Nutr Res Pract.  2018 Apr;12(2):93-100. 10.4162/nrp.2018.12.2.93.

Protective effects of perilla oil and alpha linolenic acid on SH-SY5Y neuronal cell death induced by hydrogen peroxide

• Lee AY
• Choi JM
• Lee MH
• Lee J
• Lee S
• Cho EJ

• KMID: 2407805
• DOI: http://doi.org/10.4162/nrp.2018.12.2.93

Abstract

BACKGROUND
/OBJECTIVE: Oxidative stress plays a key role in neuronal cell damage, which is associated with neurodegenerative disease. The aim of present study was to investigate the neuroprotective effects of perilla oil (PO) and its active component, alpha-linolenic acid (ALA), against hydrogen peroxide (Hâ‚‚Oâ‚‚)-induced oxidative stress in SH-SY5Y neuronal cells.
MATERIALS/METHODS
The SH-SY5Y human neuroblastoma cells exposed to 250 µM Hâ‚‚Oâ‚‚ for 24 h were treated with different concentrations of PO (25, 125, 250 and 500 µg/mL) and its major fatty acid, ALA (1, 2.5, 5 and 25 µ/mL). We examined the effects of PO and ALA on Hâ‚‚Oâ‚‚-induced cell viability, lactate dehydrogenase (LDH) release, and nuclear condensation. Moreover, we determined whether PO and ALA regulated the apoptosis-related protein expressions, such as cleaved-poly ADP ribose polymerase (PARP), cleaved caspase-9 and -3, BCL-2 and BAX.
RESULTS
Treatment of Hâ‚‚Oâ‚‚ resulted in decreased cell viability, increased LDH release, and increase in the nuclei condensation as indicated by Hoechst 33342 staining. However, PO and ALA treatment significantly attenuated the neuronal cell death, indicating that PO and ALA potently blocked the Hâ‚‚Oâ‚‚-induced neuronal apoptosis. Furthermore, cleaved-PARP, cleaved caspase-9 and -3 activations were significantly decreased in the presence of PO and ALA, and the Hâ‚‚Oâ‚‚-mediated up-regulated BAX/BCL-2 ratio was blocked after treatment with PO and ALA.
CONCLUSIONS
PO and its main fatty acid, ALA, exerted the protective activity from neuronal oxidative stress induced by Hâ‚‚Oâ‚‚. They regulated apoptotic pathway in neuronal cell death by alleviation of BAX/BCL-2 ratio, and down-regulation of cleaved-PARP and cleaved caspase-9 and -3. Although further studies are required to verify the protective mechanisms of PO and ALA from neuronal damage, PO and ALA are the promising agent against oxidative stress-induced apoptotic neuronal cell death.

Keyword

alpha-linolenic acid; apoptosis; hydrogen peroxide; neuroprotective effect; perilla

MeSH Terms

Adenosine Diphosphate Ribose
alpha-Linolenic Acid*
Apoptosis
Caspase 9
Cell Death*
Cell Survival
Down-Regulation
Humans
Hydrogen Peroxide*
Hydrogen*
L-Lactate Dehydrogenase
Neuroblastoma
Neurodegenerative Diseases
Neurons*
Neuroprotective Agents
Oxidative Stress
Perilla*
Adenosine Diphosphate Ribose
Caspase 9
Hydrogen
Hydrogen Peroxide
L-Lactate Dehydrogenase
Neuroprotective Agents
alpha-Linolenic Acid

Figure

• 

  Fig. 1 Effects of PO (A) and ALA (B) on cell viability in hydrogen peroxide-induced SH-SY5Y cells.PO, Perilla oil; ALA, Alpha-linolenic acid. Values are mean ± SD. a–dMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. The results were obtained in three independent experiments.

• 

  Fig. 2 Protective effect of PO (A) and ALA (B) on lactate dehydrogenase (LDH) release in SH-SY5Y cells treated with hydrogen peroxide.PO, Perilla oil; ALA, Alpha-linolenic acid. Values are mean ± SD. a–dMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. The results were obtained in three independent experiments.

• 

  Fig. 3 Effects of PO and ALA on DNA condensation in SH-SY5Y cells treated with hydrogen peroxide.The nuclear morphology of cells was determined by Hoechst 33342 staining. (A) Representative images of nuclei stained with Hoechst 33342. The apoptotic bodies are indicated with arrows. (B) Quantification of the nuclei demonstrating apoptotic bodies observed under a fluorescence microscope. Magnification was 400x. PO, Perilla oil; ALA, Alpha-linolenic acid. Values are mean ± SD. a–eMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. The results were obtained in three independent experiments.

• 

  Fig. 4 Effects of PO and ALA on levels of cleaved caspase-9 protein expression in SH-SY5Y cells treated with hydrogen peroxide.PO, Perilla oil; ALA, Alpha-linolenic acid. Values are mean ± SD. a–cMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. β-actin was used as a loading control. The results were obtained in three independent experiments.

• 

  Fig. 5 Effects of PO and ALA on levels of cleaved caspase-3 protein expression in SH-SY5Y cells treated with hydrogen peroxide. PO, Perilla oil; ALA, Alpha-linolenic acid.Values are mean ± SD. a–eMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. β-actin was used as a loading control. The results were obtained in three independent experiments.

• 

  Fig. 6 Effect of PO and ALA on levels of cleaved-poly ADP ribose polymerase protein expression in SH-SY5Y cells treated with hydrogen peroxide.PO, Perilla oil; ALA, Alpha-linolenic acid. Values are mean ± SD. a–fMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. β-actin was used as a loading control. The results were obtained in three independent experiments.

• 

  Fig. 7 Effect of PO and ALA on levels of BAX/BCL-2 ratio protein expression in SH-SY5Y cells treated with hydrogen peroxide.PO, Perilla oil; ALA, Alpha-linolenic acid. Values are mean ± SD. a–eMeans with different letters are significantly different (P < 0.05) as determined by Duncan's multiple range test. β-actin was used as a loading control. The results were obtained in three independent experiments.

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