Semisynthetic flavonoid 7-O-galloylquercetin activates Nrf2 and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells

Abstract

The natural flavonoid quercetin is known to activate the transcription factor Nrf2, which regulates the expression of cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, a novel semisynthetic flavonoid 7-O-galloylquercetin (or quercetin-7-gallate, 3) was prepared by direct galloylation of quercetin, and its effect on the Nrf2 pathway was examined. A luciferase reporter assay showed that 7-O-galloylquercetin, like quercetin, significantly activated transcription via the antioxidant response element in a stably transfected human AREc32 reporter cell line. In addition, 7-O-galloylquercetin caused the accumulation of Nrf2 and induced the expression of HO-1 at both the mRNA and protein levels in murine macrophage RAW264.7 cells. The induction of HO-1 by 7-O-galloylquercetin was significantly suppressed by N-acetyl-l-cysteine and SB203580, indicating the involvement of reactive oxygen species and p38 mitogen-activated protein kinase activity, respectively. HPLC/MS analyses also showed that 7-O-galloylquercetin was not degalloylated to quercetin, but it was conjugated with glucuronic acid and/or methylated in RAW264.7 cells. Furthermore, 7-O-galloylquercetin was found to increase the protein levels of Nrf2 and HO-1, and also the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. Taken together, we conclude that 7-O-galloylquercetin increases Nrf2 activity and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells.

Flavonolignan 2,3-dehydrosilydianin activates Nrf2 and upregulates NAD(P)H:quinone oxidoreductase 1 in Hepa1c1c7 cells

“…As expected, 5 μM sulforaphane (a positive control) [27] significantly increased the expression of all four genes after 6 h of exposure (Table 2), and also upregulated the protein levels of NQO1, HMOX1, GCLC and GCLM after 24 h (Fig. 4).…”

“…To further investigate the effect of 2,3-dehydrosilydianin on Hepa1c1c7 cells, we analyzed the expression of selected Nrf2 target genes, including Nqo1 , Hmox1 , Gclc and Gclm . As expected, 5 μM sulforaphane (a positive control) [27] significantly increased the expression of all four genes after 6 h of exposure ( Table 2 ), and also upregulated the protein levels of NQO1, HMOX1, GCLC and GCLM after 24 h ( Fig. 4 ).…”

“…In contrast, stress conditions associated with the oxidation or covalent modification of cysteine residues in Keap1 and/or with the phosphorylation of Nrf2 inactivate Keap1 and thus stabilize Nrf2, which in turn accumulates in the cell and increases the expression of Nrf2 target genes [26]. Using quantitative real-time PCR and Western blot analysis, we found that 50 μM 2,3-dehydrosilydianin as well as 5 μM sulforaphane, a positive control [27], elevated the protein levels of Nrf2 in Hepa1c1c7 cells after 3 and 24 h of exposure (Fig. 4), while the levels of Nrf2 mRNA remained unaffected, as shown after 6 h (Table 2).…”

“…We can, however, speculate that some effects of silymarin may be mediated by minor silymarin flavonolignans (e.g. isosilybin) or by non-flavonolignan components [3], [4], of which the flavonoids taxifolin and quercetin were recognized as Nrf2 activators [27], [33], [34]. Moreover, the formation of potentially active metabolites should also be taken into account when interpreting the in vivo bioactivity of silymarin and flavonolignans.…”

Caffeic acid phenethyl ester protects against oxidative stress and dampens inflammation via heme oxygenase 1

“…28 SB203580 also decreases HO1 expression in other in vitro models, including neuronal cells 29 and RAW264.7 macrophages. 30–32 Thus, the ability of CAPE to regulate HO1 and consequently to counteract oxidative stress and inflammation may involve p38 MAPK signalling.…”