become greater than the cell's ability to detoxify them, a series of events results in Nrf2 activation. Activated Nrf2 heterodimerizes with Jun and small musculoaponeurotic fibrosarcoma (sMaf) proteins, translocates to the nucleus and binds to the antioxidant response element (ARE) or the electrophile-response element (EpRE) in the promoter region of Nrf2 target genes. This results in the coordinated expression and activation of antioxidant, antiapoptotic, metabolic, and detoxification proteins. Among the proteins with antioxidant activity regulated by Nrf2 are superoxide dismutase (SOD), catalase (CAT), heme-oxygenase 1 (HO-1), glutathione peroxidase 1 (GPx-1) and NAD(P)H: quinone oxidoreductase 1 (reviewed in Tebay et al., 2015).

The conserved Nrf2 domains are crucial for their selfregulation and activity. In this context, the Neh-1 domain is responsible for the binding of Nrf2 to DNA through dimerization with the low molecular weight sMaf proteins of type F, G, and K (sMafF, sMafG, and sMafK), favored by the bZip domain, and by recognition of the ARE (Mohler et al., 1991; Oyake et al., 1996; reviewed in Motohashi et al., 2002). The Neh-2 domain, rich in lysines, is responsible for binding of Nrf2 to its cytoplasmic repressor protein Keap-1. This domain also has high and low affinity regions for Keap-1 binding, the ETGE and DLG motifs, respectively (Itoh et al., 1999; Kobayashi et al., 2002). The Neh-3 domain, located in the carboxy-terminal portion of Nrf2, is sumoylated. This post-translational modification is important for Nrf2 transactivation. In addition to Neh-3, the Neh-4, and Neh-5 domains, when acetylated or oxidized, in oxidative stress conditions, are responsible for the transactivation of Nrf2 (Katoh et al., 2001; Nioi et al., 2005). The Neh-6 and Neh-7 domains are responsible for the degradation of Nrf2, independently of the regulation by Keap-1 (McMahon et al., 2004; Wang et al., 2013).

In physiological conditions, when there is a balance between oxidant species and the adequate activity of antioxidant proteins, Nrf2 is suppressed in the cytoplasm. This suppression occurs adjacent to the cellular cytoskeleton by the interaction of Nrf2 with Keap-1 and Cullin3, a protein of the E3 ligase family (reviewed in Canning et al., 2015). The formation of the Nrf2- Keap-1-Cullin3 complex becomes a signal for the ubiquitination and proteasome degradation of Nrf2, maintaining its basal levels along with the genes regulated by this transcription factor (Furukawa and Xiong, 2005; reviewed in Canning et al., 2015).

Under oxidative or electrophilic stress conditions, ROS promote the breakdown of the Nrf2-Keap1-Cullin3 complex, allowing Nrf2 to translocate to the nucleus and activate the expression of antioxidant proteins. The ARE is normally suppressed by a heterodimer formed by the sMaf and Bach-1 proteins, preventing Nrf2 heterodimerization and binding to the ARE (reviewed in Tebay et al., 2015 and Tong et al., 2015). However, under conditions of oxidative stress, there is an accumulation of heme groups in the cytosol, a frequent substrate for ROS formation. In this condition, Bach-1 undergoes dissociation of sMaf proteins and migrates to the cytosol to act on the heme group metabolism, allowing Nrf2 to dimerize with the sMaf protein and initiate gene transcription (reviewed in Ryter and Choi, 2005 and Tebay et al., 2015).

Among the proteins that participate in Nrf2 regulation and activity, Keap-1 and Bach-1 stand out from the others due to their direct role in redox balance and cardiovascular homeostasis (Lopes et al., 2015). Of importance, the negative regulation of Nrf2 by Keap-1 and Bach-1 raises the possibility of new therapeutic targets to prevent oxidative stress-associated cardiac hypertrophy and vascular dysfunction (Abed et al., 2015; Jiao et al., 2018).

#### Nrf2 AND OBESITY

The mechanisms accompanying the progression of obesity and its cardiovascular comorbidities have been the focus of considerable research over the last 30 years. As the mechanistic insights clarify, it is evident that the expansion of visceral adipose tissue plays a pivotal role in the pathophysiology of obesity. Increased ROS generation by the adipocytes, which, in turn, increases expression and secretion of inflammatory adipokines, causes dramatic consequences for the regulation of energy homeostasis and vascular function (Keaney et al., 2003; reviewed in DeMarco et al., 2010 and Otani, 2011). This is a common combination associated with individual cardiovascular disease risk (Furukawa et al., 2004; reviewed in Sowers et al., 2011).

