Skip to main content
Log in

Thymoquinone Induces Mitochondrial Damage and Death of Cerebellar Granule Neurons

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Thymoquinone (TQ) exhibits a wide spectrum of biological activities. Most studies on the neurotoxic action of TQ have been carried out in cancer cell lines. Here, we studied the toxic effect of TQ in primary neuronal cultures in vitro. Incubation with 0.04-0.05 mM TQ for 24 h induced the death of cultured cerebellar granule neurons (CGNs) in a dose dependent manner. Neuronal death was preceded by an increase in the reactive oxygen species (ROS) generation, as demonstrated using CellROX Green and MitoSOX Red. Confocal and electron microscopy showed that incubation with 0.05 mM TQ for 5 h induced changes in the intracellular localization of mitochondria and mitochondria hypertrophy and cell swelling. The antioxidant N-acetyl-L-cysteine (2 mM) protected CGNs from the toxic action of TQ. Taken together, these facts suggest that TQ is toxic for normal neurons, while ROS-induced changes in the mitochondria can be one of the major causes of the TQ-induced neuronal damage and death.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

CGN:

cerebellar granule neuron; NAC

N:

acetyl-L-cysteine

ROS:

reactive oxygen species

TQ:

thymo-quinone.

Referneces

  1. Burits, M., and Bucar, F. (2000) Antioxidant activity of Nigella sativa essential oil, Phytother. Res., 14, 323–328.

    Article  CAS  Google Scholar 

  2. Hajhashemi, V., Ghannadi, A., and Jafarabadi, H. (2004) Black cumin seed essential oil, as a potent analgesic and anti-inflammatory drug, Phytother. Res., 18, 195–199; doi:10.1002/ptr.1390.

    Article  CAS  Google Scholar 

  3. Ahmed, A. M., Al-Olayan, E. M., Aboul-Soud, M. A., and Al-Khedhairy, A. A. (2010) The immune enhancer, thymo quinone, and the hope of utilizing the immune system of Aedes caspius against disease agents, Afr. J. Biotechnol., 9 3183–3195.

    Article  CAS  Google Scholar 

  4. Khader, M., Bresgen, N., and Eckl, P. M. (2010) Antimutagenic effects of ethanolic extracts from selected Palestinian medicinal plants, J. Ethnopharmacol., 127, 319–324; doi: 10.1016/j.jep.2009.11.001.

    Article  CAS  Google Scholar 

  5. Al-Majed, A. A., Al-Omar, F. A., and Nagi, M. N. (2006) Neuroprotective effects of thymoquinone against transient forebrain ischemia in the rat hippocampus, Eur. J. Pharmacol., 543, 40–47; doi: 10.1016/j.ejphar.2006.05.046.

    Article  CAS  Google Scholar 

  6. Hosseinzadeh, H., Parvardeh, S., Asl, M. N., Sadeghnia H. R., and Ziaee, T. (2007) Effect of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus, Phytomedicine, 14, 621–627; doi: 10.1016/j.phymed.2006.12.005.

    Article  CAS  Google Scholar 

  7. Alhebshi, A. H., Gotoh, M., and Suzuki, I. (2013) Thymoquinone protects cultured rat primary neurons against amyloid ß-induced neurotoxicity, Biochem. Biophys. Res. Commun., 433, 362–367; doi: 10.1016/j.bbrc.2012.11.139.

    Article  CAS  Google Scholar 

  8. Khan, A., Vaibhav, K., Javed, H., Khan, M. M., Tabassum, R., Ahmed, M. E., Srivastava, P., Khuwaja, G., Islam, F., Siddiqui, M. S., Safhi, M. M., and Islam, F. (2012) Attenuation of Aß-induced neurotoxicity by thymoquinone via inhibition of mitochondrial dysfunction and oxidative stress, Mol. Cell Biochem., 369, 55–65; doi: 10.1007/ s11010-012-1368-x.

    Article  CAS  Google Scholar 

  9. Firdaus, F., Zafeer, M. F., Anis, E., Ahmad, F., Hossain, M. M., Ali, A., and Afzal, M. (2019) Evaluation of phytomedicinal efficacy of thymoquinone against arsenic-induced mitochondrial dysfunction and cytotoxicity in SHSY5Y cells, Phytomedicine, 54, 224–230; doi: 10.1016/ j.phymed.2018.09.197.

