Korean J Physiol Pharmacol.  2019 Mar;23(2):121-130. 10.4196/kjpp.2019.23.2.121.

Neuroprotective mechanisms of dieckol against glutamate toxicity through reactive oxygen species scavenging and nuclear factor-like 2/heme oxygenase-1 pathway

• Cui Y
• Amarsanaa
• Lee JH
• Rhim JK
• Kwon JM
• Kim SH
• Park JM
• Jung SC
• Eun SY

• KMID: 2438086
• DOI: http://doi.org/10.4196/kjpp.2019.23.2.121

Abstract

Glutamate toxicity-mediated mitochondrial dysfunction and neuronal cell death are involved in the pathogenesis of several neurodegenerative diseases as well as acute brain ischemia/stroke. In this study, we investigated the neuroprotective mechanism of dieckol (DEK), one of the phlorotannins isolated from the marine brown alga Ecklonia cava, against glutamate toxicity. Primary cortical neurons (100 µM, 24 h) and HT22 neurons (5 mM, 12 h) were stimulated with glutamate to induce glutamate toxic condition. The results demonstrated that DEK treatment significantly increased cell viability in a dose-dependent manner (1-50 µM) and recovered morphological deterioration in glutamate-stimulated neurons. In addition, DEK strongly attenuated intracellular reactive oxygen species (ROS) levels, mitochondrial overload of Ca²âº and ROS, mitochondrial membrane potential (ΔΨ(m)) disruption, adenine triphosphate depletion. DEK showed free radical scavenging activity in the cell-free system. Furthermore, DEK enhanced protein expression of heme oxygenase-1 (HO-1), an important anti-oxidant enzyme, via the nuclear translocation of nuclear factor-like 2 (Nrf2). Taken together, we conclude that DEK exerts neuroprotective activities against glutamate toxicity through its direct free radical scavenging property and the Nrf-2/HO-1 pathway activation.

Keyword

Dieckol; Glutamate toxicity; Heme oxygenase-1; Mitochondria; Neurons; Reactive oxygen species

MeSH Terms

Adenine
Brain
Cell Death
Cell Survival
Cell-Free System
Glutamic Acid*
Heme Oxygenase-1
Membrane Potential, Mitochondrial
Mitochondria
Neurodegenerative Diseases
Neurons
Reactive Oxygen Species*
Adenine
Glutamic Acid
Heme Oxygenase-1
Reactive Oxygen Species

Figure

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  Fig. 1 Chemical structure and cytotoxicity test of DEK. (A) Chemical structure of DEK. (B) Cytotoxicity test of DEK in neurons. Different doses of DEK were treated for 25 h in primary cortical neurons and for 13 h in HT22 neurons. These treatment durations are the sum of co-treatment duration (24 h for primary cortical neurons and 12 h for HT22 cell line) and the DEK pretreatment duration (1 h). The cytotoxicity of DEK was examined using the MTT assay and LDH release assay. DEK below 100 µM did not show any cytotoxicity as a chemical agent for treatment, based on MTT assay and LDH release assay. Values were expressed as mean ± standard error of mean of four samples in one independent experiment. ###p < 0.001, as compared to the untreated control group. DEK, dieckol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; LDH, lactate dehydrogenase release.

• 

  Fig. 2 Neuroprotective effects of DEK on neuronal cell viabilities and morphological changes against glutamate toxicity. (A and B) Both primary cortical neurons and HT22 neuronal cell line were pretreated with DEK for 1 h prior to glutamate stimulation. Then, primary cortical neurons were stimulated with glutamate (100 µM) in the presence of different doses of DEK for 24 h. In case of HT22 neurons, cells were stimulated with glutamate (5 mM) for 12 h. Neuroprotective effects of DEK against glutamate toxicity were evaluated using MTT cell viability assay in primary cortical neurons and HT22 neurons. (C) Representative phase contrast images indicating neuroprotective effects of DEK on glutamate-induced morphological changes in HT22 neurons. Scale bar, 50 µm. Values were expressed as mean ± standard error of mean of four samples in one independent experiment. Statistical analyses were performed using one-way ANOVA followed by Bonferroni test. ###p < 0.001, as compared to untreated control group; *p < 0.05 and ***p < 0.001, as compared to glutamate alone-treated group. DEK, dieckol; Glu, glutamate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

