Relative hypoxia and oxidative stress in spleen lymphocytes of immunized Balb/c mice as indicated by HIF-1α, HIF-2α, Nrf2 expression, and glutathione peroxidase activity
Background: Lymphocytes activated by immunization must increase their metabolism to meet the energy requirements for mitosis, differentiation, and protein synthesis, which may subject the cell to conditions of relative hypoxia and oxidative stress. This study was conducted to investigate the increase in the levels of transcription factors involved in both conditions.
Methods: Male Balb/c mice were divided into the following four groups, each consisting of six animals: the control and three experimental groups. The experimental groups were immunized by injection of 0.2 ml of 2% sheep red blood cells (SRBC) suspended in phosphate-buffered saline (PBS). Lymphocytes were harvested from the spleens of each group at time intervals of 24-, 48-, and 72-h post-immunization. The buffy coat from splenocytes was separated using Ficoll Histopaque as the medium. The lymphocytes were separated from adherent cells by incubating the purified splenocytes in microtubes for 2-h. Cells were lysed by three freeze–thaw cycles (−80°C and 37°C) and used to analyze the levels of HIF-1α and HIF-2α (mRNA and protein), Nrf2 (protein), and glutathione peroxidase (GPx) activity.
Results: The treatment caused an increase in GPx activity and HIF-1α protein concentration 24-h post-immunization, whereas the HIF-1α mRNA levels remained static. Elevated Nrf2 protein levels were detected within 48-h after treatment. Meanwhile, the HIF-2α mRNA and protein levels increased within72-h after immunization.
Conclusion: Immunization with SRBC suspension induced relative hypoxia, elevated reactive oxygen species (ROS), and oxidative stress in the lymphocytes as indicated by the increase in both HIF-1α and HIF-2α protein and mRNA levels, GPx activity, and Nrf2 protein levels.
Gilkes DM, Semenza GL. Role of hypoxia-inducible factors in breast cancer metastasis. Futur Oncol. 2013;9(11):1623-36. https://doi.org/10.2217/fon.13.92
Masoud GN, Li W. HIF-1 α pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B. 2015;5(5):378-89. https://doi.org/10.1016/j.apsb.2015.05.007
Liu Z, Semenza GL, Zhang H. Hypoxia-inducible factor 1 and breast cancer metastasis. J Zhejiang Univ Sci B. 2015;16(1):32-43. https://doi.org/10.1631/jzus.B1400221
Gilkes DM, Bajpai S, Chaturvedi P, Wirtz D, Semenza GL. Hypoxia-inducible factor 1 (HIF-1) promotes extracellular matrix remodeling under hypoxic conditions by inducing P4HA1, P4HA2, and PLOD2 expression in fibroblasts. J Biol Chem. 2013;288(15):10819-29. https://doi.org/10.1074/jbc.M112.442939
Stavik B, Espada S, Cui XY, Iversen N, Holm S, Mowinkel M, et al. EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells. Biochim Biophys Acta. 2016;1862(4):670-8. https://doi.org/10.1016/j.bbadis.2016.01.017
Jusman SW, Halim A. Oxidative stress in liver tissue of rat induced by chronic systemic hypoxia. Makara Kesehatan. 2009;13(1):34-8. https://doi.org/10.7454/msk.v13i1.346
Kim SG, Lee WH, Kim YW. Nrf2. Encyclopedia of signaling molecules. In: Choi S, editor. Springer Science+Business Media; 2012. p. 80-5.
Fuse Y, Kobayashi M. Conservation of the Keap1-Nrf2 system: an evolutionary journey through stressful space and time. Molecules. 2017;22(3):436. https://doi.org/10.3390/molecules22030436
Jaramillo MC, Zhang DD. The emerging role of the Nrf2-Keap1 signaling pathway in cancer. Gene Dev. 2013;27(20):2179-91. https://doi.org/10.1101/gad.225680.113
Walker JM. The Protein Protocols Handbook. 3rd ed. New York: Springer-Verlag New York, LCC; 2009. https://doi.org/10.1007/978-1-59745-198-7
Rodwell VW, Bender DA, Botham KM, Kennelly PJ, Weil PA. Harper's Illustrated Biochemistry. 30th ed. New York: McGraw Hill; 2015.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods. 2001;408:402-8. https://doi.org/10.1006/meth.2001.1262
Ransel Glutathione Peroxidase Manual [Internet]. UK: Randox Laboratories, Ltd. 2005 [cited Oct 2017 ]. p. 1-2. Available from: https://www.sanyo-si.com/wp-content/uploads/e28aa06ef42fe6393a72a6f9052e365d.pdf
Florczyk U, Czauderna S, Stachurska A, Tertil M, Nowak W, Kozakowska M, et al. Opposite effects of HIF-1 α and HIF-2 α on the regulation of IL-8 expression in endothelial cells. Free Radic Biol Med. 2011;51(10):1882-92. https://doi.org/10.1016/j.freeradbiomed.2011.08.023
Deudero JJ, Caramelo C, Castellanos MC, Neria F, Fernández-Sánchez R, Calabia O, et al. Induction of hypoxia-inducible factor 1α gene expression by vascular endothelial growth factor*. J Biol Chem. 2008;283(17):11435-44. https://doi.org/10.1074/jbc.M703875200
Ratcliffe PJ. HIF-1 and HIF-2 : working alone or together in hypoxia ? J Clin Invest. 2007;117(4):399-402. https://doi.org/10.1172/JCI31750
Mustofa S. Penghambatan aktivitas anhidrase karbonat menurunkan proliferasi sel mononukleus darah tepi (SMDT) Manusia yang distimulasi dengan phytohaemagglutinin dan interleukin-2. Universitas Indonesia; 2016. Indonesian.
Firakania C. Penghambatan proliferasi sel mononukleus darah tepi (SMDT) manusia oleh avidin, pascastimulasi fitohemaglutinin (PHA) dan Interleukin-2 (IL-2). Universitas Indonesia; 2015. Indonesian.
Gordan JD, Bertovrt JA, Hu C, Diehl JA, Celeste MS. HIF-2 α promotes hypoxic cell proliferation by enhancing c-Myc transcriptional activity. Cancer Cell. 2007;11(4):335-47. https://doi.org/10.1016/j.ccr.2007.02.006
Asikin H. Respon makrofag limpa mencit pasca imunisasi: kajian terhadap ekspresi hypoxia-inducible factor-1a, hif-2a, sitoglobin dan peroxisome proliferator activated receptor gamma coactivator-1a. Universitas Indonesia; 2016. Indonesian.
Sarsanti PAN. Hipoksia pada makrofag peritoneum mencit Balb/c yang diimunisasi: kajian terhadap HIF-1 α , HIF-2 α , Sitoglobin, dan PGC-1 α. Universitas Indonesia; 2017. Indonesian.
Sadikin AH. Stres oksidatif pada hati tikus yang diinduksi hipoksia sistemik. Universitas Indonesia; 2008. Indonesian.
Buendia I, Michalska P, Navarro E, Gameiro I, Egea J, León R. Nrf2-ARE pathway: an emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacol Therapeut. 2016;157:84-104. https://doi.org/10.1016/j.pharmthera.2015.11.003
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