    Article  CAS  Google Scholar 

  10. Firdaus, F., Zafeer, M. F., Waseem, M., Ullah, R., Ahmad, M., and Afzal, M. (2018) Thymoquinone alleviates arsenic induced hippocampal toxicity and mitochondrial dysfunction by modulating mPTP in Wistar rats, Biomed. Pharmacother., 102, 1152–1160; doi: 10.1016/j.biopha.2018.03.159.

    Article  CAS  Google Scholar 

  11. Radad, K. S., Al-Shraim, M. M., Moustafa, M. F., and Rausch, W. D. (2015) Neuroprotective role of thymoquinone against 1-methyl-4-phenylpyridinium-induced dopaminergic cell death in primary mesencephalic cell culture, Neurosciences (Riyadh), 20, 10–16.

    Google Scholar 

  12. Genrikhs, E. E., Stelmashook, E. V., Popova, O. V., Kapay N. A., Korshunova, G. A., Sumbatyan, N. V., Skrebitsky, V. G., Skulachev, V. P., and Isaev, N. K. (2015) Mitochondria-targeted antioxidant SkQT1 decreases trauma-induced neurological deficit in rat and prevents amyloid-ß-induced impairment of long-term potentiation in rat hippocampal slices, J. Drug Target., 23, 347–352; doi: 10.3109/1061186X.2014.997736.

    Article  CAS  Google Scholar 

  13. Isaev, N. K., Stelmashook, E. V., Genrikhs, E. E., Korshunova, G. A., Sumbatyan, N. V., Kapkaeva, M. R. and Skulachev, V. P. (2016) Neuroprotective properties of mitochondria-targeted antioxidants of the SkQ-type, Rev. Neurosci., 27, 849–855; doi: 10.1515/revneuro-2016-0036.

    Article  CAS  Google Scholar 

  14. El-Najjar, N., Chatila, M., Moukadem, H., Vuorela, H., Ocker, M., Gandesiri, M., Schneider-Stock, R., and GaliMuhtasib, H. (2010) Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling, Apoptosis, 15, 183–195; doi: 10.1007/s10495-009-0421-z.

    Article  CAS  Google Scholar 

  15. Park, E. J., Chauhan, A. K., Min, K. J., Park, D. C., and Kwon, T. K. (2016) Thymoquinone induces apoptosis through downregulation of c-FLIP and Bcl-2 in renal carcinoma Caki cells, Oncol. Rep., 36, 2261–2267; doi:10.3892/or.2016.5019.

    Article  CAS  Google Scholar 

  16. Assaf, M. D., Semaan, J., El-Sabban, M., Al-Jaouni, S. K., Azar, R., Kamal, M. A., and Harakeh, S. (2018) Inhibition of proliferation and induction of apoptosis by thymoquinone via modulation of TGF family, p53, p21 and Bcl-2a in leukemic cells, Anticancer Agents Med. Chem., 18 210–215; doi: 10.2174/1871520617666170912133054.

    Article  Google Scholar 

  17. Gurung, R. L., Lim, S. N., Khaw, A. K., Soon, J. F., Shenoy, K., Mohamed Ali, S., Jayapal, M., Sethu, S., Baskar, R., and Hande, M. P. (2010) Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells, PLoS One, 5, e12124; doi: 10.1371/journal.pone.0012124.

    Article  Google Scholar 

  18. Paramasivam, A., Sambantham, S., Shabnam, J., Raghunandhakumar, S., Anandan, B., Rajiv, R., Vijayashree Priyadharsini, J., and Jayaraman, G. (2012) Anti-cancer effects of thymoquinone in mouse neuroblastoma (Neuro-2a) cells through caspase-3 activation with down-regulation of XIAP, Toxicol. Lett., 213, 151–149; doi: 10.1016/j.toxlet.2012.06.011.

    Article  CAS  Google Scholar 

  19. Paramasivam, A., Raghunandhakumar, S., Priyadharsini, J. V., and Jayaraman, G. (2015) In vitro anti-neuroblastoma activity of thymoquinone against Neuro-2a cells via cell-cycle arrest, Asian Pac. J. Cancer Prev., 16, 8313–8319; doi: 10.7314/apjcp.2015.16.18.8313.

