**5. Redox regulation of Nrf2**

Redox regulation underlines the cellular homeostasis. Regulation of redox sensitive transcription factors play a pivotal role in determining the cellular fate. Nrf2 being a master regulator is at the focal point of maintaining and regulating the cellular redox equilibrium. Perturbation in redox equilibrium is known to modulate the Nrf2 activation and hence effect the cellular fate [19]. Spatio-temporal generation of oxidative stress determines the graded activation of redox sensitive mediators. Mild oxidative stress is known to activate Nrf2 pathway and increases the cytoprotective proteins. Redox based activation of Nrf2 is attributed to the presence of more than 20 critical cysteine residues present in the KEAP-1 protein (**Figure 2**) [40]. 273 and cys288 have been shown as critical for abrogating KEAP-1 mediated inhibition of Nrf2. Mutation in these residues render activation of Nrf2 by inhibiting the Cul3-E3- KEAP-1 mediated degradation of Nrf2. The mutation did not had any effect on the

*Perturbation of Cellular Redox Status: Role of Nrf2, a Master Regulator of Cellular Redox DOI: http://dx.doi.org/10.5772/intechopen.102319*

#### **Figure 2.**

*Redox regulation of Nrf2 pathway. Under non-stressed condition, Nrf2 is sequestered by its inhibitor KEAP-1 in the cytoplasm by inducing ubiquitin mediated Nrf2 degradation. Increase in ROS and electrophiles induces oxidative stress which results in oxidation of critical cysteine residues in the redox sensitive transcription factors. Cysteine residues of KEAP-1 are oxidized to release Nrf2 which translocates to the nucleus to express the downstream genes of antioxidant and cytoprotective potential. Thus, redox perturbation and tickling induces the Nrf2 pathway that act as master regulator for maintaining cellular redox equilibrium in turn governing the cellular fate.*

detachment of the KEAP-1:Nrf2 complex, thus allowing the nuclear translocation of free Nrf2. Further, cys151 was also implicated in redox modulator-based activation of Nrf2 pathway. Perturbation in cellular redox status by prooxidants have been shown to induce cellular oxidative stress and increase the glutathionylation of proteins. 1,4 Naphthoquinone treatment induced glutathionylation of KEAP-1 for inducing Nrf2 pathway owing to its prooxidant nature. Apart from the cytoprotective nature of Nrf2, its role as a redox sensitive anti-inflammatory transcription factor has been well documented. Multiple reports have highlighted redox modulation based modulating role of Nrf2 in ameliorating immune-pathologies [41]. Nrf2 knockout mice exhibited increased bronchial inflammation, prolonged inflammation, high susceptibility for autoimmune syndrome, elevated lymphocyte proliferation and impaired redox homeostasis. Nrf2 dependent proteins including HO-1 inhibits the cytokine secretion, leukocyte migration, adhesion and suppressed LPS induced production of tumor necrosis factor-a (TNF-a) and nitric oxide (NO) in murine macrophages. Ablation in HO-1 protein increases the susceptibility towards autoimmune diseases [42].

### **6. Therapeutic potential of Nrf2**

Excessive reactive oxygen species are threat to cells redox homeostasis which are major cause of oxidative stress leading to maladies like cellular dysfunction in aging, cardiovascular disease, renal dysfunction, diabetes, cancer is some to name a few. Antioxidant therapies play an important role in combating the progress of these diseases but the results are not satisfactory therefore there is an urgent need for a solution which activates endogenous antioxidant defenses. The redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) plays a significant role in synchronizing cellular antioxidant defenses and maintaining redox homeostasis [43]. Nrf2 is the

"master regulator" of the antioxidant response, it modulates the expression of many genes, that include antioxidant enzymes, immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis. NRF2 activation by Keap1 binding is one of the major pathways that senses the oxidative stress, particularly there are Four reactive cysteine residues identified in Keap1 are most likely nominees for being the direct sensors of oxidative stress, various alternative mechanisms for Nrf2 activation were discovered, which are dependent upon kinase pathways, these include mitogen-activated protein kinases (MAPK), phosphatidylinositol-3 kinase and atypical protein kinase(s) C. Cell culture experiments report many compounds which show the ability to activate Nrf2. Large number of Nrf2 activators are principally naturally-occurring and plant-derived such as sulforaphane and curcumin and found in foods, but synthetic compounds have also shown to act as activators for instance bardoxolone methyl [44].

