Abstract
The objective of this study is to investigate the contributing effect of the nuclear transcription factor-erythroid 2-related factor 2 (Nrf2)-mediated signaling pathway on the indirect antioxidant capacity of caffeic acid phenethyl ester (CAPE) against oxidative stress in HepG2 cells. The result of an antioxidant response element (ARE)-luciferase assay showed that CAPE stimulated ARE promoter activity resulting in increased transcriptional and translational activities of heme oxygenase-1 (HO-1). In addition, CAPE treatment enhanced Nrf2 accumulation in the nucleus and the post-translational phosphorylation level of extracellular signal-regulated kinase (ERK) among several protein kinases tested. Treatment with ERK inhibitor U126 completely suppressed CAPE-induced ERK phosphorylation and HO-1 expression, but it only partly inhibited CAPE-induced Nrf2 accumulation and ARE promoter. Using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) method, the cellular antioxidant capacity of CAPE against 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)- or H2O2-induced oxidative stress also was shown to be partially suppressed by the ERK inhibitor. From the overall results it is proposed that the indirect antioxidant activity of CAPE against oxidative stress in HepG2 cells is partially attributed to induction of HO-1, which is regulated by Kelch-like erythroid-cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1)-independent Nrf2 activation relying on post-translational phosphorylation of ERK.
Highlights
The cellular protection against oxidative stress can be carried out by antioxidants in direct and indirect ways depending upon the type of working mechanism [1]
antioxidant response element (ARE) is a cis-acting DNA regulatory element present in the promoter/enhancer regions of genes encoding many antioxidant and detoxifying enzymes; and to test whether or not caffeic acid phenethyl ester (CAPE), as an indirect antioxidant, stimulates the transcription of ARE-related gene in HepG2 cells, a luciferase reporter plasmid carrying ARE promoter was introduced to HepG2 cells
On ARE prompter activity were examined over 24 h, and the results of an ARE-luciferase assay showed that CAPE treatment caused a concentration-dependent induction of ARE promoter activity with peak activity seen at 20 μM (Figure 1A); 20 μM was used as the concentration of reference in all subsequent experiments
Summary
The cellular protection against oxidative stress can be carried out by antioxidants in direct and indirect ways depending upon the type of working mechanism [1]. Direct antioxidants, which are always redox active, scavenge reactive oxygen and nitrogen radical species by being consumed or chemically modified. They have to be replenished or regenerated. In contrast with direct antioxidants, indirect antioxidants may or may not be redox active Indirect antioxidants exert their antioxidant effects through upregulating phase II detoxifying and antioxidant enzymes [1]. CAPE has been shown to stimulate heme oxygenase-1 (HO-1) through promoting inactivation of the Nrf2-Keap complex in renal epithelial cells [19], there has been no study to elucidate the induction of phase II detoxifying and antioxidant enzymes through Nrf activation in hepatic cells. We propose that CAPE exerts indirect antioxidant capacity by up-regulating the expression of phase II antioxidant and detoxifying enzyme HO-1 via the ERK-Nrf signaling pathway
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