Korean J Ophthalmol.  2015 Feb;29(1):58-65. 10.3341/kjo.2015.29.1.58.

The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells

• Song Y
• Hong S
• Iizuka Y
• Kim CY
• Seong GJ

• KMID: 2360139
• DOI: http://doi.org/10.3341/kjo.2015.29.1.58

Abstract

PURPOSE
Maltol (3-hydroxy-2-methyl-4-pyrone), formed by the thermal degradation of starch, is found in coffee, caramelized foods, and Korean ginseng root. This study investigated whether maltol could rescue neuroretinal cells from oxidative injury in vitro.
METHODS
R28 cells, which are rat embryonic precursor neuroretinal cells, were exposed to hydrogen peroxide (H2O2, 0.0 to 1.5 mM) as an oxidative stress with or without maltol (0.0 to 1.0 mM). Cell viability was monitored with the lactate dehydrogenase assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. To investigate the neuroprotective mechanism of maltol, the expression and phosphorylation of nuclear factor-kappa B (NF-kappaB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were evaluated by Western immunoblot analysis.
RESULTS
R28 cells exposed to H2O2 were found to have decreased viability in a dose- and time-dependent manner. However, H2O2-induced cytotoxicity was decreased with the addition of maltol. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. However, the cytotoxicity was reduced in the presence of 1.0 mM maltol. This H2O2-induced cytotoxicity caused apoptosis of R28 cells, characterized by DNA fragmentation. Apoptosis of oxidatively-stressed R28 cells with 1.0 mM H2O2 was decreased with 1.0 mM maltol, as determined by the TUNEL method. Western blot analysis showed that treatment with maltol reduced phosphorylation of NF-kappaB, ERK, and JNK, but not p38. The neuroprotective effects of maltol seemed to be related to attenuated expression of NF-kappaB, ERK, and JNK.
CONCLUSIONS
Maltol not only increased cell viability but also attenuated DNA fragmentation. The results obtained here show that maltol has neuroprotective effects against hypoxia-induced neuroretinal cell damage in R28 cells, and its effects may act through the NF-kappaB and mitogen-activated protein kinase signaling pathways.

Keyword

Maltol; Neuroprotection; Oxidative stress; Rat retinal neuronal cell

MeSH Terms

Animals
*Apoptosis
Blotting, Western
Cell Survival
Cells, Cultured
Disease Models, Animal
Flavoring Agents/pharmacology
In Situ Nick-End Labeling
Oxidative Stress/*drug effects
Pyrones/*pharmacology
Rats
Retinal Ganglion Cells/drug effects/metabolism/*pathology
Flavoring Agents
Pyrones

Figure

• 

  Fig. 1 Hydrogen peroxide (H2O2) induced cytotoxicity in R28 cells. (A) R28 cells were exposed to H2O2 ranging from 0.0 to 1.5 mM for 24 hours. (B) R28 cells were exposed to 1.0 mM H2O2 for up to 48 hours. Cell cytotoxicity was quantified by an lactate dehydrogenase assay. H2O2 increased lactate dehydrogenase release in a dose- and time-dependent manner. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. Data are expressed as the mean ± SD.

• 

  Fig. 2 The protective effect of maltol treatment (bright field microscopy). (A) Control. (B) R28 cells exposed to 1.0 mM hydrogen peroxide (H2O2). (C) R28 cells exposed to 1.0 mM maltol. (D) R28 cells co-cultured with 1.0 mM H2O2 and 1.0 mM maltol. (E) R28 cells were co-cultured with 1.0 mM H2O2 and different concentrations of maltol ranging from 0.1 to 1.5 mM for 24 hours. Cell cytotoxicity was quantified by an lactate dehydrogenase assay. Maltol decreased lactate dehydrogenase leakage from injured cells in a dose-dependent manner. Scale bar in panels (A) to (D) = 50 µm. Data are expressed as mean ± SD (the asterisks denote that data are significantly different from the untreated control; *1.0 mM H2O2-treated cells; **p < 0.05).

• 

  Fig. 3 Anti-apoptotic effects of maltol treatment. (A-C) Representative photographs of terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL), in which green fluorescence indicates apoptotic cells and red fluorescence indicates living cells. (A) Control. (B) 1.0 mM hydrogen peroxide (H2O2). (C) 1.0 mM H2O2 with 1.0 mM maltol. (D) Quantification of TUNEL-positive cells; data collected from 10 fields for each group; experiments were repeated three times. Data are expressed as mean ± SD of the ratio of apoptotic cells to the total number of cells. After co-culture with 1.0 mM maltol, oxidatively-stressed R28 cells became more resistant to hydrogen peroxide injury (the asterisks denote that data are significantly different from control; *p < 0.05).

• 

  Fig. 4 Western blot analysis of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase in R28 cells. (A) The expression of phospho NF-κB (pNF-κB), phospho c-Jun N-terminal kinase (pJNK), phospho extracellular signal-regulated kinase (pERK), phospho-p38 (pp38), and β-actin after 48 hours with or without 1.0 mM maltol in R28 cells exposed to 1.0 mM hydrogen peroxide (H2O2). (B) The band intensities, relative to a control in R28 cells exposed to 1.0 mM H2O2 within 2 hours. The black box indicates exposure to H2O2 without maltol treatment and the patterned box indicates exposure to H2O2 with maltol treatment. The expression of pNF-κB, pJNK, and pERK was reduced with 1.0 mM maltol treatment. However, there was no significant change of phospho-p38 in the presence or absence of maltol treatment. Data are expressed as mean ± SD (the asterisks denote that data are significantly different from 1.0 mM H2O2-treated cells, *p < 0.05).

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