Human Nrf2 (NFE2L2) protein comprises of 605 amino acid residues with molecular weight of 67.7 kDa (Cho, 2013). The Nrf2 gene consists of seven functional domains, also known as the Nrf2-ECH homology (Neh) domains (Namani et al., 2014; Canning et al., 2015). The position of each functional domain of Nrf2 is illustrated in Figure 2A. Neh 1 domain comprises of highly conserved basic region-leucine zipper (CNC-bZIP) region that dimerizes with small musculoaponeurotic fibrosarcoma (Maf) proteins and binds to ARE, a cis-acting enhancer sequence found in the promoter region of many genes encoding antioxidant and phase II detoxification enzymes or proteins. Neh 2 domain acts as a negative regulatory domain as it contains the two degrons, known as high-affinity ETGE motif and the lower-affinity DLG motif. These motifs specifically interact with Kelch-like ECH-associated protein 1 (Keap1) which mediate ubiquitination and degradation of Nrf2. The carboxy-terminal of Neh 3 domain is a transactivation domain that recruits the chromo-ATPase/helicase DNA-binding protein 6 (CHD 6) and drives ARE- gene expression. Both Neh 4 and Neh 5 also function as transactivation domains which are involved in the interaction with cAMP response element-binding protein (CREB)-binding protein (CBP) and receptor-associated coactivator 3 (RAC 3). Meanwhile, Neh 6 domain negatively regulates Nrf2 stability via glycogen synthase kinase-3 (GSK-3)/β-transducin repeat-containing protein (β-TrCP)-mediated degradation. It contains two highly conserved redox-independent degrons known as DSGIS and DSAPGS motifs. DSAPGS motif interacts with β-TrCP, which serves as a substrate receptor for the S-phase kinase-associated protein 1- Cullin 1- RING box protein-1/regulator of cullins-1 (Skp1–Cul1–Rbx1/Roc1) ubiquitin ligase complex. This results in ubiquitination and degradation of Nrf2 via the Keap1-independent pathway. Besides, suppression of Nrf2/ARE signaling pathway may be mediated via interacting with Neh 7 domain and the DNA-binding domain of retinoid X receptor α (RXRα).

A growing body of evidence showed that Nrf2 and its downstream target genes protect against CVD development, including oxidative stress-induced endothelial dysfunction and atherosclerosis. Endothelial dysfunction marks the early stages of atherosclerosis where oxidative stress enhances endothelium cell permeability, LDL oxidation, monocyte adherence, platelet activation, vascular inflammation, as well as proliferation and infiltration of vascular smooth muscle cells (VSMCs) from media to arterial intima (Hadi et al., 2005; Grover-Páez and Zavalza-Gómez, 2009). Transplantation of Nrf2-deficient bone marrow cells in LDLR^-/- mice model showed a reduction in the expression levels of antioxidant enzymes [NAD(P)H dehydrogenase, NQO1, catalase and GPX1], increased macrophage migration, production of pro-inflammatory cytokines, and atherosclerotic lesions (Collins et al., 2012). Meanwhile, silencing of Nrf2 in U937 monocytic cells led to an elevation of pro-inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and endoplasmic reticulum (ER) stress markers expression (Song et al., 2015). Further evidence showed that overexpression of Nrf2 in VSMCs of rabbit model showed increased expression of antioxidant enzymes (HO-1 and NQO1) and inhibition of VSMCs proliferation and vascular inflammation (Levonen et al., 2007). In addition, Nrf2 deficiency in macrophage promoted pro-inflammatory cytokines production (MCP-1, IL-6, and TNF-α) and enhanced oxidized low density lipoprotein (oxLDL) uptake, leading to foam cell formation (Ruotsalainen et al., 2013).