The Therapeutic potential of Nrf2 and its activators have been studied in various diseases. Some of them are as follows:

#### **6.1 Diabetic nephropathy**

It is the leading cause of chronic kidney disease, some mechanisms contribute to the onset and pathogenesis of diabetic nephropathy, including genetic and hemodynamic factors, oxidative stress, and cytokine signaling. Diabetes triggers oxidative stress through different ways, such as advanced glycation end-product accumulation and activation of polyol pathway, protein kinase C pathway, and renin angiotensinaldosterone system. Loss or decrease in expression of SOD or glutathione in renal diseases are overcome by restoring and check the progression of disease. Nrf2 regulates expression of genes through ARE (antioxidant response elements) in their promoters to neutralize free radicals and accelerate removal of environmental toxins. Protective role of Nrf2 against renal damage has been demonstrated on streptozotocin induced diabetic rats, wherein it was shown that it slows the progression of diabetic nephropathy, Nrf2-mediated protection works through the negative regulation of TGF-b1 and p21/WAF1Cip1 (p21) [45]. Reports suggest the Nrf2-dependent anti-oxidative and anti-inflammatory effects of digitoflavone in streptozotocin-induced diabetic nephropathy, *in vitro* using SV40-transformed mouse mesangial cells (SV40-Mes13), results showed that Digitoflavone activated Nrf2, reduced oxidative damage, inflammation, TGF-β1 expression, extracellular matrix protein expression and mesangial cell hyperplasia [46]. Enhancement of NRF2 activity in the renal tubules considerably improves damage related to kidney injury and prevents its progression to chronic kidney disease (CKD) by reducing oxidative stress. KEAP1-NRF2 system along with Nrf2 activators have also been proposed to be a target for renal defense, wherein KEAP 1 inhibitors like CDDO-methyl ester, a synthetic triterpenoid are the main targets for the study as they work by inhibiting KEAP1-Nrf2 bonding which lets stabilized and free Nrf2 to translocate to nucleus and activate downstream genes [47]. Another strategy employed is suppressing the degradation of Nrf2 via inhibiting proteasome activity by inhibitors like minocycline, Increasing of Nrf2 concentrations within the cells have been found to be effective against the renal damage, reports suggest that Zinc helps elevating the Nrf2 protein level within the nucleus and upregulated the expression of Nrf2 downstream enzymes by encouraging inhibition of Nrf2 nuclear promoter Fyn mediated by Akt/GSK-3β, Resveratrol and its analogue (polydatin) have reported to activated Nrf2/ARE pathway through upregulating Sirt1 (NADdependent histone deacetylase in the nucleus) in glomerular mesangial cell [48].

*Perturbation of Cellular Redox Status: Role of Nrf2, a Master Regulator of Cellular Redox DOI: http://dx.doi.org/10.5772/intechopen.102319*

#### **6.2 Cancer**

Systematic studies of carcinogenesis specify an important role of endogenous oxidative damage to DNA, and an imbalance of cellular redox homeostasis that is balanced by elaborate defense and repair processes [49]. Pancreatic cancer is the most fatal diseases, it has very high rate of metastasis, Keap1-Nrf2 pathway is an emerging target for PC prevention and therapy. Certain modulators like UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) is overexpressed in pancreatic cancer and are correlated to tumor growth. UHRF1 suppresses Keap1 expression by promoter methylation, this leads to Nrf2 activation. MBD 1 and p62 have been reported to inhibit ROS and promote tumor growth and drug resistance by inducing Nrf2 accumulation, nuclear translocation and activation. Nrf2 activation by a has also been found to inhibit PC cell growth and induce apoptosis by upregulating HO-1 [50]. The compound D3T (3*H*-1,2-dithiole-3-thione) has been shown to increase the nuclear accumulation of Nrf2, Honaucin A, natural marine-based compound, obtained from cyanobacteria, forms a covalent bond with the sulfhydryl groups on KEAP1, resulting in the activation of Nrf2, phenol, polyphenol, or triterpenoid majorly form class of compounds that activate Nrf2. Sulforaphane is highly electrophilic molecule, it non-covalently binds to sulfhydryl groups of KEAP1 resulting in Nrf2 activation. Sulforaphane can also activate antioxidant response elements (AREs) associated with Nrf2. Micro RNA's (miRNAs) miR-141, miR-432-3p, miR-200a, have also shown to modulate the activities of KEAP1 and NRF2 in ovarian carcinoma cell lines, breast cancer, esophageal squamous cell carcinoma (ESCC), endometrial cancer tissues, miR-7, directly targeted KEAP1 mRNA in neuroblastoma SH-SY5Y cells, where it activated NRF2-dependent transcription of the antioxidant genes HMOX1 and GCLM [49]. Aberrant Nrf2 activation with in cancer cells may be due to somatic mutations within the Nrf2, KEAP1, or CUL3 genes, or the increase of KEAP1 interacting proteins, such as p62/Sqstm1 and p21, or it may be due to cysteine modification by oncometabolites such as fumarate all of the above activities may confer resistance to cancer cells and hence form the major targets for the cancer therapies. Nrf2 inducers are reported to hasten the detoxification of carcinogens (often electrophiles) from the cell and hence protect from chemical carcinogenesis Nrf2 inhibitors like brusatol work as protein synthesis inhibitors, ML385, a thiazole-indoline compound binds to Neh1. Therefore, Nrf2 inducers and Nrf2 inhibitors may function as anticancer drugs, with different effects on different targets, Nrf2 inducers work in order to protect normal cells from carcinogens, whereas Nrf2 inhibitors suppress the proliferation of cancer cells that have acquired aberrant Nrf2 activation or Nrf2 addiction [51]. Keap1-deficient mice showed upregulation of detoxifying enzymes, including GST and NQO1, and higher Nrf2 activation [52]. Various epigenetic regulations like hypermethylation, histone modifications control the expression of Nrf2 and hence may form targets to different therapeutic strategies [53].

