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Epigallocatechin-3-gallate induced primary cultures of rat hippocampal neurons death linked to calcium overload and oxidative stress

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Abstract

Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is the main ingredient of green tea extract. It has been reported that EGCG is a potent antioxidant and beneficial in oxidative stress-related diseases, but others and our previous study showed that EGCG has pro-oxidant effects at high concentration. Thus, in this study, we tried to examine the possible pathway of EGCG-induced cell death in cultures of rat hippocampal neurons. Our results showed that EGCG caused a rapid elevation of intracellular free calcium levels ([Ca2+]i) in a dose-dependent way. Exposure to EGCG dose- and time-dependently increased the production of reactive oxygen species (ROS) and reduced mitochondrial membrane potential (Δψ m) as well as the Bcl-2/Bax expression ratio. Importantly, acetoxymethyl ester of 5,5′-dimethyl-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, ethylene glycol-bis-(2-aminoethyl)-N,N,N′,N′-tetraacetic acid, and vitamin E could attenuate EGCG-induced apoptotic responses, including ROS generation, mitochondrial dysfunction, and finally partially prevented EGCG-induced cell death. Furthermore, treatment of hippocampal neurons with EGCG resulted in an elevation of caspase-3 and caspase-9 activities with no significant accompaniment of lactate dehydrogenase release, which provided further evidence that apoptosis was the dominant mode of EGCG-induced cell death in cultures of hippocampal neurons. Taken together, these findings indicated that EGCG induced hippocampal neuron death through the mitochondrion-dependent pathway.

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Abbreviations

Ac-DEVD-pNA:

Acetyl-Asp-Glu-Val-Asp p-nitroanilide

Ac-LEHD-pNA:

Acetyl-Leu-Glu-His-Asp p-nitroanilide

BAPTA-AM:

Acetoxymethyl ester of 5,5′-dimethyl-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

CNS:

Central nervous system

DCF:

Dichlorofluorescein

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

Dimethyl sulfoxide

EGCG:

Epigallocatechin-3-gallate

EGTA:

Ethylene glycol-bis-(2-aminoethyl)-N,N,N′,N′-tetraacetic acid

ER:

Endoplasmic reticulum

Fluo-3-AM:

Fluo-3-acetoxymethyl ester

HEPES:

4-(2-hydroxyethyl)-1-201 piperazineethanesulfonic acid

JC-1:

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide

LDH:

Lactate dehydrogenase

mPT:

Mitochondria permeability transition

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS:

Phosphate-buffered saline

PI:

Propidium iodide

pNA:

p-nitroanilide

ROS:

Reactive oxygen species

Δψ m :

Mitochondrial membrane potential

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Acknowledgments

This work was supported by the National Nature Science Foundation of China (Nos. 30630057, 30670554, and 30670662) and Anhui High Education Natural Science Program (No. ZD2008010-2).

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Authors and Affiliations

  1. School of Life Science, University of Science and Technology of China, Hefei, Anhui, 230027, People’s Republic of China

    Shu-Ting Yin, Ming-Liang Tang, Hong-Min Deng, Tai-Ran Xing, Ju-Tao Chen, Hui-Li Wang & Di-Yun Ruan

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Yin, ST., Tang, ML., Deng, HM. et al. Epigallocatechin-3-gallate induced primary cultures of rat hippocampal neurons death linked to calcium overload and oxidative stress. Naunyn-Schmied Arch Pharmacol 379, 551–564 (2009). https://doi.org/10.1007/s00210-009-0401-4

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