    Article  Google Scholar 

  20. Elmaci, I., and Altinoz, M. A. (2016) Thymoquinone: an edible redox-active quinone for the pharmacotherapy of neurodegenerative conditions and glial brain tumors. A short review, Biomed. Pharmacother., 83, 635–640; doi:10.1016/j.biopha.2016.07.018.

    Article  CAS  Google Scholar 

  21. Mansour, M. A., Nagi, M. N., El-Khatib, A. S., and Al-Bekairi, A. M. (2002) Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action, Cell Biochem. Funct., 20, 143–151; doi:10.1002/cbf.968.

    Article  CAS  Google Scholar 

  22. Isaev, N. K., Genrikhs, E. E., Aleksandrova, O. P., Zelenova, E. A., and Stelmashook, E. V. (2016) Glucose deprivation stimulates Cu2+ toxicity in cultured cerebellar granule neurons and Cu2+-dependent zinc release, Toxicol. Lett., 250-251, 29–34; doi: 10.1016/j.toxlet.2016.04.002.

    Article  CAS  Google Scholar 

  23. Galli, C., Meucci, O., Scorziello, A., Werge, T. M., Calissano, P., and Schettini, G. (1995) Apoptosis in cere-bellar granule cells is blocked by high KCl, forskolin, and IGF-1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis, J. Neurosci., 15, 1172–1179.

    Article  CAS  Google Scholar 

  24. Stelmashook, E. V., Genrikhs, E. E., Mukhaleva, E. V., Kapkaeva, M. R., Kondratenko, R. V., Skrebitsky, V. G. and Isaev, N. K. (2019) Neuroprotective effects of methylene blue in vivo and in vitro, Bull. Exp. Biol. Med., 167, 455–445; doi: 10.1007/s10517-019-04548-3.

    Article  CAS  Google Scholar 

  25. Isaev, N. K., Avilkina, A., Golyshev, S. A., Genrikhs, E. E., Alexandrova, O. P., Kapkaeva, M. R., and Stelmashook, E. V. (2018) N-acetyl-L-cysteine and Mn2+ attenuate Cd2+-induced disturbance of the intracellular free calcium homeostasis in cultured cerebellar granule neurons, Toxicology, 393, 1–8; doi: 10.1016/j.tox.2017.10.017.

    Article  CAS  Google Scholar 

  26. Ebrahimi, S. S., Oryan, S., Izadpanah, E., and Hassanzadeh, K. (2017) Thymoquinone exerts neuroprotective effect in animal model of Parkinson’s disease, Toxicol. Lett., 276, 108–114; doi: 10.1016/j.toxlet.2017.05.018.

    Article  CAS  Google Scholar 

  27. Majdalawieh, A. F., Fayyad, M. W., and Nasrallah, G. K. (2017) Anti-cancer properties and mechanisms of action of thymoquinone, the major active ingredient of Nigella sativa, Crit. Rev. Food Sci. Nutr., 57, 3911–3928; doi:10.1080/10408398.2016.1277971.

    Article  CAS  Google Scholar 

  28. Ullah, I., Ullah, N., Naseer, M. I., Lee, H. Y., and Kim M. O. (2012) Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons, BMC Neurosci., 13 11; doi: 10.1186/1471-2202-13-11.

    Article  CAS  Google Scholar 

  29. Kanter, M. (2011) Protective effects of thymoquinone on the neuronal injury in frontal cortex after chronic toluene exposure, J. Mol. Histol., 42, 39–46; doi: 10.1007/s10735-010-9305-3.

    Article  CAS  Google Scholar 

  30. Kanter, M. (2008) Nigella sativa and derived thymoquinone prevents hippocampal neurodegeneration after chronic toluene exposure in rats, Neurochem. Res., 33, 579–588.

    Article  CAS  Google Scholar 

  31. Farkhondeh, T., Samarghandian, S., Hozeifi, S., and Azimi-Nezhad, M. (2017) Therapeutic effects of thymoquinone for the treatment of central nervous system tumors: a review, Biomed. Pharmacother., 96, 1440–1444; doi: 10.1016/j.biopha.2017.12.013.

    Article  CAS  Google Scholar 

  32. Ashour, A. E., Ahmed, A. F., Kumar, A., Zoheir, K. M., Aboul-Soud, M. A., Ahmad, S. F., Attia, S. M., Abd-Allah A. R., Cheryan, V. T., and Rishi, A. K. (2016) Thymoquinone inhibits growth of human medulloblastoma cells by inducing oxidative stress and caspase-dependent apoptosis while suppressing NF-κB signaling and IL-8 expression, Mol. Cell Biochem., 416, 141–155; doi: 10.1007/s11010-016-2703-4.

