The underlying mechanism by which local plant Piper betle (PB) stimulates Nrf2 pathway has yet not been elucidated. Our results on NQO1-ARE luciferase activity, mRNA, phase I oxidoreductase and phase II detoxifying enzymes showed that PB induced Nrf2/ARE pathway.
This study illustrated that Nrf2 deficiency in MEF cells (N0) resulted in increased cytotoxicity with PB treatment compared to WT cells. Hydroxychavicol which is found in PB was shown to exert antioxidant activity at low concentration but display pro-oxidant effect at concentration higher than 0.1 mM . Similarly, Nrf2-/- MEF cells were shown to be more sensitive to SFN treatment, peroxides, anti-cancer drugs (Displatin) and compounds that generate free radical compared to Nrf2+/+ MEF cells .
The Nrf2-Keap1-ARE signaling system is central to the induction of phase 2 genes by electrophiles and antioxidants [16, 17, 20, 21]. We showed that PB was able to activate Nrf2 and translocated into the nucleus, with increased expression of NQO1 and HO-1 genes and proteins. We postulate that the mechanism of interaction between PB and Nrf2-Keap1 complex may mimic the action of other Nrf2 inducers such as sulforaphane and Gingko biloba extract. Keap1 protein contains multiple cysteine residues and specifically C257, C273, C297 and C288 were shown to be the most reactive residues that can interact with phase 2 inducers including sulforaphane . Upon exposure to inducers, the reactive cysteine residues form intermolecular disulfide bonds and change its conformation which leads to dissociation of Nrf2 from Keap1, translocating Nrf2 into the nucleus, and activating the expression of phase 2 genes . Ginkgo biloba extract stimulated Nrf2, and translocated it into the nucleus with subsequent increase in phase 2 genes such as GCLC, GST and NQO1 through Keap1-Nrf2-ARE signaling pathway in Hepa1c1c7 and HepG2 cells . The active compound in coffee, 5-O-caffeoylquinic acid (CGA) modulates Nrf2 nuclear translocation and ARE-dependent gene expression such as HO-1, Nrf2, GST, γGCL in HT29 cells .
In this study, we showed that Nrf2 translocation is better at 10 h incubation of PB, and for ARE induction, genes and proteins expression, we incubated the cells with PB for 24 h, which is similar to other studies [27, 29]. Sulforaphane caused nuclear translocation of Nrf2 after 6 h treatment by Western blotting; however immunocytochemistry data revealed that translocation of Nrf2 in the nucleus was not seen in many cells at 6 h treatment compared to 24 h incubation that displayed high amount of Nrf2 in the nucleus of human keratinocytes cells .
The antioxidant response elements (ARE) are a cis-acting enhancer sequence located in the 5’-flaking promoter region of antioxidant and detoxifying genes such as GST, HO-1, GCLC and NQO1 [30–35]. The luciferase reporter assay further confirmed that PB induced ARE promoter region in the nucleus. Our results are supported by Shin et al.  who reported that sulforaphane, a well known Nrf2 activator, increased luciferase activity 3.5-fold in rat kidney epithelial NRK cells. Other dietary agents which activate ARE reporter gene with increased expression of Nrf2-targeted genes such as NQO1, HO-1 and GCLC include resveratrol and xanthohumol [36, 37]. Other antioxidant, curcumin activated glutathione S-transferase P1 (GSTP1) gene expression by activating the expression of the luciferase gene from a reporter construct carrying GSTP1-ARE .
The increased expressions of Nrf2, NQO1 and GSTA1 genes in WT cells but not in N0 cells further validated our claim that PB is a Nrf2/ARE inducer. McWalter et al.  reported that transcription factor, Nrf2 was activated by broccoli seeds and isothiocyanates, with induction of NQO1, GST and GCLC genes and proteins in Nrf2+/+ but not in Nrf2-/- mice. Our results are supported by the finding of Yoon et al.  who found that sulforaphane increased NQO1 and HO-1 proteins in human proximal cells. Cho et al.,  have shown that Nrf2 gene was upregulated at least 2-fold higher in Nrf2+/+ mice than in Nrf2-/- mice at basal level and also in hyperoxia-induced mice. The possibility of polyphenols in PB such as chavicol or euginol inducing Nrf2 was also reported by a study of Tsai et al., who showed that phenolic compounds from rosemary and carnosic acid induced transcription of Nrf2, ARE luciferase reporter activity and increased expression of NQO1 gene and protein in rat clone 9 cells . Lycopene metabolite, apo-8’-lycopenal that can be found in rat liver and human plasma after consuming lycopene-containing food, induced nuclear translocation, Nrf2-ARE binding activity, HO-1 and NQO1 genes and proteins expression in human hepatoma cell lines, HepG2 . The isothiocyanate, sulforaphane and the flavonoid, epigenin increased gene expression of phase II detoxifying enzyme such as glutathione S-transferase (GSTA1) and UDP-glucuronosyltransferase (UGT1A1) in human colon adenocarcinoma . L-sulforaphane modulates Nrf2-regulated genes including GSH biosynthesis (GCLC), glutathione S-transfreases (GSTA1, GSTA2, GSTA3, GSTM1) and NQO1 in small intestine of Nrf2+/+ and Nrf2-/- mice .
Our results showed decreased HO-1 mRNA expression after 24 h treatment with PB while its protein level was found to be increased. This finding is similar to a time-course study by Motterlini et al.  who showed that HO-1 mRNA expression in bovine aortic endothelial cells reached a maximum after 6 h treatment with 5 μM curcumin and declined after 24 h treatment.
Contrary to many findings, we noted that SOD1 gene and protein were not expressed in WT but were significantly expressed in N0 cells. Others who found similar results reported that the basal level of some antioxidant enzymes may not be under the influence of Nrf2 gene [45, 46]. Kwak et al.  found that MnSOD gene was 1.31 fold higher in Nrf2-disrupted mice compared to wild-type mice at constitutive levels, suggesting that Nrf2 is an essential factor to modulate many, but not all, antioxidant and phase 2 enzymes. The disruption of Nrf2 gene in Nrf2-/- may alter the basal and inducible expression of some genes including SOD1.