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

Abstract

BackgroundApoptosis 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.MethodsThus, 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.ResultsORF 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).ConclusionThese findings suggest ORF may be an effective antioxidant that could prevent oxidative stress-induced neurodegenerative disorders.

Modulation of Hydrogen Peroxide-Induced Oxidative Stress in Human Neuronal Cells by Thymoquinone-Rich Fraction and Thymoquinone via Transcriptomic Regulation of Antioxidant and Apoptotic Signaling Genes

“…An additional 37 base pairs of universal tag sequences were attached to each forward and reverse primer. Synthesis of primers was performed by First Base Ltd. (Selangor, Malaysia) and diluted in 1x TE buffer to a concentration of 500 nM for reverse primers and 200 nM for forward primers [ 18 ].…”

“…The reverse transcription reaction (RT reaction) was performed according to the GenomeLab GeXP Start Kit instructions (Beckman Coulter Inc., Miami, FL, USA) using 1 μ L of each RNA sample, 4 μ L of 5x RT buffer, 2 μ L of RT multiplex reverse primers, 1 μ L of KanR, 1 μ L of reverse transcriptase, and 11 μ L of DNAse/RNase free water. cDNA was synthesized according to the reaction protocol: 48°C for 1 min, 42°C for 60 min, 95°C for 5 min, and 4°C hold in an XP Thermal Cycler (BIOER Technology, Hangzhou, China) [ 18 ].…”

“…PCR reactions were prepared using a GeXP Start Kit (Beckman Coulter, Brea, CA, USA) consisting of cDNA samples taken from the RT reaction (9.3 μ L each), 5x PCR buffer (2 μ L), 25 mM MgCl 2 (2 μ L), PCR multiplex forward primer (1 μ L), and Thermo-Start DNA polymerase (0.7 μ L). Amplification conditions were 95°C for 10 min, followed by 34 cycles of 94°C for 30 sec, 55°C for 30 sec, 70°C for 1 min, and 4°C hold carried out in an XP Thermal Cycler (BIOER Technology, Hangzhou, China) [ 18 ].…”

“…The data were analyzed using the Fragment Analysis module of the GeXP system software and then transferred to the analysis module of the eXpress Profiler software. Normalization was performed with reference gene β -actin, according to manufacturer's instructions [ 18 ].…”

Transcriptomic profile adaptations following exposure of equine satellite cells to nutriactive phytochemical gamma-oryzanol

“…Consistent with this, significantly lower post-exercise total antioxidant status and thiobarbituric acid-reactive substance level were observed in racing horses receiving GO compared to horses from other groups [ 49 ]. GO’s influence on oxidative stress-related genes has been shown in other tissues [ 30 , 31 ]. However, in our study, gene ontology analysis did not show any significant involvement of differentially expressed genes in antioxidative processes.…”