Interestingly, some studies reported that Nrf2-mediates pro-atherogenic effects (Sussan et al., 2008; Barajas et al., 2011; Freigang et al., 2011; Harada et al., 2012; Ruotsalainen et al., 2018). Nrf2 deficiency in ApoE^-/- mice developed smaller atherosclerotic plaques by reducing CD36 expression, a type of scavenger receptor which is responsible for taking up modified LDLs (Sussan et al., 2008). Moreover, a reduction in pro-inflammatory cytokine IL-1-mediated vascular inflammation was observed in Nrf2-deficient ApoE^-/- mice (Freigang et al., 2011). Recent studies have shown that deficiency of Nrf2 in LDLR^-/- and LDLR^-/- mice expressing apoB-100 only (LDLR^-/-ApoB^100/100) reduced atherosclerotic lesion development. However, Nrf2 deficiency in aged LDLR^-/-ApoB^100/100 mice exhibit enhanced plaque inflammation and calcification (Ruotsalainen et al., 2018). These evidences suggest that Nrf2 activation plays a dual role in CVD.

Keap1 is a cysteine rich adaptor protein. Human Keap1 have a total of 27 cysteine residues which can be modified by oxidants and electrophiles. Among the cysteine residues in human Keap1, Cys 151, 273 and 288 are highly reactive and play an essential role for repression of Nrf2/ARE activation (Saito et al., 2016). Studies suggest that Keap1 cysteine residues modification by bioactive compounds derived from natural products are involved in activating Nrf2 antioxidant defense system. These bioactive compounds are reported to modify Keap1 cysteine residues via oxidation, alkylation or thiol disulfide interchange. A recent report investigating the effects of rutin, a flavonoid abundantly present in citrus fruit demonstrated that it protects against H₂O₂-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). The study showed that rutin can target the Cys 151 of Keap1, and form an adduct with Keap1 which results in Nrf2 activation and upregulation of glutamate cysteine ligase, a glutathione biosynthesis rate-limiting enzyme which plays an important role in the endogenous antioxidant system (Sthijns et al., 2017). Honaucin A, an anti-inflammatory compound isolated from marine filamentous cyanobacterium Leptolyngbya crossbyana has been reported to induce alkylation of Keap1 cysteine thiols and thereby activating Nrf2/ARE pathway (Mascuch et al., 2018). Similarly, treatment with withaferin A, a steroidal lactone significantly increased expression of HO-1 in HUVECs and EA.hy926 endothelial cells by enhancing nuclear translocation of Nrf2. Analysis of the in vitro and in silico model suggested that withaferin A can interact with Cys 151, Cys 319, Cys 434, Cys 489, and Cys 613 (Heyninck et al., 2016). Apart from that, a number of bioactive compounds including xanthohumol, sulforaphane, falcarindiol, carnosic acid and (6)-shogaol have also been showed to interact with cysteine residues on Keap1, thereby stimulating its dissociation from Nrf2 and promoting Nrf2 nuclear accumulation which induces antioxidant proteins and phase II detoxification enzymes (Satoh et al., 2008; Ohnuma et al., 2010; Hu et al., 2011; Chen et al., 2014; Yao et al., 2015). The activation of Nrf2 and stimulation of downstream antioxidants and phase II detoxification enzymes expression suggest that these natural bioactive compounds represent a potential therapeutic source for prevention and treatment of CVDs.

Apart from modification of Keap1 cysteine residues, studies have demonstrated that bioactive compounds from natural products can disrupt the Nrf2/Keap1 interaction, thereby promoting Nrf2 nuclear translocation. For instance, EGCG is a well-known Nrf2 activator that promotes the dissociation of Nrf2/Keap1 and activate ARE genes transcription, thereby inhibiting TNF-α-induced NF-κB activation in human THP-1 cells (Jiang et al., 2012). Similar study also found that ECGC protects mice model against diabetic nephropathy through inhibiting the function of Keap1 by forming hydrogen bonds with specific residues such as Ser 508, Ser 555, Ser 602, Tyr 525, Tyr 572, Gln 530, and Arg 483 (Sun et al., 2017). Besides, α-linolenic acid (ALA) has been found to protect against DOX-induced cardiotoxicity by exerting anti-oxidative and anti-apoptosis properties in rat model. The underlying mechanism is associated with the enhancement of antioxidant defense system through Nrf2/Keap1 pathway by promoting the degradation of Keap1 and thus facilitating nuclear translocation of Nrf2, as well as activation of AKT/ERK pathway (Yu et al., 2013). Similarly, khayandirobilide A (KLA) also exhibits anti-inflammatory effect via elevated expression of HO-1 by inducing Keap1 autophagic degradation and thus facilitating Nrf2 nuclear translocation (Zhou et al., 2018).

