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(−)-Epicatechin, a Natural Flavonoid Compound, Protects Astrocytes Against Hemoglobin Toxicity via Nrf2 and AP-1 Signaling Pathways

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Abstract

(−)-Epicatechin is a brain-permeable, natural product found at high concentrations in green tea and cocoa. Our previous research has shown that (−)-epicatechin treatment reduces hemorrhagic stroke injury via nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in vivo. However, the mechanism of action of this compound in modulation of oxidant stress and in protection against hemoglobin-induced astrocyte injury is unclear. Therefore, we explored the cellular and molecular mechanisms that underlie these protective effects in vitro. Mouse primary astrocytes isolated from wild-type mice and Nrf2 knockout (KO) mice were preconditioned with hemoglobin to simulate intracerebral hemorrhage (ICH) in vitro. Effects of (−)-epicatechin were measured by Western blotting, immunostaining, MTT assay, and reactive oxidant stress (ROS) assay. (−)-Epicatechin increased Nrf2 nuclear accumulation and cytoplasmic levels of superoxide dismutase 1 (SOD1) in wild-type astrocytes but did not increase SOD1 expression in Nrf2 knockout (KO) astrocytes. Furthermore, (−)-epicatechin treatment did not alter heme oxygenase 1 (HO1) expression in wild-type astrocytes after hemoglobin exposure, but it did decrease HO1 expression in similarly treated Nrf2 KO astrocytes. In both wild-type and Nrf2 KO astrocytes, (−)-epicatechin suppressed phosphorylated JNK and nuclear expression of JNK, c-jun, and c-fos, indicating that inhibition of activator protein-1 (AP-1) activity by (−)-epicatechin is Nrf2-independent. These novel findings indicate that (−)-epicatechin protects astrocytes against hemoglobin toxicity through upregulation of Nrf2 and inhibition of AP-1 activity. These cellular and molecular effects may partially explain the cerebroprotection as we previously observed for (−)-epicatechin in animal models of ICH.

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Abbreviations

AP-1:

Activator protein-1

DAPI:

4,6-diamidino-2-phenylindole

EC:

(−)-Epicatechin

HO1:

Heme oxygenase 1

ICH:

Intracerebral hemorrhage

Nrf2:

Nuclear factor erythroid 2-related factor 2

ROS:

Reactive oxygen species

SOD1:

Superoxide dismutase 1

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Acknowledgements

This work was supported by the National Institute of Health R01NS078026 and R01AT007317 (JW) and by the American Heart Association Mid-Atlantic Affiliate Grant-in-Aid (13GRNT15730001 to JW) and Postdoctoral Fellowship Awards (15POST25090114 to X. Lan and 14POST20140003 to X. Han). The authors thank Tian Cheng and Jieru Wan for the Western blotting technical support, Wenzhu Wang for the blind analysis of immunostaining, and Claire Levine for the assistance with manuscript preparation. We thank Raymond Koehler, Zengjin Yang, and all the Wang lab members for their constructive suggestions.

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  1. Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Ross Bldg 370B, Baltimore, MD, 21205, USA

    Xi Lan, Xiaoning Han, Qian Li & Jian Wang

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  1. Xi Lan
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  2. Xiaoning Han
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Lan, X., Han, X., Li, Q. et al. (−)-Epicatechin, a Natural Flavonoid Compound, Protects Astrocytes Against Hemoglobin Toxicity via Nrf2 and AP-1 Signaling Pathways. Mol Neurobiol 54, 7898–7907 (2017). https://doi.org/10.1007/s12035-016-0271-y

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