Sains Malaysiana 50(5)(2021): 1457-1466

http://doi.org/10.17576/jsm-2021-5005-24

Palm Tocotrienol-Rich Fraction Protects Neonatal Rat Cardiomyocytes from H₂O₂-Induced Oxidative Damage

(Fraksi Kaya Tocotrienol Sawit Melindungi Kardiomiosit Tikus Neonatal daripada Induksi Kerosakan Pengoksidaan H₂O₂)

NOOR SHAREENA AISHA ABDUL KHALID, KHUZAIDATUL AZIDAH AHMAD NAZRI & ZAKIAH JUBRI*

Department of Biochemistry, UKM Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

Diserahkan: 8 Julai 2020/Diterima: 30 September 2020

ABSTRACT

Oxidative stress plays an important role in the pathogenesis of heart disease. Tocotrienol-rich fraction (TRF) is an antioxidant and that has the potential to reduce the risk of heart disease. This study is to determine the protective effects of palm TRF against H₂O₂-induced oxidative damage in neonatal rat cardiomyocytes (NRCM). The NRCM were divided into control, treated with TRF (10 µg/mL), H₂O₂ (0.5 mM) and treated with TRF prior to H₂O₂ induction (pre-treatment). Cell viability was determined by the MTS assay,while the presence of reactive oxygen species (ROS) was determined using fluorescent dihydroethidium (DHE) and 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA) dye. Mitochondrial integrity and cell death were determined using JC-1 and Annexin V-FITC staining, respectively. Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity were determined by colorimetric assay kit. The concentration of H₂O₂ from 0.5 to 5 mM reduced the cell viability and the H₂O₂ IC₅₀ value of 0.5 mM was used in the experiment. H₂O₂ induction increased the intensity of carboxy-H₂DCFDA and DHE-stains; and also the intensity of green fluorescence of J-monomers in JC-1 staining compared to the control group. The activity of LDH increased while the activity of SOD decreased in the H₂O₂ group. Pre-treatment with TRF reduced the intensities of carboxy-H₂DCFDA and DHE-stains, as well as the green fluorescence of J-monomers in JC-1. Meanwhile, the LDH activity was reduced in the pre-treatment group but no changes were recorded in SOD activity compared to the H₂O₂ group. Palm TRF protects cardiomyocytes from oxidative damage by reducing ROS production and maintaining the mitochondrial membrane integrity thus reducing cell death.

Keywords: Cardiomyocytes; H₂O₂; oxidative damage; tocotrienol-rich fraction

ABSTRAK

Tekanan oksidatif memainkan peranan penting dalam patogenesis penyakit jantung. Fraksi kaya tokotrienol (TRF) adalah antioksidan dan berpotensi mengurangkan risiko penyakit jantung. Kajian ini adalah untuk mengetahui kesan pelindung TRF sawit terhadap kerosakan oksidatif aruhan H₂O₂ pada kardiomiosit tikus neonatal (NRCM). NRCM dibahagi kepada kawalan, dirawat dengan TRF (10 µg/mL), H₂O₂ (0.5 mM) dan dirawat dengan TRF sebelum induksi dengan H₂O₂ (pra-rawatan). Kebolehhidupan sel ditentukan dengan ujian MTS. Kehadiran ROS ditentukan menggunakan pewarna dihidroetidium (DHE) dan pewarna 5-(dan-6)-karboksi-2',7′-diklorodihidrofluorescein diasetat (carboxy-H2DCFDA). Integriti mitokondria dan kematian sel ditentukan menggunakan pewarnaan JC-1 dan Annexin V-FITC masing-masing. Aktiviti laktat dehidrogenase (LDH) dan superoksid dismutase (SOD) ditentukan menggunakan kit esei kalorimetrik. Kepekatan H₂O₂ bermula daripada 0.5 hingga 5 mM menurunkan kebolehhidupan sel dan nilai IC₅₀ H₂O₂ 0.5 mM digunakan di dalam kajian ini. Aruhan H₂O₂ meningkatkan keamatan karboksi-H₂DCFDA dan pewarnaan DHE; dan juga keamatan pendarfluor hijau monomer-J dalam pewarnaan JC-1 berbanding kumpulan kawalan. Aktiviti LDH meningkat sementara aktiviti SOD menurun dalam kumpulan H₂O₂. Pra-rawatan dengan TRF menurunkan keamatan karboksi-H₂DCFDA dan pewarnaan DHE; dan juga keamatan pendarfluor hijau monomer-J dalam pewarnaan JC-1. Manakala aktiviti LDH menurun dalam kumpulan pra-rawatan tetapi tiada perubahan ditunjukkan dalam aktiviti SOD berbanding kumpulan H₂O₂. TRF sawit melindungi kardiomiosit daripada kerosakan oksidatif melalui pengurangan penghasilan ROS dan mengekalkan integriti membran mitokondria seterusnya mengurangkan kematian sel.

Kata kunci: Fraksi kaya tokotrienol; H₂O₂; kardiomiosit; kerosakan oksidatif

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*Pengarang untuk surat-menyurat; email: zakiah.jubri@ppukm.ukm.edu.my