J Nutr Health.  2017 Oct;50(5):415-425. 10.4163/jnh.2017.50.5.415.

Neuroprotective effects of Momordica charantia extract against hydrogen peroxide-induced cytotoxicity in human neuroblastoma SK-N-MC cells

Affiliations
  • 1Research Institute, Seoul Medical Center, Seoul 02053, Korea. nostoi72@naver.com
  • 2Department of Neurology, Seoul Medical Center, Seoul 02053, Korea.
  • 3Department of Neurosurgery, Seoul Medical Center, Seoul 02053, Korea.

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 Hâ‚‚Oâ‚‚-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 ICâ‚…â‚€ value for DPPH radical scavenging activity was 506.95 µg/ml for MCE. Pre-treatment with MCE showed protective effects against Hâ‚‚Oâ‚‚-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 Hâ‚‚Oâ‚‚. Pre-treatment with MCE inhibited phosphorylation of p38 and the JNK pathway and down-regulated cleaved caspase-3 and cleaved PARP by Hâ‚‚Oâ‚‚.
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.

Keyword

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

MeSH Terms

Apoptosis
Blotting, Western
Caspase 3
Catechin
Cell Death
Cell Survival
Down-Regulation
Ethanol
Gallic Acid
Gene Expression
Humans*
Hydrogen*
MAP Kinase Signaling System
Mitogen-Activated Protein Kinases
Momordica charantia*
Momordica*
Neuroblastoma*
Neurons
Neuroprotective Agents*
Oxidative Stress
Phenol
Phosphorylation
Reactive Oxygen Species
Real-Time Polymerase Chain Reaction
RNA, Messenger
Sincalide
Caspase 3
Catechin
Ethanol
Gallic Acid
Hydrogen
Mitogen-Activated Protein Kinases
Neuroprotective Agents
Phenol
RNA, Messenger
Reactive Oxygen Species
Sincalide

Figure

  • 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

  • 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.

  • 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.

  • 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.

  • 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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