Sains Malaysiana 52(1)(2023): 35-46

http://doi.org/10.17576/jsm-2023-5201-03

Cytoskeletal Morphological Changes of Mesenchymal Stem Cells after Oxidant Damage and its Prevention by Thymoquinone

(Perubahan Morfologi Sitokerangka Sel Stem Mesenkima selepas Kerosakan Oksidan dan Pencegahannya oleh Timoquinon)

SUHAIMI DRAMAN¹, NURUL KABIR^1,* & DURRIYYAH SHARIFAH HASAN ADLI²

¹Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

²Centre for Civilisational Dialogue, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

Received: 29 September 2021/Accepted: 3 November 2022

Abstract

The functional integrity of the cytoskeleton of mesenchymal stem cells (MSCs) is essential for its differentiation into multiple cell lineages including adipocytes, chondrocytes, and osteoblasts. Abnormalities in the cytoskeletal proteins such as actin and microtubule can cause disrupted cell signalling and irregular movements of organelles leading to cell death. This study investigated cytoskeletal and nuclear morphological changes of the MSC due to oxidative damage by hydrogen peroxide (H₂O₂) and the possible prevention of these changes by the antioxidant thymoquinone (TQ). Bone marrow MSCs from Sprague Dawley rats were cultured and treated with different concentrations of H₂O₂ with or without TQ to observe the potential protective activity. Triple-label fluorescence immunocytochemistry was performed post-treatment to observe the nucleus, actin and microtubules using 4’,6-diamidino-2-phenylindole (DAPI), Alexa Fluor 488-labelled phalloidin and Cy3-labelled anti-tubulin antibody, respectively. The normal stem cell cytoskeleton demonstrated intact actin and microtubule structures along with normal appearance of the nucleus. However, oxidative damage by H₂O₂ caused a severe disruption of the cytoskeletal morphology of the actin and microtubule along with apoptosis and necrosis of the nucleus. Interestingly, both immunocytochemical and Fluorescence-Activated Cell Sorting (FACS) results showed that these morphological changes were prevented by TQ at low concentrations while higher concentrations of TQ were harmful. This study suggested that TQ could save MSCs from oxidative-induced cell death.

Keywords: Cytoskeleton; oxidative damage; stem cell; thymoquinone

Abstrak

Keutuhan fungsi sitokerangka sel induk mesenkima (MSCs) adalah penting untuk pembezaannya kepada pelbagai keturunan sel termasuk adiposit, kondrosit dan osteoblas. Keabnormalan dalam protein sitokerangka seperti aktin dan mikrotubul boleh mengakibatkan isyarat sel terganggu dan pergerakan organel tidak teratur yang membawa kepada kematian sel. Penyelidikan ini mengkaji perubahan morfologi sitokerangka dan nukleus MSC akibat kerosakan oksidatif oleh hidrogen peroksida (H₂O₂) dan kemungkinan pencegahan perubahan ini oleh antioksidan, timoquinon (TQ). MSC sumsum tulang daripada tikus Sprague Dawley telah dikulturkan dan dirawat dengan kepekatan H₂O₂ yang berbeza dengan atau tanpa TQ untuk mencerap potensi aktiviti perlindungan. Imunositokimia triple-label fluorescence telah dilakukan selepas rawatan untuk mencerap nukleus, aktin dan mikrotubul sel menggunakan masing-masing 4’,6-diamidino-2-fenilindol (DAPI), faloidin terlabel Alexa Fluor 488 dan antibodi anti-tubulin terlabel Cy3. Sitokerangka sel induk normal menunjukkan struktur aktin dan mikrotubul yang utuh berserta dengan penampilan normal nukleus. Walau bagaimanapun, kerosakan oksidatif oleh H₂O₂ mengakibatkan gangguan teruk ke atas morfologi sitokerangka aktin dan mikrotubul berserta dengan apoptosis dan nekrosis nukleus. Menariknya, kedua-dua hasil imunositokimia dan Fluorescence-activated Cell Sorting (FACS) menunjukkan bahawa perubahan morfologi ini dihalang oleh TQ pada kepekatan rendah manakala kepekatan TQ yang lebih tinggi adalah berbahaya. Kajian ini menyarankan bahawa TQ boleh menyelamatkan MSC daripada kematian sel yang disebabkan oleh oksidan.