Recent evidence shows that Nrf2 is involved in the control of energy metabolism and might be a promising therapeutic target for obesity. The underlying mechanism of Nrf2 in obesity has been investigated using various experimental approaches, including Nrf2 gene deletion, Nrf2 pharmacological activators and Nrf2 gene overexpression, but only few of these approaches were clinically tested. Specific pharmacological activators of Nrf2, including epigallocatechin 3-gallate, oltipraz, sulforaphane, curcumin and 1-[2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28 oyl] imidazole (CDDO-Im), induce the expression and activity of Nrf2 both in vitro and in vivo (Shin et al., 2009; Yu et al., 2011; Xu et al., 2012; Gao et al., 2013). The epigallocatechin 3-gallate-induced activation of Nrf2 in liver and adipose tissue of obese mice improves lipidemic control, decreases oxidative products generation, and reduces body mass, insulin and glucose levels (Sampath et al., 2017). Interestingly, knockout mice for the Keap-1 protein exhibit similar features, including suppression of high-fat diet-induced obesity and decreased deposition of lipids and cholesterol in the liver (Slocum et al., 2016). Of importance, improvement of metabolic profile is closely associated with improved cardiovascular function (da Costa et al., 2016; Neves et al., 2018).

A recent study demonstrated that the Nrf2 activator sulforaphane, during revascularization procedures in metabolically compromised individuals, has the potential to suppress the progression of intimal hyperplasia. In addition, Nrf2 activation attenuates leptin-induced proliferation of VSMCs in the diet-induced obesity scenario (Shawky et al., 2016).

Natural compounds are also promising elements in Nrf2 activation during obesity. As an example, Zeng et al. (2015) observed that curcumin, a natural Nrf2 activator, suppresses oxidative stress, inflammation and hypertrophy induced by treatment of cardiac cells with free fatty acids. Similarly, high-fat diet induced oxidative stress, inflammation, fibrosis, hypertrophy and tissue remodeling are attenuated by curcumin treatment. These benefits are closely associated with increased Nrf2 expression and activity, as well as reduced ROS generation (Zeng et al., 2015).

Deletion of the Nrf2 gene is expected to increase ROS generation and to aggravate the phenotypes of obesity. Nrf2 knockout mice show increased ROS generation, deposition of fatty acids in the liver and increased expression of genes related to the synthesis of lipids and cholesterol (Tanaka et al., 2008). Consistent with these findings, our group showed that obesity in mice favors vascular oxidative stress by increasing the expression of the downregulatory proteins of Nrf2, Keap-1 and Bach-1 (Costa et al., 2017).

Nrf2 in adipose tissue function and metabolic syndrome has also been examined. Xue et al. (2013) determined the role of Nrf2 in the development of obesity and associated metabolic disorders, using Nrf2-knockout mice on a leptindeficient ob/ob background, a model with an extremely positive energy balance. In obese mice, ablation of Nrf2, globally or specifically in adipocytes, reduces white adipose tissue mass. However, Nrf2 deletion results in even more severe metabolic syndrome with aggravated insulin resistance, hyperglycemia, and hypertriglyceridemia. In addition, when compared to wild-type mice, the white adipose tissue of obese mice expresses substantially higher levels of many genes related to antioxidant response, inflammation, adipogenesis, lipogenesis, glucose uptake, and lipid transport (Xue et al., 2013). These findings support a role for Nrf2 in regulating adipose tissue development and function, insulin sensitivity, glucose and lipid homeostasis.

In contrast to the above-mentioned study, Nrf2 knockout mice treated with a high-fat diet tend to gain less body mass and display increased insulin sensitivity and glucose tolerance. In addition, they do not exhibit increased glucose, cholesterol, or plasma triglycerides. Of importance, the altered metabolic phenotype of Nrf2- knockout mice on high-fat diet is associated with higher expression and abundance of fibroblast growth factor 21 (FGF21), a novel hormone that regulates energy metabolism, glucose tolerance and adipose tissue expansion (Chartoumpekis et al., 2011).

The complex roles of Nrf2 in adipogenesis and adipose tissue functions were recently examined by adipose tissue-specific ablation of Nrf2 in mice. This condition is associated with a transient delayed increase of body weight in high-fat dietfed mice. However, the benefit is eventually suppressed after prolonged feeding. The phenotypic changes induced by adipose tissue-specific ablation of Nrf2 also extend to the whole-body level, reducing blood glucose and altering the expression of genes involved in glucose, lipid and energy metabolism (Zhang et al., 2016). These findings are consistent with those of previous studies using Nrf2 knockout mice (Pi et al., 2010; Xu J. et al., 2015) and provide additional mechanistic insights into the role of Nrf2 as an important mediator of glucose, lipid and energy metabolism. To date there are no studies showing direct effects of Nrf2 deletion on cardiovascular function.