Epigenetic mechanisms have been reported to be associated with the pathogenesis of CVD. Epigenomics study in atherosclerotic human aorta demonstrated a genome-wide increase in DNA methylation during the onset and progression of atherosclerosis (Zaina et al., 2014). Studies have demonstrated that DNA demethylation and histone (de)acetylation can trigger or increase Nrf2 expression. Natural compounds derived from plants (such as sulforaphane, corosolic acid, taxifolin, reserpine, quercetin, Z-ligustilide and curcumin) and marine constituents (fucoxanthin) have been shown to activate Nrf2 signaling through epigenetic regulation (Khor et al., 2011; Su et al., 2013; Liu et al., 2015; Hong et al., 2016; Muta et al., 2016; Kuang et al., 2017; Jie et al., 2018; Yang et al., 2018c; Zhao F. et al., 2018). Quercetin, a natural flavonoid, significantly inhibited nickel-induced inflammation in mouse liver by decreasing Nrf2 DNA methylation and inhibiting the p38 MAPK signaling pathway (Liu et al., 2015). In addition, some natural compounds are involved in regulating miRNAs expression, which in turn activate Nrf2 pathway. For instance, treatment with dioscin, a natural steroid saponin markedly decreased the expression level of miRNA-140-5p, and subsequently activates Nrf2 and silent information regulator factor 2-related enzyme 2 (Sirt2) in cardiac H9c2 cells, thereby upregulating downstream target genes such as HO-1, NQO1, GST, GCLM, and forkhead box O3 (FOXO3a) (Zhao L. et al., 2018). However, the mechanism underlying epigenetic pathway responsible for the cardioprotective effects of natural product remains to be elucidated.

Protein kinases such as ERK, AMPK, GSK3β, p38 MAPK, and PI3K/AKT can mediate Nrf2 phosphorylation which enhance Nrf2 stability, thereby promoting nuclear Nrf2 translocation and transactivation activity (Nguyen et al., 2003; Bryan et al., 2013). Several natural compounds, including sodium tanshinone IIA sulfonate (STS), dihydromyricetin (DMY), methyleugenol (MLG), and astaxanthin have been shown to phosphorylate ERK, and upregulate Nrf2 expression (Wei et al., 2013; Luo et al., 2017; Zhou et al., 2017; Niu et al., 2018). STS, a water-soluble derivative of tanshione IIA, was found to protect against isoproterenol (ISO)-induced myocardial infarction (MI) in rat model. Pre-treatment with STS has dramatically increased ERK phosphorylation, and subsequently enhanced the expressions of Nrf2 and HO-1 (Wei et al., 2013). DMY has also been shown to ameliorate oxLDL-induced oxidative injury in HUVECs through activation of Akt/ERK/Nrf2/HO-1 (Luo et al., 2017). Similarly, MLG protects against t-BHP-triggered cytotoxicity and attenuated ROS generation by inducing antioxidant enzymes expression and ERK phosphorylation in RAW 264.7 and J774A.1 murine macrophage cell lines. Interestingly, MLG has also shown to phosphorylate AMPK and GSK3β, which leads to upregulation of Nrf2 and its downstream target genes (Zhou et al., 2017). Besides targeting ERK pathway, other cardioprotective natural products such as butin, xanthohumol, botulin, fisetin, paeonol and danshensu combination (PDSS), punicalagin and 3-caffeoyl, 4-dihydrocaffeoyl quinic acid can activate Nrf2 through phosphorylation AMPK, GSK3, p38 MAPK, and PI3K/AKT.