Kata kunci: Kerosakan oksidatif; sel induk; sitokerangka; timoquinon

REFERENCES

Abd-Elkareem, M., Abd-El-Rahman, M., Khalil, N. & Amer, A. 2021. Antioxidant and cytoprotective effects of Nigella sativa L. seeds on the testis of monosodium glutamate challenged rats. Scientific Reports 11(1): 13519.

Acharya, B., Chatterjee, A., Ganguli, A., Bhattacharya, S. & Chakrabarti, G. 2014. Thymoquinone inhibits microtubule polymerization by tubulin binding and causes mitotic arrest following apoptosis in A549 cells. Biochimie 97: 78-91.

Ahlatci, A., Kuzhan, A., Taysi, S., Demirtas, O., Alkis, H., Tarakcioglu, M., Demirci, A., Caglayan, D., Saricicek, E. & Cinar, K. 2014. Radiation-modifying abilities of Nigella sativa and Thymoquinone on radiation-induced nitrosative stress in the brain tissue. Phytomedicine 21(5): 740-744.

Ardiana, M., Pikir, B., Santoso, A., Hermawan, H. & Al-Farabi, M. 2020. Effect of Nigella sativa supplementation on oxidative stress and antioxidant parameters: A meta-analysis of randomized controlled trials. The Scientific World Journal 2020: 2390706.

Ates, M.B. & Ortatatli, M. 2021. The effects of Nigella sativa seeds and Thymoquinone on aflatoxin phase-2 detoxification through glutathione and glutathione-s-transferase alpha-3, and the relationship between aflatoxin B1-DNA. Toxicon 193: 86-92.

Ayuob, N., Balgoon, M., El-Mansy, A., Mubarak, W. & Firgany, A. 2020. Thymoquinone upregulates catalase gene expression and preserves the structure of the renal cortex of propylthiouracil-induced hypothyroid rats. Oxidative Medicine and Cellular Longevity 2020: 3295831.

Badary, O., Taha, R., Gamal El-Din, A. & Abdel-Wahab, M. 2003. Thymoquinone is a potent superoxide anion scavenger. Drug and Chemical Toxicology 26(2): 87-98.

Badr, G., Mohany, M. & Abu-Tarboush, F. 2011. Thymoquinone decreases F-actin polymerization and the proliferation of human multiple myeloma cells by suppressing STAT3 phosphorylation and Bcl2/Bcl-XL expression. Lipids in Health and Disease 10(1): 236.

Bellion, P., Olk, M., Will, F., Dietrich, H., Baum, M., Eisenbrand, G. & Janzowski, C. 2009. Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29. Molecular Nutrition & Food Research 53(10): 1226-1236.

Ben-Shmuel, A., Batel, S., Guy, B. & Mira, B. 2021. The role of the cytoskeleton in regulating the natural killer cell immune response in health and disease: From signaling dynamics to function. Frontiers in Cell and Developmental Biology 9: 609532.

Bestetti, S., Mauro, G., Ilaria, S., Paolo, P., Alessandro, R., Roberto, S. & Iria, M.F. 2020. Human aquaporin-11 guarantees efficient transport of H₂O₂ across the endoplasmic reticulum membrane. Redox Biology 28: 101326.

Boveris, A. & Cadenas, E. 2001. Mitochondrial production of hydrogen peroxide regulation by nitric oxide and the role of ubisemiquinone. International Union of Biochemistry and Molecular Biology Life 50(4): 245-250.