The apparent contradictory role of Nrf2 protecting mice from obesity and insulin resistance in conditions of Nrf2 deficiency in comparison to Nrf2 pharmacological activation, may be explained by the observations that Nrf2 deficiency leads to a mild increase in the levels of ROS, which stimulate the antioxidant system in a manner similar to the Nrf2 activators (reviewed in Bocci et al., 2014). Another possible explanation is that Nrf2 activators also regulate non-Nrf2 signaling pathways to modulate glucose and lipid metabolism. The same is true for Keap-1, the Nrf2 repressor protein, which may also have Nrf2-independent effects on transcription factors and, consequently, on metabolic homeostasis (Huang et al., 2012). Controversial results on the role of Nrf2 in obesity may be linked to differences in the pathophysiological characteristics of obesity (such as diet content or time under obesity conditions), as well as specific genetic characteristics. **Table 1** summarizes the contribution of Nrf2 signaling in different obesity conditions. Further studies are required to explore this apparent discrepancy on the role of Nrf2 in obesity.

The Nrf2/Keap1/ARE signaling pathway also represents a mechanism by which the gut microbiome activates a wide range of host signaling and homeostatic processes (reviewed in Murota et al., 2018). The intestinal microbiome and its metabolites display a pivotal role in host physiological processes including immune, metabolic, neurological, and nutritional homeostasis (reviewed in Lynch and Pedersen, 2016). Many of these physiological processes are under influence of ROS generation in the gut epithelia. Alterations in the gut microbiota have recently emerged as major triggers of abnormalities in the integrity of the intestinal barrier, facilitating blood translocation of bacteria and uremic toxins, systemic inflammation and adverse outcomes in obesity and diabetes (reviewed in Belizário et al., 2018). Nrf2 activation in intestinal barrier leads to upregulation of antioxidant enzymes, thereby strengthening the cell's ability to neutralize several types of free radicals (Zheng et al., 2018). In addition, the relationship between bacterial-dependent ROS generation and Nrf2 pathway activity was recently revealed by observations that lactobacilli-induced and Nox1 mediated generation of ROS evokes Nrf2-dependent activation of cytoprotective antioxidants genes (Jones et al., 2015). In fact, microbe-induced ROS generation and activation of Keap1/Nrf2/ ARE signaling may contribute to our understanding on the mechanisms involved in the genesis of obesity and diabetes, comorbidities associated with increased cardiovascular risk.

### Nrf2 AND TYPE 2 DIABETES MELLITUS

There is abundant evidence that oxidative damage caused by free radicals contributes to the pathogenesis and progression of type 2 diabetes mellitus and its complications (reviewed in Dandona et al., 1996; Brownlee, 2001 and Giacco and Brownlee, 2010). Only recently, however, has the role of the Nrf2/Keap-1/ARE pathway in the pathophysiology of this condition and the wide range of its complications, such as diabetic nephropathy and impaired cutaneous wound healing begun to be elucidated (Jiménez-Osorio et al., 2014; reviewed in Uruno et al., 2015; Long et al., 2016). Furthermore, as noted in an excellent and recent review on Nrf2 (David et al., 2017), this pathway is implicated in diabetic damage to the pancreas (Yagishita et al., 2014) and heart (Li et al., 2009). There are promising results provided by animal studies and clinical trials suggesting that activation of this pathway can delay or even reverse type 2 diabetes mellitus-associated dysfunctions (Ichikawa et al., 2009; Uruno et al., 2013).

A consistent alteration in diabetic patients is the presence of endothelial dysfunction, which precedes the development of diabetes-associated vascular complications and may explain, in part, the increased cardiovascular risk in this condition. Endothelial dysfunction in diabetes is associated with enhanced vascular contractility, oxidative stress and vascular inflammation (Zakkar et al., 2009; Tabit et al., 2010

TABLE 1 | Nrf2 signaling and actions in obesity and atherosclerosis animals models.