• 

  Fig. 3 ROS scavenging activities of DEK in glutamate-stimulated neurons. (A) Free radical scavenging activities of DEK in a cell-free system were performed using the DPPH assay. (B and C) Both primary cortical neurons and HT22 neuronal cell line were pretreated with DEK for 1 h prior to glutamate stimulation. Then, primary cortical neurons were stimulated with glutamate (100 µM) in the presence of different doses of DEK for 24 h. In case of HT22 neurons, cells were stimulated with glutamate (5 mM) for 12 h. Effects of DEK on glutamate-induced intracellular ROS generation were examined using a spectrofluorometer with the ROS-sensitive fluorescent dye DCF-DA. Values were expressed as mean ± standard error of mean of four samples in one independent experiment. Statistical analyses were performed using one-way ANOVA followed by Bonferroni test. ##p < 0.01 and ###p < 0.001, as compared to the untreated control group; *p < 0.05 and ***p < 0.001, as compared to the glutamate alone-treated group. ROS, reactive oxygen species; DPPH, 1,1-diphenyl-2-picrylhydrazyl; DEK, dieckol; Glu, glutamate.

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  Fig. 4 DEK attenuates glutamate-induced mitochondria dysfunction. HT22 neurons were pretreated with DEK (50 µM) for 1 h and then stimulated with glutamate (5 mM) in the presence of DEK for 12 h. Effects of DEK on glutamate-induced mitochondrial dysfunction were examined. (A) Intracellular ATP levels were measured using a luciferase/luciferin ATP determination kit. (B) The effects of DEK on glutamate-induced ΔΨmm disruption were analyzed by flow cytometry using JC-1. Figure shows the representative data of flow cytometry in the lower panel and their quantitative analyses in the upper panel. (C) The effects of DEK on glutamate-induced mitochondrial Ca2+ levels were analyzed by flow cytometry using Rhod-2. (D) The effects of DEK on glutamate-induced mitochondrial ROS generation were analyzed by flow cytometry using MitoSOX. Figure shows the representative data of flow cytometry in the lower panel and their quantitative analyses in the upper panel. (E) Representative confocal images of HT22 neurons loaded with MitoSOX were shown to demonstrate neuroprotective effects of DEK on glutamate-induced mitochondrial ROS generation. Mitochondria were stained with Mito Tracker Green. Scale bar, 50 µm. Values were expressed as mean ± standard error of mean of four samples in one independent experiment. ##p < 0.01 and ###p < 0.001, as compared to untreated control group; *p < 0.05 and ***p < 0.001, as compared to glutamate alone-treated group. ATP, adenine triphosphate; Glu, glutamate; DEK, dieckol; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide; Rhod-2, rhod-2 acetoxymethyl ester; ROS, reactive oxygen species; ΔΨm, mitochondrial membrane potential. MitoSOX and Mito Tracker Green; Invitrogen, Carlsbad, CA, USA.

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  Fig. 5 DEK increases nuclear translocation of Nrf2 and HO-1 expression. Effects of DEK on HO-1 expression (A) and nuclear Nrf2 translocation (B) were examined using Western blot analysis. HT22 neurons were exposed to different doses of DEK for 12 h. Total proteins were isolated for HO-1 and nuclear fractions were isolated at different time points for Nrf2. β-Actin and TBP were used as controls for equal protein loading in whole cells and nuclear fraction, respectively. Values were expressed as mean ± standard error of mean of four samples in one independent experiment. Statistical analyses were performed using one-way ANOVA followed by Bonferroni test. #p < 0.05, ##p < 0.01, and ###p < 0.001, as compared to untreated control group. DEK, dieckol; Nrf2, nuclear factor-like 2; HO-1, heme oxygenase-1; TBP, TATA binding protein.

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  Fig. 6 Schematic diagram on neuroprotective effects of DEK against glutamate-induced neuronal cell death. There are two pathways underlying glutamate toxicity mechanism integrated in parallel in neurons; receptor-mediated excitotoxicity pathway and nonreceptor-mediated oxidative stress pathway. Increases of intracellular Ca2+ and ROS are triggering events in mitochondrial (mt) dysfunction and neuronal cell death against glutamate toxicity. Glutamate-induced intracellular Ca2+ increase is evoked mainly through NMDAR and G protein-coupled mGluR. Intracellular glutamate-induced ROS generation is induced through NOX and cystine/glutamate antiporters in the initial state. DEK exerted neuroprotective activities against glutamate toxicity through direct ROS scavenging and activation of the Nrf-2/HO-1 pathway as a cellular anti-oxidant defense system in this study. Reference numbers are shown in the known signaling pathways. NMDAR, N-methyl-D-aspartate receptors; mGluR, metabotropic glutamate receptors; NADPH, nicotinamide adenine dinuclelotide phosphate; NOX, NADPH oxidase; GSH, glutathione; ROS, reactive oxygen species; DEK, dieckol; Nrf2, nuclear factor-like 2; HO-1, heme oxygenase-1; ΔΨm, mitochondrial membrane potential; ATP, adenine triphosphate; MMP, mitochondrial membrane permeabilization.

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