Cheeseman, K. & Slater, T. 1993. An introduction to free radical biochemistry. British Medical Bulletin 49(3): 481-493.

Chen, H., Ousheng, L., Sijia, C. & Yueying, Z. 2021. Aging and mesenchymal stem cells: Therapeutic opportunities and challenges in the older group. Gerontology 68(3): 339-352.

Ciftci, E., Turkoglu, V. & Bas, Z. 2021. Inhibition effect of thymoquinone and lycopene compounds on glutathione reductase enzyme activity purified from human erythrocytes. Journal of Biomolecular Structure and Dynamics 40(20): 10086-10093.

Demain, A. & Vaishnav, P. 2010. Natural products for cancer chemotherapy. Microbial Biotechnology 4(6): 687-699.

Dupré, L., Boztug, K. & Pfajfer, L. 2021. Actin dynamics at the T cell synapse as revealed by immune-related actinopathies. Frontiers in Cell and Developmental Biology 9: 665519.

El-Mahdy, M., Zhu, Q., Wang, Q., Wani, G. & Wani, A. 2005. Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells. International Journal of Cancer 117(3): 409-417.

Emerson, P., Tejas, D., Luke, S., Shehane, M., Megan, H., Paula, B., Shyam, P., Mikhail, A., David, G. & Liza, T. 2022. Left atrial strain in cardiac surveillance of bone marrow transplant patients with prior anthracycline exposure. International Journal of Cardiology 354: 68-74.

Eteraf-Oskouei, T. & Moslem, N. 2013. Traditional and modern uses of natural honey in human diseases: A review. Iranian Journal of Basic Medical Sciences 16(6): 731-742.

Fanoudi, S., Alavi, M., Hosseini, M. & Sadeghnia, H. 2019. Nigella sativa and thymoquinone attenuate oxidative stress and cognitive impairment following cerebral hypoperfusion in rats. Metabolic Brain Disease 34(4): 1001-1010.

Feeney, C., Frantseva, M., Carlen, P., Pennefather, P., Shulyakova, N., Shniffer, C. & Mills, L. 2008. Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices. Brain Research 1198: 1-15.

Folmer, V., Pedroso, N., Matias, A., Lopes, S., Antunes, F., Cyrne, L. & Marinho, H. 2008. H₂O₂ induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae. Biochimica et Biophysica Acta (BBA) - Biomembranes 1778(4): 1141-1147.

Galluzzi, L., Bravo-San Pedro, J., Vitale, I., Aaronson, S., Abrams, J., Adam, D., Alnemri, E., Altucci, L., Andrews, D., Annicchiarico-Petruzzelli, M., Baehrecke, E., Bazan, N., Bertrand, M., Bianchi, K., Blagosklonny, M., Blomgren, K., Borner, C., Bredesen, D., Brenner, C., Campanella, M., Candi, E., Cecconi, F., Chan, F., Chandel, N., Cheng, E., Chipuk, J., Cidlowski, J., Ciechanover, A., Dawson, T., Dawson, V., De Laurenzi, V., De Maria, R., Debatin, K., Di Daniele, N., Dixit, V., Dynlacht, B., El-Deiry, W., Fimia, G., Flavell, R., Fulda, S., Garrido, C., Gougeon, M., Green, D., Gronemeyer, H., Hajnoczky, G., Hardwick, J., Hengartner, M., Ichijo, H., Joseph, B., Jost, P., Kaufmann, T., Kepp, O., Klionsky, D., Knight, R., Kumar, S., Lemasters, J., Levine, B., Linkermann, A., Lipton, S., Lockshin, R., López-Otín, C., Lugli, E., Madeo, F., Malorni, W., Marine, J., Martin, S., Martinou, J., Medema, J., Meier, P., Melino, S., Mizushima, N., Moll, U., Muñoz-Pinedo, C., Nuñez, G., Oberst, A., Panaretakis, T., Penninger, J., Peter, M., Piacentini, M., Pinton, P., Prehn, J., Puthalakath, H., Rabinovich, G., Ravichandran, K., Rizzuto, R., Rodrigues, C., Rubinsztein, D., Rudel, T., Shi, Y., Simon, H., Stockwell, B., Szabadkai, G., Tait, S., Tang, H., Tavernarakis, N., Tsujimoto, Y., Vanden Berghe, T., Vandenabeele, P., Villunger, A., Wagner, E., Walczak, H., White, E., Wood, W., Yuan, J., Zakeri, Z., Zhivotovsky, B., Melino, G. & Kroemer, G. 2014. Essential versus accessory aspects of cell death: Recommendations of the NCCD 2015. Cell Death & Differentiation 22(1): 73.

