Background: Oxidative stress plays an important role in the pathogenesis of heart diseases. Antioxidants such as palm tocotrienol-rich fraction (TRF) can reduce oxidative stress, hence preventing and reducing the risk of heart disease. This study was aimed to determine the protective effects of TRF against hydrogen peroxide (H2O2 ) - induced oxidative stress in neonatal rat cardiomyocytes (NRCM). Methods: The NRCM were divided into five groups: (1) control, (2) cells treated with TRF (10 µg/ml) for 24 hours, (3) cells subjected to H2O2 (0.5 mM ) for 30 minutes, (4) cells pre-treated with TRF, and (5) cells post-treated with TRF. The IC50 of H2O2 (0 – 5 mM ) and the effective dose of TRF (0 – 25 µg/ml) were determined using the MTS cell viability assay. Meanwhile, ELISA was used to measure the level of reactive oxygen species (ROS). The presence of superoxides and H2O2 were detected by dihydroethidium and 5-(and-6 ) - carboxy -2′,7′-dichlorodihydrofluorescein diacetate respectively. Flowcytometry analysis was conducted to determine the presence of apoptosis and measure the mitochondrial membrane potential, whereby the former involved the use of Annexin V-FITC stain while the latter JC-1 stain. The gene expressions of antioxidant (SOD, CAT, GPx) and apoptosis (Bax, Bcl-2, Caspase-3) enzymes were studied using qRT -PCR. Results: The IC50 of H2O2 was 0.5 mM while the effective dose of TRF 10 µg/ml. The cells which were subjected to H2O2 showed a decrease in NRCM viability and significant increase (p < 0.05) in ROS production. LDH activity and green fluorescence intensity (which indicated mitochondrial depolarisation ) were increased following H2O2 induction . With reference to the control, the H2O2- induced group had a higher percentage of late apoptotic cells, which was associated with the upregulation of the pro-apoptotic gene, Bax, and downregulation of the anti-apoptotic gene, Bcl-2 (p < 0.05). H2O2 also upregulated GPx expression , apart from downregulating CAT and Cu/Zn SOD expression (p < 0.05). The pre- and post-treatment groups had increased cell viability and reduced ROS production. Pre-treatment with TRF protected the cell membranes and mitochondria from H2O2- induced injury, as reflected by the reduction in extracellular LDH activity and apoptosis (the latter of which was associated with the downregulation of Bax). Meanwhile, the expression of GPx, Cat, and Cu/Zn SOD was reduced in the post-treatment group. Conclusion: By scavenging for ROS, palm TRF directly protects the cell membrane from H2O2- induced injury, leading to a decrease in oxidative stress. Thus, palm TRF maintains the mitochondrial membrane potential and prevents apoptosis secondary to decreased Bax expression.
Fucoxanthin protects neonatal rat cardiomyocytes and attenuates high glucose-mediated oxidative stress via the AMPK pathway
