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

Received: 8 July 2020/Accepted: 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

REFERENCES

Akyol, S., Yükselten, Y., Çakmak, Ö., Uğurcu, V., Altuntaş, A., Gürler, M., Akyol, Ö. & Demircan, K. 2014. Hydrogen peroxide-induced oxidative damage in human chondrocytes: the prophylactic effects of Hypericum perforatum Linn extract on deoxyribonucleic acid damage, apoptosis and matrix remodeling by a disintegrin-like and metalloproteinase with thrombospondin motifs proteinases. Archives of Rheumatology 29: 203-214.

Ali, S.F. & Woodman, O.L. 2015. Tocotrienol rich palm oil extract is more effective than pure tocotrienols at improving endothelium-dependent relaxation in the presence of oxidative stress. Oxidative Medicine and Cellular Longevity 2015: 150829.

Berbee, M., Fu, Q., Boerma, M., Pathak, R., Zhou, D., Kumar, K.S. & Hauer-Jensen, M. 2011. Reduction of radiation-induced vascular nitrosative stress by the vitamin E analog γ-tocotrienol: Evidence of a role for tetrahydrobiopterin. International Journal of Radiation Oncology Biology Physics 79(3): 884-891.

Bester, D.J., Kupai, K., Csont, T., Szucs, G., Csonka, C., Esterhuyse, A.J., Ferdinandy, P. & Van Rooyen, J. 2010. Dietary red palm oil supplementation reduces myocardial infarct size in an isolated perfused rat heart model. Lipids in Health and Disease 9(1): 64.

Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S. & Kalayci, O. 2012. Oxidative stress and antioxidant defense. World Allergy Organization Journal 5(1): 9-19.

Biswas, S.K. 2016. Does the interdependence between oxidative stress and inflammation explain the antioxidant paradox. Oxidative Medicine and Cellular Longevity 2016: 5698931.

Brand, M.D., Affourtit, C., Esteves, T.C., Green, K., Lambert, A.J., Miwa, S., Pakay, J.L. & Parker, N. 2004. Mitochondrial superoxide: Production, biological effects, and activation of uncoupling proteins. Free Radical Biology and Medicine 37: 755-767.

Casey, T.M., Arthur, P.G. & Bogoyevitch, M.A. 2007. Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress. Biochimica et Biophyica Acta - Molecular Cell Research 1773: 342-351.

Condorelli, G., Roncarati, R,, Ross, J., Pisani, A., Stassi, G., Todaro, M., Trocha, S., Drusco, A., Gu, Y. & Russo, M.A. 2001. Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function. Proceedings of the National Academy of Sciences 98: 9977-9982.

Dieterich, S., Bieligk, U., Beulich, K., Hasenfuss, G. & Prestle, J. 2000. Gene expression of antioxidative enzymes in the human heart: Increased expression of catalase in the end-stage failing heart. Circulation 101(1): 33-39.

Dröge, W. 2002. Free radicals in the physiological control of cell function. Physiological Reviews 82: 47-95.

Esterhuyse, A.J., Du Toit, E.F., Benade, A.J.S. & Van Rooyen, J. 2005. Dietary red palm oil improves reperfusion cardiac function in the isolated perfused rat heart of animals fed a high cholesterol diet. Prostaglandins, Leukotrienes and Essential Fatty Acids 72(3): 153-161.

Fauconnier, J., Meli, A.C., Thireau, J., Roberge, S., Shan, J., Sassi, Y., Reiken, S.R., Rauzier, J.M., Marchand, A., Chauvier, D., Cassan, C., Crozier, C., Bideaux, P., Lompre, A.M., Jacotot, E., Marks, A.R. & Lacampagne, A. 2011. Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion. Proceedings of the Natlional Academy of Sciences U.S.A 108: 13258-13263.

Fu, J.Y., Che, H.L., Tan, D.M. & Teng, K.T. 2014. Bioavailability of tocotrienols: Evidence in human studies. Nutrition & Metabolism 11(1): 5.

Howard, A.C., McNeil, A.K. & McNeil, P.L. 2011. Promotion of plasma membrane repair by vitamin E. Nature Communications 2: 597.

Hrelia, S., Fiorentini, D., Maraldi, T., Angeloni, C., Bordoni, A., Biagi, P.L. & Hakim, G. 2002. Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes. Biochimica et Biophysica Acta (BBA)-Biomembranes 1567: 150-156.

Kamal-Eldin, A. & Appelqvist, L.A. 1995. The effects of extraction methods on sasame oil stability. Journal of the American Oil Chemists' Society 72: 967-969.

Khor, H.T., Chieng, D.Y. & Ong, K.K. 1995. Tocotrienols inhibits HMG-CoA reductase activity in the guinea pig. Nutrition Research 15: 537-544.

Kourouma, A., Quan, C., Duan, P., Qi, S., Yu, T., Wang, Y. & Yang, K. 2015. Bisphenol A induces apoptosis in liver cells through induction of ROS. Advances in Toxicology 2015: Article ID. 901983.

Krager, K.J., Pineda, E.N., Kharade, S.V., Kordsmeier, M., Howard, L., Breen, P.J. & Aykin-Burns, N. 2015. Tocotrienol-rich fraction from rice bran demonstrates potent radiation protection activity. Evidence-Based Complementary and Alternative Medicine 2015: 148791.

Kuhad, A. & Chopra, K. 2009. Tocotrienol attenuates oxidative-nitrosative stress and inflammatory cascade in experimental model of diabetic neuropathy. Neuropharmacology 57(4): 456-462.

Lane, R.K., Hilsabeck, T. & Rea, S.L. 2015. The role of mitochondrial dysfunction in age-related diseases. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1847: 1387-1400.