Ganie, S., Zargar, B., Masood, A. & Zargar, M. 2012. Effect of long dose exposure of Podophyllum hexandrum methanol extract on antioxidant defense system and body and organ weight changes of albino rats. Asian Pacific Journal of Tropical Biomedicine 2(3):  S1600-S1605.

Gille, J. & Joenje, H. 1992a. Cell culture models for oxidative stress: superoxide and hydrogen peroxide versus normobaric hyperoxia. Mutation Research/DNAging 275(3-6): 405-414.

Gille, J. & Joenje, H. 1992b. Cell culture models for oxidative stress: Superoxide and hydrogen peroxide versus normobaric hyperoxia. Mutation Research/DNAging 275(3-6): 405-414.

Grishagin, I.V. 2015. Automatic cell counting with ImageJ. Analytical Biochemistry 473: 63-65.

Gülden, M., Jess, A., Kammann, J., Maser, E. & Seibert, H. 2010. Cytotoxic potency of H₂O₂ in cell cultures: Impact of cell concentration and exposure time. Free Radical Biology and Medicine 49(8): 1298-1305.

Häcker, G. 2000. The morphology of apoptosis. Cell and Tissue Research 301(1): 5-17.

Haron, A.S., Sharifah, S.S.A., Latifah, S.Y., Rohaina, A.R., Yong, S.O., Fatin, H.Z.A. & Henna, R.A. 2018. Cytotoxic effect of thymoquinone-loaded nanostructured lipid carrier (TQ-NLC) on liver cancer cell integrated with hepatitis b genome, Hep3b. Evidence-Based Complementary and Alternative Medicine 2018: 1549805.

Hu, W. & Lu, Q. 2014. Impact of oxidative stress on the cytoskeleton of pancreatic epithelial cells. Experimental and Therapeutic Medicine 8(5): 1438-1442.

Hu, X., Ma, J., Vikash, V., Li, J., Wu, D., Liu, Y., Zhang, J. & Dong, W. 2017. Thymoquinone augments cisplatin-induced apoptosis on oesophageal carcinoma through mitigating the activation of JAK2/STAT3 pathway. Digestive Diseases and Sciences 63(1): 126-134.

Ichwan, S. 2014. Apoptotic activities of thymoquinone, an active ingredient of black seed (Nigella sativa), in cervical cancer cell lines. The Chinese Journal of Physiology 57(5): 249-255.

Isaev, N.K., Chetverikov, N.S., Stelmashook, E.V., Genrikhs, E.E., Khaspekov, L.G. & Illarioshkin, S.N. 2020. Thymoquinone as a potential neuroprotector in acute and chronic forms of cerebral pathology. Biochemistry (Moscow) 85(2): 167-176.

Jehan, S., Zhong, C., Li, G., Zulqarnain Bakhtiar, S., Li, D. & Sui, G. 2020. Thymoquinone selectively induces hepatocellular carcinoma cell apoptosis in synergism with clinical therapeutics and dependence of p53 status. Frontiers in Pharmacology 11: 555283.

Kabir, N., Schaefer, A., Nakhost, A., Sossin, W. & Forscher, P. 2001. Protein Kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes. Journal of Cell Biology 152(5): 1033-1044.

