Journal List > J Nutr Health > v.50(5) > 1081518

J Nutr Health. 2017 Oct;50(5):415-425. Korean.
Published online October 31, 2017.  https://doi.org/10.4163/jnh.2017.50.5.415
© 2017 The Korean Nutrition Society
Neuroprotective effects of Momordica charantia extract against hydrogen peroxide-induced cytotoxicity in human neuroblastoma SK-N-MC cells
Kkot Byeol Kim,1 Seonah Lee,1 Jae Hyeok Heo,1,2 and Jung hee Kim1,3
1Research Institute, Seoul Medical Center, Seoul 02053, Korea.
2Department of Neurology, Seoul Medical Center, Seoul 02053, Korea.
3Department of Neurosurgery, Seoul Medical Center, Seoul 02053, Korea.

To whom correspondence should be addressed. tel: +82-2-2276-8601, Email: nostoi72@naver.com
Received July 20, 2017; Revised August 15, 2017; Accepted September 19, 2017.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Purpose

Many studies have suggested that neuronal cells protect against oxidative stress-induced apoptotic cell death by polyphenolic compounds. We investigated the neuroprotective effects and the mechanism of action of Momordica charantia ethanol extract (MCE) against H2O2-induced cell death of human neuroblastoma SK-N-MC cells.

Methods

The antioxidant activity of MCE was measured by the quantity of total phenolic acid compounds (TPC), quantity of total flavonoid compounds (TFC), and 2,2-Diphenyl-1-pycrylhydrazyl (DPPH) radical scavenging activity. Cytotoxicity and cell viability were determined by CCK-8 assay. The formation of reactive oxygen species (ROS) was measured using 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Antioxidant enzyme (SOD-1,2 and GPx-1) expression was determined by real-time PCR. Mitogen-activated protein kinases (MAPK) pathway and apoptosis signal expression was measured by Western blotting.

Results

The TPC and TFC quantities of MCE were 28.51 mg gallic acid equivalents/extract g and 3.95 mg catechin equivalents/extract g, respectively. The IC50 value for DPPH radical scavenging activity was 506.95 µg/ml for MCE. Pre-treatment with MCE showed protective effects against H2O2-induced cell death and inhibited ROS generation by oxidative stress. SOD-1,2 and GPx-1 mRNA expression was recovered by pre-treatment with MCE compared with the presence of H2O2. Pre-treatment with MCE inhibited phosphorylation of p38 and the JNK pathway and down-regulated cleaved caspase-3 and cleaved PARP by H2O2.

Conclusion

The neuroprotective effects of MCE in terms of recovery of antioxidant enzyme gene expression, down-regulation of MAPK pathways, and inhibition apoptosis is associated with reduced oxidative stress in SK-N-MC cells.

Keywords: Momordica charantia ethanol extract (MCE); antioxidant; MAPK pathway; apoptosis; SK-N-MC

Figures


Fig. 1
Protective effects of Momordica charantia ethanol extracts (MCE) on H2O2-induced cell death and cytotoxicity in SK-N-MC cells. (A) Cell viability was determined by CCK-8 assay. SK-N-MC cell wre treated with increasing concentrations (100~600 µM) of H2O2 500 µM for 24 hr. (B) SK-N-MC cell were treated with increasing concentrations (1~50 µg/ml) of MCE for 24 hr. (C) The cell pretreated with MCE at the indicated concentration (1~50 µg/ml) for 6hr, and then treated with H2O2 for 18 hr. Value represent the mean ± SD. *p < 0.05, **p < 0.01 compared with control. #p < 0.05 vs compared with H2O2 treated cell
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Fig. 2
Intracellular ROS scavenging activity of MCE. Cells were treated with H2O2 and/ or MCE as described in the legend of Fig. 1C. The generation of intracellular ROS was determined by DCF-DA methods using a fluorescence spectrophotometer with excitation and emission wavelengths of 485 nm and 530 nm, respectively. Value represent the mean ± SD. Means with different letters (a~c) at each mRNA are significantly different (p < 0.05) by Duncan's multiple range test.
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Fig. 3
Effects of MCE on H2O2-induced mRNA expression levels of antioxidant enzymes in SK-N-MC cells. Cells were treated with H2O2 and/ or MCE as described in the legend of Fig. 1C. The mRNA levels of antioxidant enzymes were determined by real-time PCR analysis. (A) SOD-1, superoxide dismutase-1; (B) SOD-2, superoxide dismutase-2; (C) GPx1, glutathione peroxidase-1. Value represent the mean ± SD. Means with different letters (a~d) at each mRNA are significantly different (p < 0.05) by Duncan's multiple range test.
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Fig. 4
Effects of MCE on H2O2-induced phosphorylation of MAPK (p38 and JNK). Pathway in SK-N-MC cells. Cells were treated with H2O2 and/ or MCE as described in the legend of Fig. 1C. The protein levels of MAPKs pathway signals were analyzed by western blotting, and normalizes to the GAPDH level. The density of each protein band was quantified by using Bio-1D imaging software (Vilber Lourmat, Marne-la-Vallée, France). Value represent the mean ± SD. Means with different letters (a~d) at each mRNA are significantly different (p < 0.05) by Duncan's multiple range test.
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Fig. 5
MCE attenuates H2O2-induced cleavage of caspase-3 and PARP in SK-N-MC cells. Cells were treated with H2O2 and/ or MCE as described in the legend of Fig. 1C. The protein levels of caspase-3, cleaved caspase-3 (A), PARP and cleaved PARP (B) were analyzed by western blotting, and normalizes to the GAPDH level. Value represent the mean ± SD. Means with different letters (a~e) at each mRNA are significantly different (p < 0.05) by Duncan's multiple range test.
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Tables


Table 1
Primer sequences used for RT-PCR
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Table 2
Total polyphenols and flavonoids contents of Momordica charantia
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Table 3
DPPH radical scavenging activity of 70% ethanol extracts from Momordica charantia
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Notes

This study was supported by grants of Seoul Medical Center Research Institute (#17-C21).

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