Glutathione (GSH) is a sulfhydryl group tripeptide composed of glutamate, cysteine and glycine. It plays an important role in cellular redox homeostasis. Several studies suggested that GSH can protect cells against oxidative stress due to its capability in interacting with antioxidant enzymes. Examples of GSH-linked defense enzymes include GSH peroxidase (GPX), GST, glutathione reductase (GRD), thioredoxins (Trx), glutaredoxin (GRX2), and PRX (Ribas et al., 2014; Ye et al., 2015). Glutathione is present mainly in reduced form and it is only oxidized into glutathione disulfide (GSSG) in the presence of oxidative stress. Thus, the ratio of GSH and GSSG within the cells is often used as the index for intracellular oxidative stress. Furthermore, clinical evidences have shown an association between GSH level and CVD. Low GSH level was observed in patients with the most severe cases of heart failure. Patients with cardiac diseases showed 21% depletion in blood GSH than healthy controls (Damy et al., 2009). Chronic depletion of myocardial GSH levels was reported in GCLM-deficient mice after transverse aortic constriction-induced pressure overload, and this condition leads to increased left ventricular dilation, myocardial fibrosis, and dysfunction (Watanabe et al., 2013).

Recent studies have reported the role of natural products at inducing glutathione-linked enzymes via Nrf2 signaling pathway. Azafrin, a natural carotenoid, have shown to induce Nrf2 downstream target genes such as HO-1, GCLC, GCLM, Trx1 and GST (Yang et al., 2018a). This is in consistent with another study showing that pre-treatment with triptolide, a diterpenoid epoxide protected Wistar rat from myocardial ischemia/reperfusion injuries by suppressing the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and inducing Nrf2-regulated antioxidant enzymes (SOD, GSH, GPx, and HO-1) (Yu et al., 2016). Besides, coffee constituent 5-O-caffeoylquinic acid (CGA) has also been shown to enhance Nrf2 nuclear translocation and increase the transcriptional expression of γ-GCL, HO-1 and GSTA1 (Boettler et al., 2011). Curcumin on the other hand has been found to attenuate hemin-induced ROS production and increase the ratio of GSH/GSSG in primary cultures of rat cerebellar granule neurons (CGNs). Furthermore, it also increased the cytoprotective enzymes such as HO-1, GR, GST, and SOD via inducing Nrf2 nuclear translocation (Gonzlez-Reyes et al., 2013).

The transcription factor NF-κB plays a crucial role in regulating innate immunity and inflammatory responses, and it is also involved in the pathogenesis of atherosclerotic plaques formation (Maracle et al., 2018). Recent studies showed that there are potential crosstalk between NF-κB and Nrf2. Nrf2 deficient mice were found to have increased NF-κB activation, inflammatory cytokines TNF-α, IL-1β, and IL-6 production, and ICAM-1 expression in brain after traumatic brain injury compared to wild-type mice (Jin et al., 2008). Evidence suggests that Nrf2/Keap1 pathway can inactivate NF-κB activity through ubiquitin-mediated degradation of IKKβ (Lee et al., 2009). Besides, it has been reported that p65, a canonical NF-κB subunit antagonized the transcriptional activity of Nrf2 by depriving Nrf2 transcriptional co-activator CBP (Liu et al., 2008). These data suggest that there is a crosstalk between Nrf2 and NF-κB in regulating the transcription of its downstream target proteins.