Kalamegam, G., Saadiah, M.A., Afnan, O.B., Etimad, A.A., Mamdouh, A.G., Mohammed, M.A., Farid, A., Muhammed, A.E. & Peter, N.P. 2020. In vitro evaluation of the anti-inflammatory effects of thymoquinone in osteoarthritis and in silico analysis of inter-related pathways in age-related degenerative diseases. Frontiers in Cell and Developmental Biology 8: 646.

Kanner, J. 2020. Polyphenols by generating H₂O₂, affect cell redox signaling, inhibit Ptps and activate Nrf2 axis for adaptation and cell surviving: in vitro, in vivo and human health. Antioxidants 9 (9): 797.

Kanter, M., Coskun, O., Korkmaz, A. & Oter, S. 2004. Effects of Nigella sativa on oxidative stress and beta-cell damage in streptozotocin-induced diabetic rats. The Anatomical Record 279A(1): 685-691.

Kawamori, D., Kajimoto, Y., Kaneto, H., Umayahara, Y., Fujitani, Y., Miyatsuka, T., Watada, H., Leibiger, I., Yamasaki, Y. & Hori, M. 2003. Oxidative stress induces nucleo-cytoplasmic translocation of pancreatic transcription factor PDX-1 through activation of c-Jun NH2-terminal kinase. Diabetes 52(12): 2896-2904.

Khan, M.A., Chen, H.C., Tania, M. & Zhang, D.Z. 2011. Anticancer activities of Nigella sativa (black cumin). African Journal of Traditional, Complementary and Alternative Medicines 8(5 Suppl): 226-232.

Kocsis, Á., Pasztorek, M., Rossmanith, E., Djinovic, Z., Mayr, T., Spitz, S., Zirath, H., Ertl, P. & Fischer, M.B. 2021. Dependence of mitochondrial function on the filamentous actin cytoskeleton in cultured mesenchymal stem cells treated with cytochalasin B. Journal of Bioscience and Bioengineering 132(3): 310-320.

Kodavanti, P.R., Royland, J.E., Richards, J.E., Besas, J. & Macphail, R.C. 2011. Toluene effects on oxidative stress in brain regions of young-adult, middle-age, and senescent brown Norway rats. Toxicology and Applied Pharmacology 256(3): 386-398.

Korak, T., Emel, E. & Ali, S. 2020. Nigella sativa and Cancer: A review focusing on breast cancer, inhibition of metastasis and enhancement of natural killer cell cytotoxicity. Current Pharmaceutical Biotechnology 21(12): 1176-1185.

Kuang, F., Liu, J., Tang, D. & Kang, R. 2020. Oxidative damage and antioxidant defense in ferroptosis. Frontiers in Cell and Developmental Biology 8: 586578.

Leong, X.F., Mohd, R.M. & Kamsiah, J. 2013. Nigella sativa and its protective role in oxidative stress and hypertension. Evidence-Based Complementary and Alternative Medicine 2013: 253479.

Li, Y.J. & Chen, Z. 2022. Cell-based therapies for rheumatoid arthritis: Opportunities and challenges. Therapeutic Advances in Musculoskeletal Disease 14: 1759720X221100294.

Liu, T., Li, S., Yubin, Z., Yonglin, W. & Jiang, Z. 2021. Imbalanced GSH/ROS and sequential cell death. Journal of Biochemical and Molecular Toxicology 36: e22942.

Long, L.H., Clement, M.V. & Halliwell, B. 2000. Artifacts in cell culture: Rapid generation of hydrogen peroxide on addition of (−)-epigallocatechin, (−)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochemical and Biophysical Research Communications 273(1): 50-53.

Mahmoud, Y.K. & Abdelrazek, H.M.A. 2019. Cancer: Thymoquinone antioxidant/pro-oxidant effect as potential anticancer remedy. Biomedicine & Pharmacotherapy 115: 108783.

