**3. Redox modulation of NF-**κ**B**

Imbalance in redox state is redox modulation. The redox state of cells controls the activation and inhibition of NF-κB, as in the state of oxidative stress that can both activate and inhibit NF-κB by targeting the upstream kinases [30]. Activation of NF-κB by regular signaling is well known, however NF-κB activation also depends on redox state of cells in three possible ways: (i) many NF-κB -activating substances cause the production of reactive oxygen species (ROS) superoxide, H2O2, lipoxygenase products or act as oxidants on their own, (ii) NF-κB activation can be caused by superoxide H2O2 or organic hydroperoxide in some cell lines when no physiological stimulation is present, and (iii) A wide range of NF-κB inhibitors inhibit NF-κB – activation and antioxidants that are chemically unrelated. These observations have led to a consensus that NF-κB activation is related to some oxidative reaction. Molecules like thioredoxin, escalates the activity of NF-κB to bind DNA under oxidative stress [31]. A component of dynein motor complex LC-8 also participates in redox regulation of NF-κB. It activates NF-κB on exposure of TNFα and results in ROS production which oxidizes LC-8 and its dissociation from IκBα thus leading to NF-κB activation. Reportedly, NF-κB activation has anti- oxidant and pro-oxidant roles, the former involves the suppression of ROS accumulation, autophagy promotion, Inhibition of JNK activation and increased anti-oxidant targets whereas the pro-oxidant role includes the induction of pro-oxidation genes. One of the most important molecules in regulating redox modulation is hydrogen peroxide, it has been a question of debate, if H2O2 is involved in redox activation of NF-κB. As indicated in literature, TNFα induced activation of NF-κB mediated by H2O2. TNFα is a strong activator of NF-κB, that induces superoxide formation in mitochondria. As in Wurzberg cells where H2O2 directly activates NF-κB. The findings were found to be inefficient when lymphoblastoid cell lines, Jurkat cells showed no results of NF-κB activation by H2O2 [31, 32]. Various exogenous and endogenous sources can enhance the redox reaction. Redox reactions play a huge role in inflammation specifically in lung inflammation where oxidative injury is most common due to its structure and function. ROS production is an immune response against inhaled pathogens and pollutants like cigarette smoke, automobile exhaust. Excess production of endogenous ROS leads to chronic inflammatory lung disease such as chronic obstructive pulmonary disease, asthama and pulmonary fibrosis. Oxidative stress produced by cigarette smoke activates NF-κB by activating IKK complex which interferes with the chromatin modifications that escalate the transcription of pro-inflammatory genes [33].

### **4. Nrf2: the master regulator**

The nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a member of the cap 'n' collar (CNC) subfamily of basic region leucine zipper (bZip) transcription factors including nuclear factor erythroid-derived 2 (NFE2) and NRF1, NRF2, and NRF3. There are seven conserved NRF2-ECH homology (Neh) domains within NRF2 gene, with different functions to control NRF2 transcriptional activity. The bZip in the Neh1 domain acts to activate gene transcription by forming dimer with small

musculoaponeurotic fibrosarcoma proteins (sMAF). Neh2 domain mediates NRF2 ubiquitination and degradation as it contains ETGE and DLG motifs which act together with Kelch domain of Kelch-like-ECH-associated protein 1 (KEAP1) [34]. The Neh3-5 domains find their role as transcriptional activation domains, Neh6 domain works to mediates Nrf2 degradation in cells experiencing oxidative stress. Neh7 domain mediates interaction with retinoic X receptor alpha (RXRα), which represses Nrf2 activity. It is involved in the control of development of labial and mandibular segment of Drosophila by basic leucine zipper DNA binding domain (bZip) homeotic gene [35].

Removal of Nrf2 alters the defense machinery of the cell against oxidative stress. Knocking out Nrf2 has no effect on the mortality of the mice. Basal level expression of Nrf2 in the cytoplasm ensures the production of cytoprotective proteins to exert normal physiological redox homeostasis [36]. Modulation in redox status is known to activate prosurvival redox sensitive Nrf2 pathway. Under normal condition Nrf2 is sequestered in cytoplasm by the inhibitor KEAP-1. Abrogation of KEAP-1 binding leads to translocation of Nrf2 to the nucleus mediated by nuclear localization signal. In nucleus Nrf2 forms a heterodimer with the co-transcription factor MAF. The heterodimers bind to the corresponding antioxidant response element and induces the expression of downstream cytoprotective and antioxidant enzymes [37]. The Nrf2 system is considered to be a major cellular defense mechanism against cellular oxidative stress. Nrf2 plays an important role in cellular defense and in improving the removal of ROS by activating downstream genes that encode phase II detoxifying enzymes and antioxidant enzymes, such as GCLM, NQO1, HMOX1, GPX, and glutathione S-transferases (GST) [38]. Nrf2 controls the expression of key components of the glutathione (GSH) and thioredoxin (TXN) antioxidant system, as well as enzymes involved in NADPH regeneration, ROS and xenobiotic detoxification, heme metabolism, thus playing a fundamental role in maintaining the redox homeostasis of the cell. Excessive ROS production causes oxidative stress to increase mitochondrial DNA damage, further promotes the activation of oncogenes or the inactivation of anti-oncogenes, which facilitates its tumorigenic signaling pathways and tumor progression. Nrf2/ARE pathway protects cells against oxidative stress via regulating the expression of Sestrin 2 gene as evident by monitoring the expression of downstream antioxidants. Sestrin 2 has strong antioxidant capacity and can provide cell with cytoprotective against various harmful stimuli. Sestrin blocks mTOR expression and mitigates the accumulation of ROS [39].
