J Nutr Health.  2015 Oct;48(5):451-456. 10.4163/jnh.2015.48.5.451.

Anti-apoptotic effect of fermented Citrus sunki peel extract on chemical hypoxia-induced neuronal injury

• Ko WC
• Lee SR

• KMID: 2209879
• DOI: http://doi.org/10.4163/jnh.2015.48.5.451

Abstract

PURPOSE
Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimer's disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride (CoCl2)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells.
METHODS
Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2',7'-dichlorofluorescin diacetate (DCF-DA) and 4',6-diamidino-2-phenylindole (DAPI) staining.
RESULTS
Exposure to CoCl2, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the CoCl2-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel.
CONCLUSION
Based on these data, fermented C. sunki peel extract might have a protective effect against CoCl2-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.

Keyword

Citrus sunki; apoptosis; hypoxia; stroke; neuronal cells

MeSH Terms

Aging
Alzheimer Disease
Anoxia
Apoptosis
Blotting, Western
Caspase 3
Cell Death
Cell Survival
Citrus*
Cobalt
Cytochromes c
Cytoprotection
Cytosol
Humans
Ischemia
Medicine, Traditional
Mitochondria
Neuroblastoma
Neurodegenerative Diseases
Neurons*
Reactive Oxygen Species
Risk Factors
Stroke
Trypan Blue
Caspase 3
Cobalt
Cytochromes c
Reactive Oxygen Species
Trypan Blue

Figure

• 

  Fig. 1 Induction of caspase-3 mediated cell death. Effects of CoCl2 on the cell viability by trypan blue exclusion (A) and expression of apoptosis-related proteins by western blot analysis (B). Data are represented as mean ± SD of three independent experiments. *p < 0.05 vs control. C: control

• 

  Fig. 2 Protective Effect of fermented C. sunki peel extract on the neurotoxicity (A), activities of caspases and PARP (B), formation of apoptotic body (C) in CoCl2-treated SH-SY5Y cells. Data are presented as mean ± SD of three independent experiments. *p < 0.05 vs control. #p < 0.05 vs CoCl2-treated cells. FCS: Fermented C. sunki peel extract

• 

  Fig. 3 Effect of fermented C. sunki peel extract on the CoCl2-induced ROS generation (A) and release of mitochondrial cytochrome c to cytosol (B). Data are represented as mean ± SD of three independent experiments. *p < 0.05 vs control. #p < 0.05 vs CoCl2-treated cells. FCS: Fermented C. sunki peel extract, NAC: N-acetyl-cystein, Cyt c: Cytochrome c

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