Mansour, M.A., Mahmoud, N.N., Aiman, S.E. & Abdullah, M.A. 2002. Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: A possible mechanism of action. Cell Biochemistry and Function 20(2): 143-151.

Maraldi, T., Cristina, A., Cecilia, P. & Silvana, H. 2021. NADPH oxidases: Redox regulators of stem cell fate and function. Antioxidants 10(6): 973.

Marnett, L.J. 2000. Oxyradicals and DNA damage. Carcinogenesis 21(3): 361-370.

Mattia, C.J., Ali, S.F. & Bondy, S.C. 1993. Toluene-induced oxidative stress in several brain regions and other organs. Molecular and Chemical Neuropathology 18(3): 313-328.

Mohammadi, S., Abolfazl, B., Alireza, D., Jaleh, B. & Yadollah, O. 2021. Astaxanthin protects mesenchymal stem cells from oxidative stress by direct scavenging of free radicals and modulation of cell signalling. Chemico-Biological Interactions 333: 109324.

Mondal, S., Bandyopadhyay, S., Ghosh, M.K., Mukhopadhyay, S., Roy, S. & Mandal, C. 2012. Natural products: Promising resources for cancer drug discovery. Anti-Cancer Agents in Medicinal Chemistry 12(1): 49-75.

Mu, X., Tseng, C., Hambright, W.S., Matre, P., Lin, C.Y., Chanda, P., Chen, W., Gu, J., Ravuri, S., Cui, Y., Zhong, L., Cooke, J.P., Niedernhofer, L.J., Robbins, P.D. & Huard, J. 2020. Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome. Aging Cell 19(8): e13152.

Negi, P., Sharma, I., Hemrajani, C., Rathore, C., Bisht, A., Raza, K. & Katare, O.P. 2019. Thymoquinone-loaded lipid vesicles: A promising nanomedicine for psoriasis. BMC Complementary Medicine and Therapies 19(1): 334.

Ng, W.K., Latifah, S.Y. & Maznah, I. 2011. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein. Toxicology in Vitro 25(7): 1392-1398.

Rahmani, A.H., Mohammad, A.A., Masood, A.K. & Salah, M.A. 2014. Therapeutic implications of black seed and its constituent thymoquinone in the prevention of cancer through inactivation and activation of molecular pathways. Evidence-Based Complementary and Alternative Medicine 2014: 724658.

Ray, P.D., Huang, B.W. & Tsuji, Y. 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signalling. Cell Signal 24(5): 981-990.

Robinson, M.M., Bergen, K.S., Emily, R.B., Sarah, E.E., Harrison, D.S., Maria, C.F. & Sean, A.N. 2019. Robust intrinsic differences in mitochondrial respiration and H₂O₂ emission between L6 and C2C12 cells. American Journal of Physiology-Cell Physiology 317(2): C339-C347.

Sahak, M.K.A., Kabir, N., Abbas, G., Draman, S., Hashim, N.H. & Hasan Adli, D.S. 2016. The role of Nigella sativa and its active constituents in learning and memory. Evidence-Based Complementary and Alternative Medicine 2016: 6075679.

Samarghandian, S., Mohsen, A.N. & Tahereh, F. 2019. Thymoquinone-induced antitumor and apoptosis in human lung adenocarcinoma cells. Journal of Cellular Physiology 234(7): 10421-10431.

Sankaranarayanan, C. & Pari, L. 2011. Thymoquinone ameliorates chemical induced oxidative stress and β-cell damage in experimental hyperglycemic rats. Chemico-Biological Interactions 190: 148-54.

Sassoli, C., Pierucci, F., Tani, A., Frati, A., Chellini, F., Matteini, F., Vestri, A., Anderloni, G., Nosi, D., Zecchi-Orlandini, S. & Meacci, E. 2018. Sphingosine 1-Phosphate Receptor 1 is required for MMP-2 function in bone marrow mesenchymal stromal cells: Implications for cytoskeleton assembly and proliferation. Stem Cells International 2018: 5034679.

