Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

Abstract

In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography–tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2–/– mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.

Synthesis, evaluation, and metabolism of novel [6]-shogaol derivatives as potent Nrf2 activators

“…27 In the current study, we found that 6S (1) is even more active than SFN (2.40 vs. 2.03 times higher than vehicle) (Figure 1), a well-known potent activator of Nrf2. 37,38 Using quantitative microscopy, we showed GFP fluorescence intensity at the olfactory sensory neural epithelia of 6S-treated zebrafish embryos is stronger than those of SFN-treated individuals ( Figure 2).…”

“…25 In a recent study, we identified [6]-shogaol (6S), a major component of dry ginger, as an activator of Nrf2 in colon epithelial cells in vitro and in vivo. 26 Our data obtained with gene expression profiling, Western blotting and immunostaining clearly demonstrated that 6S induced nuclear translocation of Nrf2 and activated Nrf2 target genes in an Nrf2-dependent manner. 27 With an α,β-unsaturated carbonyl group in the alkyl tail, 6S is a typical Michael acceptor.…”

“…26 Our data obtained with gene expression profiling, Western blotting and immunostaining clearly demonstrated that 6S induced nuclear translocation of Nrf2 and activated Nrf2 target genes in an Nrf2-dependent manner. 27 With an α,β-unsaturated carbonyl group in the alkyl tail, 6S is a typical Michael acceptor. We hypothesized that 6S could activate Nrf2 via alkylation of cysteine residues of Keap1 protein.…”

“…Also, three conjugates 37, 37b, and 38 were detected in 26-treated zebrafish embryos ( Figure 6F) We have reported that 6S could activate Nrf2 via modification of Keap1 at 17 cysteine residues and these cysteine residues were located in all 5 domains of Keap1 protein. 26 In light of our findings above, we proposed that both ,-unsaturated carbonyl entities and catechol moieties in 6S derivatives could react with the reactive sulfhydryl groups of cysteine residues of Keap1, disrupting the Keap1-Nrf2 complex in the cytoplasm, thereby releasing Nrf2. However, this needs to be proved experimentally.…”

Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol

“…The pleasurable aroma of ginger is from the constituents present in the volatile oil, while the non-volatile pungent phytochemicals of ginger consisting of gingerols, shogaols, paradols, and zingerone give the warm pungent sensation and are also reported to account for the most of its pharmacological effects 12,13 . Activation of Nrf2 in epithelial cells, HepG2 cells and HEK293 cells by 6-S was disclosed recently [22][23][24] . Of all the gingerols, the compound 6-gingerol (6-G, Fig.…”