UPREGULATION OF NEUROGLOBIN PROMOTES TM3 LEYDIG CELL VIABILITY

Globin ailesi proteinlerinden bir tanesi olan Neuroglobin (NGB) sinir sisteminde ifade edilmektedir ve oksidatif stres ve apoptoz kaynaklı nöron ölümünü engellemektedir. Oksidatif stres ve reaktif oksijen türlerine karşı gözlemlenen anti-oksidan aktivite, NGB’nin diğer dokulardaki potansiyel koruyucu rolünü gündeme getirmektedir. Testosteron üreten Leydig hücrelerinde NGB’nin fazla ifade edilmesi hipoksi ile ilişkili erkek infertilitesi için bir çözüm olabilecektir. Bu çalışmada, NGB proteinin viral aktarım metodu ile Leydig hücrelerinde aşırı ifade ettirilmiş ve hücre çoğalması, gen anlatımı ve anti-oksidan enzim seviyeleri analiz edilmiştir. Artan NGB, anti-apoptotik Bcl-2, hücre çoğalması ve anti-oksidan enzim seviyelerini arttırmış, BAX, p53 ve kaspaz 3 gibi apoptoz genlerinin ifadesini azaltmıştır. Ek olarak, H2O2 uygulaması sonrasında NGB aktarılan hücreler çoğalmış ve apoptotik genlerin ifadesi azalmıştır. Sonuç olarak, NGB androjen eksikliğine bağlı erkek infertilitesi için hedef olabilir ve gelecekte klinikte kullanılabilir.

Anahtar Kelimeler:

Neuroglobin, Leydig cell, oxidative stress, anti-oxidant enzymes, male fertility

UPREGULATION OF NEUROGLOBIN PROMOTES TM3 LEYDIG CELL VIABILITY

Neuroglobin (NGB) is a globin family member protein expressed in the nerve system and prevents oxidative stress and apoptosis induced death in neurons. The anti-oxidant role of NGB against oxidative stress and reactive oxygen species (ROS) brings up its promising protective role in other tissues. The overexpression of NGB in testosterone producing Leydig cells might be a solution to hypoxia related male infertility. In the current study, NGB was overexpressed in Leydig cells by using viral transduction methods and the cell proliferation, gene expression and anti-oxidant enzyme levels were analyzed. Upregulation of NGB increased anti-apoptotic Bcl-2, cell proliferation and anti-oxidant enzyme levels and decreased the expression of apoptotic genes BAX, p53 and caspase 3. In addition, NGB transduced cells proliferated and expressed less apoptotic genes after H2O2 exposure. In conclusion, NGB might be a target for androgen deficiency related male infertility and could be used in clinics in the future.

Keywords:

Neuroglobin, Leydig cell, oxidative stress, anti-oxidant enzymes, male fertility,

