Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

https://doi.org/10.1016/j.taap.2013.10.018Get rights and content

Highlights

  • E7G activates Nrf2 in astrocytes.

  • E7G stimulates expression of Nrf2-mediated cytoprotective proteins in astrocytes.

  • E7G protects astrocytes against OGD-induced cell death and apoptosis.

  • The neuroprotective effect of E7G involves the Nrf2/ARE pathway.

  • E7G protects rats against cerebral ischemic injury.

Abstract

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke.

Introduction

Oxidative stress is a major contributor to cerebrovascular dysfunction and one of the main causes of tissue damage following ischemic insults in the brain (Chen et al., 2011). Phase II detoxifying and antioxidant enzymes are the primary means by which neuronal cells protect themselves from toxic reactive oxygen species (ROS). The induction of such enzymes is governed by the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2). Activation of Nrf2 represents a key step in endogenous cellular protection and is becoming a promising therapeutic target for neuroprotection. There is increasing evidence that induction of the Nrf2/Antioxidant response element (ARE) signaling pathway confers protection against cerebral ischemia-reperfusion injury (Alfieri et al., 2011, Son et al., 2010).

Astrocytes, the major glial non-neuronal cells, play an important role in the cellular antioxidant defense in the brain. They are the main source of glutathione (GSH) and supply the neurons with substrate for GSH synthesis to improve the neuronal antioxidative reserves (Dringen et al., 1999). ARE-regulated genes are preferentially activated in astrocytes, which consequently have more efficient detoxification and antioxidant defense than neurons. Activation of Nrf2 in astrocytes protects neurons from a wide array of potentially toxic insults. Nrf2 activation in astrocytes has thus been proposed as a novel therapeutic target for neuroprotection (Bell et al., 2011, Escartin et al., 2011, Vargas and Johnson, 2009).

Over the past two decades, epidemiological studies have linked the consumption of flavonoid-containing dietary sources to reduced risk of cardiovascular disease and stroke (Arab and Liebeskind, 2010, Leonardo and Dore, 2011). Eriodictyol-7-O-glucoside (E7G), one of the most abundant flavonoids isolated from the Chinese herb Dracocephalum rupestre, has been identified as a novel Nrf2 activator and can confer protection against cisplatin-induced toxicity through activation of the Nrf2 pathway (Hu et al., 2012). Our previous paper also showed that its aglycone eriodictyol protected PC12 cells against H2O2-induced oxidative stress (Lou et al., 2012). However, whether it is also protective in a cerebral ischemic model remains unknown. Based on the previous study, we hypothesized that E7G may confer neuroprotection against cerebral ischemia through Nrf2-coordinated induction of endogenous cytoprotective proteins. The present study reveals that E7G might be beneficial in mitigating cerebral ischemic injury in cellular and animal stroke models by upregulating phase II and antioxidant gene expression via Nrf2 activation.

Section snippets

Animals and reagents

Adult male Sprague–Dawley (SD) rats (280 g ~ 320 g) were used in the in vivo experiments. Newborn SD rats (day 0–1) were used for primary cortical astrocyte cultures. All experiments were approved by the Institutional Animal Care and Use Committee of Shandong University. E7G was provided by the Department of Natural Products Chemistry of Shandong University (Jinan, China) where its purity was confirmed to be > 98%. Dulbecco's modified Eagle medium (DMEM), penicillin, streptomycin and fetal bovine

E7G activates the Nrf2 pathway and stimulates expression of Nrf2-mediated cytoprotective gens in astrocytes

Examination of Nrf2 nuclear expression by western blot revealed that E7G induce Nrf2 nuclear translocation in a dose- and time-dependent manner (Figs. 1A, B). We next determined whether E7G induces the expression of endogenous ARE-regulated genes. As shown in Fig. 1C, exposure of cells to E7G strongly induced HO-1 (heme oxygenase-1) and gamma glutamate cysteine ligase (γ-GCS) protein expression in a concentration dependent manner with little effect on NAD(P)H:quinine oxidoreductase 1 (NQO-1)

Discussion

Stroke is the second most common cause of death and the leading cause of adult disability for which no effective neuroprotective treatment is currently available (Balami et al., 2011, Donnan et al., 2008, Endres et al., 2008, Lopez et al., 2006). Oxidative stress is an important contributing factor in the pathogenesis of stroke, suggesting that therapeutic strategies directed against ROS might be particularly valuable for the prevention of stroke. There is increasing evidence showing that

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

This study was supported by grants from the National Natural Science Foundation of China (No. 81274124, No. 81200982), the Foundation for Excellent Young and Middle-Aged Scientists of Shandong Province (No. BS2010YY036) and the Postdoctoral Innovation Research Program of Shandong Province (No. 201102021).

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    Xu Jing and Dongmei Ren contribute equally to this work.

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