Lobo, V., Patil, A., Phatak, A. & Chandra, N. 2010. Free radicals, antioxidants and functional foods: impact on human health. Pharmacognosy Reviews 4: 118.

Miura, T., Tanno, M. & Sato, T. 2010. Mitochondrial kinase signalling pathways in myocardial protection from ischaemia/reperfusion-induced necrosis. Cardiovascular Research 88: 7-15.

Muharis, S.P., Top, A.G.M., Murugan, D. & Mustafa, M.R. 2010. Palm oil tocotrienol fractions restore endothelium dependent relaxation in aortic rings of streptozotocin-induced diabetic and spontaneously hypertensive rats. Nutrition Research 30(3): 209-216.

Nakamura, T., Wang, L., Wong, C.C., Scott, F.L., Eckelman, B.P., Han, X., Tzitzilonis, C., Meng, F., Gu, Z. & Holland, E.A. 2010. Transnitrosylation of XIAP regulates caspase-dependent neuronal cell death. Molecular Cell 39: 184-195.

Nazrun, A.S., Khairunnur, A., Norliza, M., Norazlina, M. & Ima Nirwana, S. 2008. Effects of palmt tocotrienols on oxidative stress and bone strength in ovariectomised rats. Medicine and Health 3(2): 247-255. 

Nowak, G., Bakajsova, D., Hayes, C., Hauer-Jensen, M. & Compadre, C.M. 2012. γ-Tocotrienol protects against mitochondrial dysfunction and renal cell death. Journal of Pharmacology and Experimental Therapeutics 340: 330-338.

Obal, D., Dai, S., Keith, R., Dimova, N., Kingery, J., Yu-Ting, Z., Zweier, J., Velayutham, M., Prabhu, S.D., Li, Q., Conklin, D., Yamg, D., Bhatnagar, A., Bolli, R. & Rokosh, G. 2012. Cardiomyocyte-restricted overexpression of extracellular superoxide dismutase increases nitric oxide bioavailability and reduces infarct size after ischemia/reperfusion. Basic Research in Cardiology 107(6): 305.

Panth, N., Paudel, K.R. & Parajuli, K. 2016. Reactive oxygen species: A key hallmark of cardiovascular disease. Advances in Medicine 2016: 9152732.

Pinto, A., Immohr, M.B., Jahn, A., Jenke, A., Boeken, U., Lichtenberg, A. & Akhyari, P. 2016. The extracellular isoform of superoxide dismutase has a significant impact on cardiovascular ischaemia and reperfusion injury during cardiopulmonary bypass. European Journal of Cardio-Thoracic Surgery 50: 1035-1044.

Sabbah, H.N. 2016. Targeting mitochondrial dysfunction in the treatment of heart failure. Expert Review of Cardiovascular Therapy 14(12): 1305-1313.

Sahhugi, Z., Hasenan, S.M. & Jubri, Z. 2014. Protective effects of gelam honey against oxidative damage in young and aged rats. Oxidative Medicine and Cellular Longevity 2014: 673628.

Salameh, A. & Dhein, S. 2005. Culture of neonatal cardiomyocytes. In Practical Methods in Cardiovascular Research, edited by Dhein, S., Mohr, F.W. & Delmar, M. Springer, Berlin, Heidelberg. pp. 568-576.

Sambanthamurthi, R., Sundram, K. & Tan, Y.A. 2000. Chemistry and biochemistry of palm oil. Progress in Lipid Research 39: 507-558.

Sharma, P., Jha, A.B., Dubey, R.S. & Pessarakli, M. 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany 2012: Article ID. 217037.

Sugamura, K. & Keaney, J.F. 2011. Reactive oxygen species in cardiovascular disease. Free Radical Biology and Medicine 51: 978-992.

Taverne, Y.J., Bogers, A.J., Duncker, D.J. & Merkus, D. 2013. Reactive oxygen species and the cardiovascular system. Oxidative Medicine and Cellular Longevity 2013: 862423.

Tham, Y.K., Bernardo, B.C., Ooi, J.Y., Weeks, K.L. & McMullen, J.R. 2015. Pathophysiology of cardiac hypertrophy and heart failure: Signaling pathways and novel therapeutic targets. Archives of Toxicology 89: 1401-1438.

Vasanthi, H.R., Parameswari, R. & Das, D.K. 2012. Multifaceted role of tocotrienols in cardioprotection supports their structure: Function relation. Genes and Nutrition 7: 19-28.

Wang, X., Dong, W., Yuan, B., Yang, Y., Yang, D., Lin, X., Chen, C. & Zhang, W. 2016. Vitamin E confers cytoprotective effects on cardiomyocytes under conditions of heat stress by increasing the expression of metallothionein. International Journal of Molecular Medicine 37(5): 1429-1436.

Webster, K.A. 2012. Mitochondrial membrane permeabilization and cell death during myocardial infarction: Roles of calcium and reactive oxygen species. Future Cardiology  8(6): 863-884.

World Health Organization. 2016. The World Health Report 2002. Reducing Risk, Promoting Healthy Life. World Health Organization (WHO).

Wu, A., Ying, Z. & Gomez-Pinilla, F. 2010. Vitamin E protects against oxidative damage and learning disability after mild traumatic brain injury in rats. Neurorehabilitation and Neural Repair 24: 290-298.

Zhang, Y., Chen, X., Gueydan, C. & Han, J. 2018. Plasma membrane changes during programmed cell deaths. Cell Research 28(1): 9-21.

Zhou, T., Chia-Chen, C. & Zuo, L. 2015. Molecular characterization of reactive oxygen species in myocardial ischemia-reperfusion injury. BioMed Research International 2015: Article ID. 864946.

*Corresponding author; email: zakiah.jubri@ppukm.ukm.edu.my