#### **6.3 Pulmonary fibrosis or lung injury and inflammation**

Pulmonary fibrosis is a progressive and irreversible disease; it is characterized by an increase in differentiation and of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. A study reports antifibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on a long noncoding RNA [54]. Therapeutic potential of thymoquinone (TQ ) in bleomycininduced lung fibrosis (BMILF) were also investigated and it was seen that it decreases expressions of Nrf2, Ho-1 and TGF-β. Nrf2/Ho-1 signaling pathway is a principal target for TQ protective effect against BMILF in rats [55]. The protective role of Nrf2 is mediated by PPARγ in hypoxia-induced Acute Lung Injury (ALI). Reports reveal that overexpression of Brg1 increases Nrf2 activity and reduces ROS and inflammatory factors in lung tissues. In lipopolysaccharide (LPS)-induced lung inflammation the defensive role of the PI3K/Akt-dependent activation of the Nrf2-HO-1 pathway was revealed in mice treated with desoxyrhapontigenin. Nrf2 knockout resulted in a worsening of asthma symptoms. Protective role of Nrf2 in emphysema induced mice can be correlated by its activation in alveolar macrophages. The role of Nrf2 dysfunction in COPD may be the result of loss of DJ-1. DJ-1 overexpression activates Nrf2 and inhibits apoptosis of alveolar type II cells that are undergoing Cigarette smokinginduced oxidative stress and inflammatory response. DJ-1 induces the activation of Nrf2 and increases the expression of downstream antioxidant machinery to reduce the oxidative stress. The underlines anti-inflammatory effects are attributed to the expression of HO-1. These findings highlight the role of DJ-1 as putative target for cigarette smoking induced lung diseases [56].

#### **6.4 Neurodegeneration**

Neurodegenerative conditions may be results of various primary causes which include including expression of certain gene alleles, toxicant administration, aging, protein aggregation, proteasomal or autophagic dysfunction, inflammation, neuronal apoptosis, oxidative stress, mitochondrial dysfunction, and interactions between neurons and glia. The earliest degenerative condition to be associated with oxidative stress was aging. Some diseases characterized as neurodegenerative are Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease, Huntington's disease, Friedrich's ataxia, multiple sclerosis, and stroke. Nrf2 activation provides neuroprotection against oxidative stressors and mitochondrial toxins, including hydrogen peroxide, tert-butyl hydroperoxide, 6-hydroxydopamine, 3-nitropropionic acid (3-NP), 1-methyl-4-phenylpyridinium (MPP), and rotenone [57]. In various studies conducted water derivative of artemisinin namely artesunate and lipid soluble derivative artemisinin, artemether both show to enhance the activation of Nrf2 via increasing its nuclear translocation and binding to downstream antioxidant response elements, as well as through suppressing ROS-dependent p38 MARK and NF-κB pathways [58]. Report suggests that SFN, an isothiocyanate compound that occurs naturally and can be derived from cruciferous vegetables such as broccoli is capable of activating Nrf2, Results show that SFN is able to cross the blood brain barrier, activate Nrf2-dependent gene expression in the basal ganglia, eventually protecting nigral dopaminergic neurons from cell death induced by MPTP. Wide variety of bioactive compounds like resveratrol, curcumin, naphthazarin, genistein, and carnosic acid and berberin have been reported as Nrf2 activators that show positive effects in neurodegenerative disorders by protecting dorsal root ganglion (DRG) neurons from glucose-induced injury also by antioxidant activity in primary spinal cord astrocytes exposed to H2O2 [59].