    Article  CAS  Google Scholar 

  33. Kolli-Bouhafs, K., Boukhari, A., Abusnina, A., Velot, E., Gies, J. P., Lugnier, C., and Ronde, P. (2012) Thymoquinone reduces migration and invasion of human glioblastoma cells associated with FAK, MMP-2 and MMP-9 down-regulation, Invest. New Drugs, 30, 2121–2131; doi: 10.1007/s10637-011-9777-3.

    Article  CAS  Google Scholar 

  34. Thangnipon, W., Kingsbury, A., Webb, M., and Balazs, R. (1983) Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurons and astrocytes, Brain Res., 313, 177–189; doi: 10.1016/0165-3806(83)90215-8.

    Article  CAS  Google Scholar 

  35. Costa, L. G., Tagliaferri, S., Roque, P. J., and Pellacani, C. (2016) Role of glutamate receptors in tetrabrominated diphenyl ether (BDE-47) neurotoxicity in mouse cerebellar granule neurons, Toxicol. Lett., 241, 159–166; doi:10.1016/j.toxlet.2015.11.026.

    Article  CAS  Google Scholar 

  36. Robinson, K. M., Janes, M. S., Pehar, M., Monette, J. S., Ross, M. F., Hagen, T. M., Murphy, M. P., and Beckman, J. S. (2006) Selective fluorescent imaging of superoxide in vivo using ethidium-based probes, Proc. Natl. Acad. Sci. USA, 103, 15038–15043; doi: 10.1073/pnas.0601945103.

    Article  CAS  Google Scholar 

  37. Johnson-Cadwell, L. I., Jekabsons, M. B., Wang, A., Polster, B. M., and Nicholls, D. G. (2007) “Mild uncoupling” does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress, J. Neurochem., 101, 1619–1631.

    Article  CAS  Google Scholar 

  38. Zhang, M., Du, H., Huang, Z., Zhang, P., Yue, Y., Wang, W., Liu, W., Zeng, J., Ma, J., Chen, G., Wang, X., and Fan, J. (2018) Thymoquinone induces apoptosis in bladder cancer cell via endoplasmic reticulum stress-dependent mitochondrial pathway, Chem. Biol. Interact., 292, 65–75; doi:10.1016/j.cbi.2018.06.013.

    Article  CAS  Google Scholar 

  39. Gokce, E. C., Kahveci, R., Gokce, A., Cemil, B., Aksoy, N., Sargon, M. F., Kisa, U., Erdogan, B., Guvenc, Y., Alagoz, F., and Kahveci, O. (2016) Neuroprotective effects of thymoquinone against spinal cord ischemia-reperfusion injury by attenuation of inflammation, oxidative stress, and apoptosis, J. Neurosurg. Spine, 24, 49–59; doi: 10.3171/2015.10.SPINE15612.

    Article  Google Scholar 

  40. Stelmashook, E. V., Genrikhs, E. E., Kapkaeva, M. R., Zelenova, E. A., and Isaev, N. K. (2017) N-Acetyl-L-cysteine in the presence of Cu2+ induces oxidative stress and death of granule neurons in dissociated cultures of rat cerebellum, Biochemistry (Moscow), 82, 1176–1182; doi:10.1134/S0006297917100108.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to N. K. Isaev.

Ethics declarations

Compliance to ethical norms. All experimental protocols were approved by the Animal Ethics Committee of the Research Center of Neurology (Protocol registration no. 25/16) and were in accordance with the Council Directive 2010/63EU of the European Parliament and the Council of September 22, 2010 on the protection of animals used for scientific purposes.

Additional information

Conflict of interest. The authors declare no conflict of interest in financial or any other area.

Published in Russian in Biokhimiya, 2020, Vol. 85, No. 2, pp. 239–247.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM19-215, December 23, 2019.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stelmashook, E.V., Chetverikov, N.S., Golyshev, S.A. et al. Thymoquinone Induces Mitochondrial Damage and Death of Cerebellar Granule Neurons. Biochemistry Moscow 85, 205–212 (2020). https://doi.org/10.1134/S0006297920020078

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297920020078

Keywords

Navigation