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1. Aitken, R.J. & Roman, S.D. (2008). Antioxidant systems and oxidative stress in the testes. Oxidative Medicine and Cellular Longevity, 1: 15-24.
2. Amri, F., Ghouili, I., Amri, M., Carrier, A. & Masmoudi‐Kouki, O. (2017). Neuroglobin protects astroglial cells from hydrogen peroxide‐induced oxidative stress and apoptotic cell death. Journal of Neurochemistry, 140: 151-169.
3. Apdik, H., Doğan, A., Demirci, S., Aydın, S. & Şahin F (2015). Dose-dependent effect of boric acid on myogenic differentiation of human adipose-derived stem cells (hADSCs). Biological Trace Element Research, 165: 123-130.
4. Asadi, N., Bahmani, M., Kheradmand, A. & Rafieian-Kopaei, M. (2017). The impact of oxidative stress on testicular function and the role of antioxidants in improving it: A review. Journal of Clinical and Diagnostic Research: JCDR, 11: IE01.
5. Baek, I-J., Seo, D-S., Yon, J-M., Lee, S-R., Jin, Y., Nahm, SS., Jeong, J-H., Choo, Y-K., Kang, J-K. & Lee, BJ. (2007). Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice. Journal of Molecular Histology, 38: 237-244.
6. Bisht, S., Faiq, M., Tolahunase, M. & Dada, R. (2017). Oxidative stress and male infertility. Nature Reviews Urology, 14: 470.
7. Brittain, T., Skommer, J., Henty, K., Birch, N. & Raychaudhuri, S. (2010). A role for human neuroglobin in apoptosis. IUBMB life, 62: 878-885.
8. Burmester, T. & Hankeln, T. (2009). What is the function of neuroglobin? Journal of Experimental Biology, 212: 1423-1428.
9. Burmester, T., Weich, B., Reinhardt, S. & Hankeln, T. (2000). A vertebrate globin expressed in the brain. Nature, 407: 520.
10. De Marinis, E., Fiocchetti, M., Acconcia, F., Ascenzi, P. & Marino, M. (2013). Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells. Cell Death & Disease, 4: e508.
11. Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V., Aerts, T., Marden, M.C., Caubergs, R. & Moens, L. (2001). Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family. Journal of Biological Chemistry, 276: 38949-38955.
12. Doğan, A., Demirci, S., Çağlayan, A.B., Kılıç, E., Günal, M.Y., Uslu, Ü., Cumbul, A. & Şahin, F. (2014). Sodium pentaborate pentahydrate and pluronic containing hydrogel increases cutaneous wound healing in vitro and in vivo. Biological Trace Element Research, 162: 72-79.
13. Dohle, G., Smit, M. & Weber, R. (2003). Androgens and male fertility. World Journal of Urology, 21: 341-345.
14. Fiocchetti, M., Cipolletti, M. & Marino, M. (2017). Compensatory role of Neuroglobin in nervous and non-nervous cancer cells in response to the nutrient deprivation. PloS One, 12: e0189179.
15. Fiocchetti, M., De Marinis, E., Ascenzi, P. & Marino, M. (2013). Neuroglobin and neuronal cell survival. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1834: 1744-1749.
16. Greenberg, D.A., Jin, K. & Khan, A.A. (2008). Neuroglobin: an endogenous neuroprotectant. Current Opinion in Pharmacology, 8: 20-24.
17. Haines, B., Demaria, M., Mao, X., Xie, L., Campisi, J., Jin, K. & Greenberg, D.A. (2012). Hypoxia-inducible factor-1 and neuroglobin expression. Neuroscience Letters, 514: 137-140.
18. Hundahl, C., Fago, A., Dewilde, S., Moens, L., Hankeln, T., Burmester, T. & Weber, R.E. (2006). Oxygen binding properties of non‐mammalian nerve globins. The FEBS Journal, 273: 1323-1329.
19. Husain, K. & Somani, S. (1998). Interaction of exercise training and chronic ethanol ingestion on testicular antioxidant system in rat. Journal of Applied Toxicology, 18: 421-429.
20. Jin, K., Mao, Y., Mao, X., Xie, L. & Greenberg, D.A. (2010). Neuroglobin expression in ischemic stroke. Stroke, 41: 557-559.
21. Kaur, P., Kaur, G. & Bansal, M.P. (2006). Tertiary-butyl hydroperoxide induced oxidative stress and male reproductive activity in mice: role of transcription factor NF-κB and testicular antioxidant enzymes. Reproductive Toxicology, 22: 479-484.
22. Khan, A.A., Wang, Y., Sun, Y., Mao, X.O., Xie, L., Miles, E., Graboski, J., Chen, S., Ellerby, L.M. & Jin, K. (2006). Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia. Proceedings of the National Academy of Sciences, 103: 17944-17948.
23. Lan, W-B., Lin, J-H., Chen, X-W., Wu, C-Y., Zhong, G-X., Zhang, L-Q., Lin, W-P., Liu, W-N., Li, X. & Lin, J-L. (2014). Overexpressing neuroglobin improves functional recovery by inhibiting neuronal apoptosis after spinal cord injury. Brain Research, 1562: 100-108.
24. Li, R.C., Guo, S.Z., Lee, S.K. & Gozal, D. (2010). Neuroglobin protects neurons against oxidative stress in global ischemia. Journal of Cerebral Blood Flow & Metabolism, 30: 1874-1882.
25. Liu, X., Gao, Y., An, Y., Fu, X., Li, Y., Sun, D. & Wang, J. (2015). Neuroglobin plays a protective role in arsenite-induced cytotoxicity by inhibition of CDC42 and rac1gtpases in rat cerebellar granule neurons. Cellular Physiology and Biochemistry, 36: 1613-1627.
26. Petersen, M.G., Dewilde, S. & Fago, A. (2008). Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions. Journal of Inorganic Biochemistry, 102: 1777-1782.
27. Reuss, S., Saaler-Reinhardt, S., Weich, B., Wystub, S., Reuss, M., Burmester, T. & Hankeln, T. (2002). Expression analysis of neuroglobin mRNA in rodent tissues. Neuroscience, 115: 645-656.
28. Schmidt-Kastner, R., Haberkamp, M., Schmitz, C., Hankeln, T. & Burmester, T. (2006). Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture. Brain Research, 1103: 173-180.
29. Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U. & Burmester, T. (2003). How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina. Journal of Biological Chemistry, 278: 1932-1935.
30. Sun, Y., Jin, K., Peel, A., Mao, X.O., Xie, L. & Greenberg, D.A. (2003). Neuroglobin protects the brain from experimental stroke in vivo. Proceedings of the National Academy of Sciences, 100: 3497-3500.
31. Ye, S-q., Zhou, X-y., Lai, X-j., Zheng, L. & Chen, X-q. (2009). Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3γ. Acta Pharmacologica Sinica, 30: 913.
32. Zhang, B., Liu, Y., Li, Y., Zhe, X., Zhang, S. & Zhang, L. (2018). Neuroglobin promotes the proliferation and suppresses the apoptosis of glioma cells by activating the PI3K/AKT pathway. Molecular Medicine Reports, 17: 2757-2763.
33. Zhang, C., Wang, C., Deng, M., Li, L., Wang, H., Fan, M., Xu, W., Meng, F., Qian, L. & He, F. (2002). Full-length cDNA cloning of human neuroglobin and tissue expression of rat neuroglobin. Biochemical and Biophysical Research Communications, 290: 1411-1419.