Korean J Physiol Pharmacol.  2009 Aug;13(4):281-285. 10.4196/kjpp.2009.13.4.281.

Naringin Protects against Rotenone-induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

• Kim HJ
• Song JY
• Park HJ
• Park HK
• Yun DH
• Chung JH

• KMID: 2071682
• DOI: http://doi.org/10.4196/kjpp.2009.13.4.281

Abstract

Rotenone, a mitochondrial complex I inhibitor, can induce the pathological features of Parkinson's disease (PD). In the present study, naringin, a grapefruit flavonoid, inhibited rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We assessed cell death and apoptosis by measuring mitogen-activated protein kinase (MAPKs) and caspase (CASPs) activities and by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Naringin also blocked rotenone-induced phosphorylation of Jun NH2-terminal protein kinase (JNK) and P38, and prevented changes in B-cell CLL/lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) expression levels. In addition, naringin reduced the enzyme activity of caspase 3 and cleavages of caspase 9, poly (ADP-ribose) polymerase (PARP), and caspase 3. These results suggest that naringin has a neuroprotective effect on rotenone-induced cell death in human neuroblastoma SH-SY5Y cells.

Keyword

Apoptosis; Naringin; Parkinson's disease; Rotenone; SH-SY5Y

MeSH Terms

Apoptosis
B-Lymphocytes
bcl-2-Associated X Protein
Caspase 3
Caspase 9
Cell Death
Citrus paradisi
Flavanones
Humans
Indoles
Neuroblastoma
Neuroprotective Agents
Parkinson Disease
Phosphorylation
Protein Kinases
Rotenone
Tetrazolium Salts
Thiazoles
Caspase 3
Caspase 9
Flavanones
Indoles
Neuroprotective Agents
Protein Kinases
Rotenone
Tetrazolium Salts
Thiazoles
bcl-2-Associated X Protein

Figure

• 

  Fig. 1. Effect of naringin on rotenone-induced cell death in SH-SY5Y cells. (A) MTT cell viability assay at 0.1, 1, 5, 10, and 20 μM rotenone for 24 h in SH-SY5Y cells. (B) Naringin inhibits rotenone-induced cytotoxicity in SH-SY5Y cells, treated with 10 μM rotenone for 24 h. Naringin (1, 5, and 10 μM) was added 4 h before rotenone treatment. Independent experiments were repeated three times. Values are presented as mean±SEM. Con, control. ∗p<0.05 compared to non-treated cells; †p<0.05 compared to rotenone-treated cells.

• 

  Fig. 2. Effect of naringin on rotenone-induced apoptotic features in SH-SY5Y cells pretreated with 10 μM rotenone for 24 h. Naringin (10 μM) was treated 4 h prior to rotenone treatment. Condensed chromatin was stained dark brown (lower). Scale bar, 100 μm. Three independent experiments were performed for this study.

• 

  Fig. 3. The effect of rotenone on the phosphorylation of JNK and P38 in SH-SY5Y cells. (A) The effect of rotenone on the phosphorylation of JNK and P38 by Western blot analysis. Cells were treated with 10 μM rotenone for 120 min. (B) Western blot to determine the effect of naringin on rotenone-induced phosphorylation of JNK and P38 in SH-SY5Y cells. The cells were pretreated with 10 μM rotenone for 120 min, with 10 μM naringin added 4 h prior to rotenone treatment. The cells were harvested for the phosphorylation assay at 30 and 120 min after rotenone treatment. β-Actin was used as an internal standard protein. Three independent experiments were performed for this assay.

• 

  Fig. 4. The effect of rotenone on the expression of apoptotic proteins BCL2, BAX, CASP9, PARP, and cleaved CASP3 in SH-SY5Y cells. (A) The effect of rotenone on the expression of BCL2, BAX, CASP9, PARP, and cleaved CASP3 by Western blot analysis. Cells were treated with 10 μM rotenone for 24 h. (B) Western blot assay to determine the effect of naringin on rotenone-induced expression of the apoptotic proteins, BCL2, BAX, cleaved CASP9, cleaved PARP, and cleaved CASP3 in SH-SY5Y cells. Cells were pretreated with 10 μM rotenone for 24 h, with 10 μM naringin added 4 h prior to rotenone treatment. β-Actin was detected as an internal standard protein. (C) Effect of naringin on rotenone-induced activity of CASP3 in SH-SY5Y cells. Cells were pretreated with 100 μM rotenone for 24 h and 10 μM naringin was then added 4 h prior to rotenone treatment. The cleaved CASP3 substrate acetyl-AspGlu-Val-Asp-p-Nitroanilide (Ac-DEVD-p-NA) was measured at 405 nm. CASP3 was used as a positive control. The CASP3 inhibitor (Ac-DEVD-CHO) added with CASP3 was used as negative control. Values are presented as mean±SEM. Three independent experiments were performed for this assay. ∗p<0.05 compared to non-treated cells; †p<0.05 compared to rotenone-treated cells.

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