Several natural compounds have been reported to protect against CVDs by targeting both Nrf2 and NF-κB signaling pathways. For instance, Ligustilide, a phthalide compound inhibited VCAM-1, ICAM-1 and E-selectin expression by suppressing the NF-κB activation as well as inducing Nrf2-mediated HO-1 expression in TNF-α-stimulated HUVECs (Choi et al., 2018). Similarly, bioactive compounds such as cyanidin-3-O-glucoside and isothiocyanates have been reported to counteract the pathogenesis of endothelial dysfunction, including upregulation of Nrf2-dependent antioxidant response elements (HO-1, GCLC and GCLM) and downregulation of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) via inhibition of NF-κB activation (Huang et al., 2013; Fratantonio et al., 2015). Furthermore, Antrodia salmonea, a medicinal fungal species exerts an anti-angiogenic and anti-atherogenic activity in human vascular endothelial cell line (EA.hy 926) and human leukemic monocyte lymphoma cell line (U937). It significantly suppressed TNF-α-induced matrix metalloproteinase-9 (MMP-9) and ICAM-1 expression via suppressing I-κB degradation and I-κB kinase phosphorylation, as well as upregulating the expression of HO-1 and γ-GCLC through Nrf2 signaling pathway (Yang et al., 2014).

Macrophage foam cell formation represents the early hallmarks of atherosclerosis lesion formation. Foam cells formation is closely associated with abnormal cholesterol metabolism that results from imbalanced cholesterol uptake and efflux. Macrophages may take up modified LDL via scavenger receptors or through the pinocytosis process. Scavenger receptors such as scavenger receptors class A (SR-A), scavenger receptor class B (CD36), and lectin-type oxidized LDL receptor (LOX-1) have been implicated in the pathogenesis of atherosclerosis (Kunjathoor et al., 2002; Schaeffer et al., 2009). Several studies suggested that SR-A and CD36 exert pro-atherogenic properties due to their ability to interact with modified LDL, thereby contributing to the foam cell formation. Silencing SR-A or CD36 alone in LDLR^-/- ApoB100 mice was shown to profoundly protect against atherosclerosis (Mäkinen et al., 2010). Besides, studies have shown that cholesterol efflux transporters such as scavenger receptor class B type 1 (SR-B1), ABCA1 and ABCG1 promote efflux of free cholesterol to apolipoproteinA-1 (apoA-1) and HDL. Deficiency of either one or both efflux transporters (ABCA1 or ABCG1) have shown to enhance lipopolysaccharide (LPS)-induced inflammatory gene expression, reduce aortic endothelial NO synthase (eNOS), increase monocyte adhesion and infiltration into atherosclerotic plaque (Westerterp et al., 2016). Similarly, double-knockout ABCA1 and ABCG1 mice administered with a high cholesterol diet exhibited extensive infiltration of macrophage foam cells in the myocardium and spleen and have shown accelerated progression in atherosclerosis (Yvan-Charvet et al., 2007; Westerterp et al., 2013).

The role of Nrf2 in the transcriptional regulation of these scavenger receptors and cholesterol efflux transporters has been established. Many recent studies have reported the role of natural compounds in modulating cholesterol uptake and efflux receptors by targeting the Nrf2 signaling pathway. Oleanolic acid (OA), a natural pentacyclic triterpenoid, exerts anti-atherosclerotic effect in quail models and HUVECs where it was shown to inhibit oxLDL-induced LOX-1 and NADPH oxidase subunits expression while increasing the expression of Nrf2 and HO-1 (Jiang Q. et al., 2015). Furthermore, salidroside protects against foam cells formation by upregulating ABCA1 and downregulation of LOX-1 via activation of MAPK/Akt/Nrf2 pathways (Ni et al., 2017). This is consistent with another study which showed that Tanshinone IIA (Tan) inhibit atherosclerotic plaque formation in ApoE^-/- mice and it is suggested that Tan regulates cholesterol metabolism by reducing the expression of SR-A, while further enhancing ABCA1 and ABCG1 expression in human THP-1 cells via activation of ERK/Nrf2/HO-1 pathway (Liu et al., 2014). Other plant constituents (as shown in Table 2) such as Tanshindiol C, epigallocatechin-3-gallate, and 4-O-methylhonokiol (MH) can regulate SR-A and CD36 receptors via Nrf2 activation and its downstream effects.