Nrf2⁻ARE Signaling Acts as Master Pathway for the Cellular Antioxidant Activity of Fisetin.

Huihui Zhang,Wan Zheng,Fei Yang,Jihua Chen,Hong Qin,De-Xing Hou,Xiangling Feng,Shusong Wu

doi:10.3390/molecules24040708

Abstract

Fisetin, a dietary flavonoid, is reported to have cellular antioxidant activity with an unclear mechanism. In this study, we investigated the effect of fisetin on the nuclear factor, erythroid 2-like 2 (Nrf2) signaling pathway in HepG2 cells to explore the cellular antioxidant mechanism. Fisetin upregulated the mRNA expression of heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H quinone oxidoreductase-1 (NQO1), and induced the protein of HO-1 but had no significant effect on the protein of GCLC, GCLM and NQO1. Moreover, nuclear accumulation of Nrf2 was clearly observed by immunofluorescence analysis and western blotting after fisetin treatment, and an enhanced luciferase activity of antioxidant response element (ARE)-regulated transactivation was obtained by dual-luciferase reporter gene assays. In addition, fisetin upregulated the protein level of Nrf2 and downregulated the protein level of Kelch-like ECH-associated protein 1 (Keap1). However, fisetin had no significant effect on Nrf2 mRNA expression. When protein synthesis was inhibited with cycloheximide (CHX), fisetin prolonged the half-life of Nrf2 from 15 min to 45 min. When blocking Nrf2 degradation with proteasome inhibitor MG132, ubiquitinated proteins were enhanced, and fisetin reduced ubiquitination of Nrf2. Taken together, fisetin translocated Nrf2 into the nucleus and upregulated the expression of downstream HO-1 gene by inhibiting the degradation of Nrf2 at the post-transcriptional level. These data provide the molecular mechanism to understand the cellular antioxidant activity of fisetin.

Highlights

Reactive oxygen species (ROS) are chemical species containing oxygen, such as peroxides, superoxide, hydroxyl radical, singlet oxygen [1]
1a)h,atand thea intracellular level, HepG2 cells weremechanism exposed toof different of fisetin for 24 intracellular level, HepG2 cells were exposed to different concentrations of fisetin for h, and a methyl thiazolyl tetrazolium (MTT) assay was subsequently performed
We found that the growth methyl thiazolyl tetrazoliumincreased (MTT) assay was subsequently performed

Summary

Introduction

Reactive oxygen species (ROS) are chemical species containing oxygen, such as peroxides, superoxide, hydroxyl radical, singlet oxygen [1]. During times of environmental stress (for instance, ultraviolet radiation or heat exposure), ROS levels can increase dramatically [2]. If excessive ROS are not removed effectively, this will cause oxidative stress including DNA damage, lipid peroxidation, or protein damage. Molecules 2019, 24, x FOR PEER REVIEW diseases [3], especially chronic diseases, as Parkinson’s. Alzheimer’s disease, cancer, leads to pathological changes, and ROS aresuch associated with the disease, early onset of more than 200 clinical cardiovascular disease,chronic diabetes, etc. [3], especially diseases, such as Parkinson’s disease, Alzheimer’s disease, cancer, Phase II enzymes play a key in defending against oxidative damage and in scavenging cardiovascular disease, diabetes, etc.role [4,5,6,7,8]. [3], especially diseases, such as Parkinson’s disease, Alzheimer’s disease, cancer, Phase II enzymes play a key in defending against oxidative damage and in scavenging cardiovascular disease, diabetes, etc.role [4,5,6,7,8]. excess ROS.

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