Elsevier

Brain Research

Volume 956, Issue 2, 29 November 2002, Pages 319-322
Brain Research

Research report
(−)-Epigallocatechin gallate protects against NO stress-induced neuronal damage after ischemia by acting as an anti-oxidant

https://doi.org/10.1016/S0006-8993(02)03564-3Get rights and content

Abstract

The present study investigated the effects of (−)-epigallocatechin gallate (EGCG), which is the major component of polyphenol in green tea, on nitric oxide (NO) stress-induced neuronal damage, by monitoring NO mobilizations in the intact rat hippocampus and assaying the viability of cultured rat hippocampal neurons. A 10-min ischemia increased NO (NO3/NO2) concentrations in the intact rat hippocampus, while EGCG (50 mg/kg i.p.) inhibited the increase by 77% without affecting hippocampal blood flow. The NO donor, sodium nitroprusside (SNP; 50 μM), produced NO (NO3/NO2), while EGCG inhibited it in a dose-dependent manner at concentrations ranging from 50 to 200 μM. Treatment with SNP (100 μM) reduced the viability of cultured rat hippocampal neurons to 22% of control levels, while EGCG caused it to recover to 51% for 10 μM, 73% for 20 μM, and 70% for 50 μM. Taken together, it appears that EGCG could protect against ischemic neuronal damage by deoxidizing peroxynitrate/peroxynitrite, which is converted to NO radical or hydroxy radical.

Introduction

Nitric oxide (NO) is synthesized by the NO synthases, endothelial NO synthase (eNOS), neuronal NO synthase (nNOS), and inducible NO synthase (iNOS). NO originated from nNOS exerts a neurotoxic action, while NO from eNOS exhibits a vasodilative effect and is employed in the protective actions against neurotoxicity [13]. Recent studies suggest that the neurotoxic action of NO is also relevant to the pathogenesis of neurodegenerative diseases, such as Alzheimer disease [9], Parkinson disease [5], [6], and prion disease [4]. In addition, NO is thought to accelerate ischemic brain damage [3].

(−)-Epigallocatechin gallate (EGCG) is the major component of polyphenol in green tea. EGCG is shown to inhibit the growth and metastasis of malignant tumor [8], to protect against ischemic brain damage [10], and to decrease the risk of cardiovascular disease [12]. Little, however, is known about the mechanism underlying the protective action against ischemic brain damage. To understand this, we monitored real-time NO mobilizations in the intact rat hippocampus by using an NO sensitive microsensor and assayed cell viability with the dye, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). The results of the present study suggest that EGCG reduces NO3/NO2 conversion from NO, which is produced after ischemia, thereby protecting against ischemic neuronal damage.

Section snippets

Materials and methods

An ischemic model was made using adult Wistar rats by the method previously described [7]. Briefly, bilateral common carotid arteries and vertebral arteries, with the head being fixed with a stereotactic frame, were occluded for 10 min by ligation, and subsequently the occlusion was released. During experiments, rats were under general anesthesia with urethane (1.2 g/kg i.p.), with ventilation controlled with an artificial respirator and rectal temperature maintained at 37–38 °C with a heating

Results

For saline-injected rats, NO currents decreased during ischemia, but the ensuing reperfusion caused an increase in the currents, with a maximum of 2232±169 pA (∼268±20 μM) (Fig. 1A,B). Pre-treatment with EGCG (50 mg/kg i.p.) reduced the increase by 77% (524±113 pA, ∼63±14 μM) without affecting basal NO currents before or without ischemia (Fig. 1A,B), suggesting that EGCG inhibits NO synthesis or reduces NO3/NO2 production from NO. Hippocampal blood flow transiently elevated after ischemia,

Discussion

NO radical or hydroxy radical from NO as well as other free radicals is well-recognized to perturb cells. We found earlier that brain ischemia causes a rise in NO (NO3/NO2) concentrations and that NO originated from nNOS, but not eNOS, exhibits a neurotoxic effect in the intact rat hippocampus [7]. In the present study, EGCG drastically attenuates a rise in hippocampal NO3/NO2 concentrations after ischemia without affecting hippocampal blood flow. This raises the possibility that EGCG is

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