Schaefer, A.W., Nurul, K. & Forscher, P. 2002. Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones. The Journal of Cell Biology 158(1): 139-152.

Schneider, C., Rasband, W. & Eliceiri, K. 2012. NIH image to ImageJ: 25 years of image analysis. Nature Methods 9(7): 671-675.

Shanmugam, M., Ahn, K., Hsu, A., Woo, C., Yuan, Y., Tan, K., Chinnathambi, A., Alahmadi, T., Alharbi, S., Koh, A., Arfuso, F., Huang, R., Lim, L., Sethi, G. & Kumar, A. 2018. Thymoquinone inhibits bone metastasis of breast cancer cells through abrogation of the CXCR4 signaling axis. Frontiers in Pharmacology 9: 1294.

Sharifi-Rad, M., Nanjangud, V.A.K., Paolo, Z., Elena, M.V., Luciana, D., Elisa, P., Jovana, R., Tsouh Fokou, P.V., Azzini, E., Peluso, I., Prakash, M.A., Nigam, M., El Rayess, Y., Beyrouthy, M.E., Polito, L., Iriti, M., Martins, N., Martorell, M., Docea, A.O., Setzer, W. N., Calina, D., Cho, W.C. & Sharifi-Rad, J. 2020. Lifestyle, oxidative stress, and antioxidants: Back and forth in the pathophysiology of chronic diseases. Frontiers in Physiology 11: 694.

Sies, H. 2019. Chapter 13 - Oxidative stress: Eustress and distress in redox homeostasis. In Stress: Physiology, Biochemistry, and Pathology, edited by Fink, G. Massachusetts: Academic Press. pp. 153-163.

Smajilagić, A., Mufida, A., Amira, R., Selma, F. & Alena, L. 2013. Rat bone marrow stem cells isolation and culture as a bone formative experimental system. Bosnian Journal of Basic Medical Sciences 13(1): 27-30.

Stadtman, E.R. & Levine, R.L. 2000. Protein oxidation. Annals of the New York Academy of Sciences 899: 191-208.

Szanto, I., Marc, P. & Maria, M. 2019. H₂O₂ metabolism in normal thyroid cells and in thyroid tumorigenesis: Focus on NADPH oxidases. Antioxidants 8(5): 126.

Uttara, B., Singh, A.V., Zamboni, P. & Mahajan, R.T. 2009. Oxidative stress and neurodegenerative diseases: A review of upstream and downstream antioxidant therapeutic options. Current Neuropharmacology 7(1): 65-74.

Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunological Methods 184(1): 39-51.

Vijayamalini, M. & Manoharan, S. 2004. Lipid peroxidation, vitamins C, E and reduced glutathione levels in patients with pulmonary tuberculosis. Cell Biochemistry and Function 22(1): 19-22.

Wei, H., Zongwei, L., Shengshou, H., Xi, C. & Xiangfeng, C. 2010. Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases, p38 and JNK. Journal of Cellular Biochemistry 111(4): 967-978.

Zakaria, A.F., Latifah, S., Wan, K., Khong, K.C., Ng, Y., Foong, J.N., Gopalsamy, B., Ng, W.K., How, C.W., Ong, Y.S., Abdullah, R. & Aziz, M.Y. 2020. Pharmacokinetics and biodistribution of thymoquinone-loaded nanostructured lipid carrier after oral and intravenous administration into rats. International Journal of Nanomedicine 15: 7703-7717.

Zubair, H., Khan, H.Y., Sohail, A., Azim, S., Ullah, M.F., Ahmad, A., Sarkar, F.H. & Hadi, S.M. 2013. Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death: Putative anticancer mechanism of antioxidants. Cell Death and Disease 4(6): e660.

^*Corresponding author; email: nurul.kabir@um.edu.my