Mechanistic basis for protection of differentiated SH-SY5Y cells by oryzanol-rich fraction against hydrogen peroxide-induced neurotoxicity

Abstract

Background: Apoptosis is often the end result of oxidative damage to neurons. Due to shared pathways between oxidative stress, apoptosis and antioxidant defence systems, an oxidative insult could end up causing cellular apoptosis or survival depending on the severity of the insult and cellular responses. Plant bioresources have received close attention in recent years for their potential role in regulating the pathways involved in apoptosis and oxidative stress in favour of cell survival. Rice bran is a bioactive-rich by-product of rice milling process. It possesses antioxidant properties, making it a promising source of antioxidants that could potentially prevent oxidative stress-induced neurodegenerative diseases.

Methods: Thus, the present study investigated the neuroprotective properties of oryzanol-rich fraction (ORF) against hydrogen peroxide (H2O2)-induced neurotoxicity in differentiated human neuroblastoma SH-SY5Y cells. ORF was extracted from rice bran using a green technology platform, supercritical fluid extraction system. Furthermore, its effects on cell viability, morphological changes, cell cycle, and apoptosis were evaluated. The underlying transcriptomic changes involved in regulation of oxidative stress, apoptosis and antioxidant defence systems were equally studied.

Results: ORF protected differentiated SH-SY5Y cells against H2O2-induced neurotoxicity through preserving the mitochondrial metabolic enzyme activities, thus reducing apoptosis. The mechanistic basis for the neuroprotective effects of ORF included upregulation of antioxidant genes (catalase, SOD 1 and SOD 2), downregulation of pro-apoptotic genes (JNK, TNF, ING3, BAK1, BAX, p21 and caspase-9), and upregulation of anti-apoptotic genes (ERK1/2, AKT1 and NF-Kβ).

Conclusion: These findings suggest ORF may be an effective antioxidant that could prevent oxidative stress-induced neurodegenerative disorders.

Figure 1

Cell viability (MTT assay) of SH-SY5Y cells pretreated with Oryzanol-rich fraction (ORF) for 24 h followed by subsequent exposure to 250 μM H ₂ O ₂ for 24 h. (A) ORF alone; (B) ORF + 250 μM H₂O₂. Results are mean ± SD. ^# p <0.05 versus control, *p < 0.05 versus H₂O₂.

Figure 2

Acridine orange (AO)–propidium iodide (PI) double staining cell morphological assessment. Morphological changes in SH-SY5Y cells pretreated with Oryzanol-rich fraction (ORF) for 24 h followed by subsequent exposure to 250 μM H₂O₂ for 24 h. (A) untreated cells (control); (B) 250 μM H₂O₂ alone; (C) 100 μ g/mL ORF +250 μM H₂O₂. Viable cells are stained green by acridine orange; Late apoptotic and necrotic cells are stained orange and red by propidium iodide.

Figure 3

Cell cycle analysis. Flow cytometric measurement of cell death and cell cycle on SH-SY5Y cells pretreated with Oryzanol-rich fraction (ORF) (100 μg/mL) before exposure to 250 μM H₂O₂ over 24 h. Results are mean ± SD. ^# p <0.05 versus control, *p <0.05 versus H₂O₂.

Figure 4

Annexin V-FITC and propidium iodide staining assay of SH-SY5Y cells following exposure to 250 μM H ₂ O ₂ over 24 h, in the presence or absence of 24 h Oryzanol-rich fraction (ORF) (100 μg/mL) pretreatment. Results are mean ± SD. ^# p < 0.05 versus control, *p <0.05 versus H₂O₂.

Figure 5

Expression of (A) Antioxidant genes (catalase, SOD 1 and SOD 2), (B) Downstream apoptotic genes (BAD, TNF, ING3, BAK1, BAX, p21, caspase-9 and BCL-2), and (C) Upstream apoptotic genes (ERK1/2, p53, JNK, PARP1, AKT1, NF-Kβ, and p38), following treatment with 100 μg/mL Oryzanol-rich fraction (ORF) and subsequent exposure with 250 μM H ₂ O ₂ . Results are the mean ± SD. ^# p < 0.05 versus control, *p <0.05 versus H₂O₂.

Figure 6

Schematic presentation of the proposed mechanistic basis for the neuroprotective effects of ORF against H ₂ O ₂ -induced neurotoxicity in human differentiated SH-SY5Y cells. Oxidative stress likely activates pro-apoptotic genes (i.e. JNK, TNF, ING3, BAK1, BAX, p21 and caspase-9) and downregulates anti-apoptotic genes (i.e. ERK1/2, AKT1 and NF-Kβ), resulting in cellular apoptosis. In contrast, ORF likely upregulate endogenous antioxidant defences (i.e. Catalase, SOD1 and SOD2) that can enhance cell survival. Additionally, ORF may downregulate pro-apoptotic genes thereby preventing mitochondrial malfunction and caspase activation. Activation of anti-apoptotic genes likely also contributes to enhanced cell survival.