Frontiers in Pharmacology | www.frontiersin.org

(Continued)

#### TABLE 1 | Continued

fphar-10-00382 April 10, 2019 Time: 20:3 # 6

↔, not altered; ↓, decreased; ↑, increased; Adipocyte-specific Nrf2-KO, mice with Nrf2 deletion only in adipose tissue; ApoE−/−, knockout mice to apolipoprotein E; BM, bone marrow; cLDL, cholesterol fraction of the Low density lipoprotein; E-25 and E-75, treatment with Epigallocatechin-3-gallate 25 and 75 mg/kg, respectively; Gpx-1, glutathione peroxidase 1; HFD, high fat diet; HFHS + SFN, HFHS diet-fed mice that receive SFN treatment; HFHS, high fat high sugar diet; SFN, sulforaphane; HO-1, Hemeoxygenase 1; Keap1-hypo, hypomorphic Keap1 allele mice; LDL, low density lipoprotein; LDLr−/−, knockout mice to low density lipoprotein receptor; Lepob/ob or ob/ob, leptin-deficient mice; LFD, low fat diet; NQO-1, NAD(P)H:Quinone Oxidoreductase 1; Nrf2, Nuclear factor-like 2; Nrf2−/−:ob/ob mice, Nrf2 knockout and leptin-deficient mice; Nrf2-KO or NRF2−/−, knockout mice to Nrf2; oxPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine; SD, standard diet; WT, wild type mice.

and Hamilton and Watts, 2013). The importance of Nrf2 and its downstream elements in the control of vascular function in diabetes has become increasingly apparent and is reinforced by multiple studies using many of the same agents for protection from conditions other than diabetes. Bardoxolone methyl, a synthetic small molecule activator of the Nrf2/Keap-1 pathway, improves structural and functional changes in animal models of renal disease (Wu et al., 2011; Chin et al., 2013). Its therapeutic potential was extended to clinical trials in type 2 diabetic patients with chronic kidney disease (Pergola et al., 2011). The recent studies published by Tan and Sharma's group demonstrated that the bardoxolone analog, dh404, at low doses, attenuates atherosclerosis in diabetic Apolipoprotein E (ApoE) knockout mice (Tan et al., 2014) and protects against diabetes-induced endothelial dysfunction, both in vivo and in vitro (Sharma et al., 2017). Considering that endothelial dysfunction, which is the first step in the development of vascular complications in diabetes, is accompanied by pro-oxidative and pro-inflammatory processes, the atheroprotective effects of dh404 could be mediated by improvement of the endothelial function. In agreement with these observations, increased Nrf2 activity induced, for example, by bardoxolone or sulforaphane, abrogates augmented vascular contraction (Alves-Lopes et al., 2016; Sharma et al., 2017) and attenuates reduced vasodilation in diabetic mice (Lu et al., 2017).

One proposed mechanism for the improvement of endothelial function following Nrf2 activation is the increased expression of a subunit of the BK (big potassium or large conductance, Ca2+ activated potassium) channel, the BK-β1, and its attenuated degradation (Lu et al., 2017), a process commonly accelerated by diabetes-induced oxidative stress (Li et al., 2017). Other mechanisms include reduced systemic and vascular oxidative stress as well as increased nitric oxide (NO) bioavailability (Liu et al., 2016; Pereira et al., 2017). Beyond this immediate homeostatic response, long-term consequences of Nrf2 activity have also been described as important culprits of microand macrovascular complications associated with diabetes. Accordingly, the literature describe functional connections between Nrf2 and signaling pathways involving nuclear factorκB (NF-κB), p53, ERK5, mTOR46, heat shock proteins, activator protein-1 (AP-1) and Notch homolog 1, translocation-associated (Drosophila) (NOTCH1) (Hwang et al., 2017). This implies that Nrf2 modulates many cellular activities, beyond its immediate homeostatic and cytoprotective actions, influencing processes as diverse as inflammation, proliferation, apoptosis, cell differentiation, tissue regeneration and even metabolism. In fact, decreased activation of Nrf2 is observed in experimental diabetic cardiomyopathy, along with a decrease in the downstream activity of antioxidant enzymes and increased oxidative stress

(Wang et al., 2017). In this sense, activation of the Nrf2 system attenuates vascular remodeling by decreasing proliferation, migration, and fibrotic processes. These effects are mediated by reduced metalloproteinase activity and decreased protein expression of adhesion molecules and TNF-α (Wang et al., 2014; Choi et al., 2015).

Such widespread protective effects of Nrf2 might constitute the underlying mechanism involved in the progression of diabetes-associated complications. These results have also provided strong support for the development of new potent enhancers of Nrf2 activity for the prevention and treatment of many diseases in which both inflammatory and oxidative processes have a key pathogenic role.

#### Nrf2 AND ATHEROSCLEROSIS