The Effects of ATP sensitive potassium channel (KATP) opener and blockers on Bcl-2, Bax, and Cyt-c gene expression levels in epileptic rats

Abstract

In this study, it was aimed to investigate the gene expression levels of Bcl-2, Bax, and cytochrome c (Cyt-c), in the cortex region of pinacidil as a KATP channel opener and glibenclamide as a blocker on penicillin model epilepsy. Male Wistar-Albino rats were used. A total of 4 main groups were formed: Control, Epilepsy, Epilepsy-opener, and Epilepsy-blocker groups, then three-time points were formed subgroups (1st day, 4th, and 8th). 48 rats were used in total. The epileptic focus was created by intracortical administration of penicillin at a dose of 500 IU/2 μl. Cortex is removed from all animals and cyt c, Bcl-2, and Bax gene expression levels were determined by qPCR. The SPSS 21 program was used for statistics. Bcl-2 and Bax gene expression levels were increased in the cortex regions of rats with epilepsy (p<0.05). Bcl-2, Bax gene expression levels, which increased due to epilepsy with the effect of KATP channels opened with pinacidil, returned to normal levels in the epilepsy opener group (p<0.05). Bcl-2 gene expression level, which was increased as a result of epilepsy due to the effect of KATP channels closed with glibenclamide, was higher than in the control and epilepsy-opener groups (p<0.05). Bcl-2 and Bax gene expression levels are increased in the cortex region due to epilepsy indicates that the apoptotic pathway could be activated. This study also It has been shown that the apoptotic pathway activated by epilepsy can be inactivated by pinacidil.

Keywords

• Epilepsy
• Bcl-2
• Bax
• Cytochrome c
• KATP channels

References

1. 1. Trinka E, Cock H, Hesdorffer D, Rossetti AO, Scheffer IE, Shinnar S, et al. A definition and classification of status epilepticus - Report of the ILAE Task Force on Classification of Status Epilepticus. Epilepsia. 2015;56(10):1515–23. 2. Zhang X, Cui S Sen, Wallace AE, Hannesson DK, Schmued LC, Saucier DM, et al. Relations between brain pathology and temporal lobe epilepsy. J Neurosci. 2002;22(14):6052–61.
2. 3. Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, et al. Essential versus accessory aspects of cell death: Recommendations of the NCCD 2015. Cell Death Differ. 2015;22(1):58–73.
3. 4. Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC. Neuronal cell death. Physiol Rev. 2018;98(2):813–80.
4. 5. Bengzon J, Kokaia Z, Elmér E, Nanobashvili A, Kokaia M, Lindvall O. Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci U S A. 1997;94(19):10432–7.
5. 6. Sankar R, Shin DH, Liu H, Mazarati A, De Vasconcelos AP, Wasterlain CG. Patterns of status epilepticus-induced neuronal injury during development and long-term consequences. J Neurosci. 1998;18(20):8382–93.
6. 7. Festjens N, Van Gurp M, Van Loo G, Saelens X, Vandenabeele P. Bcl-2 family members as sentinels of cellular integrity and role of mitochondrial intermembrane space proteins in apoptotic cell death. Acta Haematol. 2004;111(1–2):7–27.
7. 8. Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, et al. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci U S A. 1995;92(17):7834–8.
8. 9. Jean-Claude M, Douglas R. G. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol. 2001;2(January):63–7.

9. 10. Zamzami N, Kroemer G. The mitochondrion in apoptosis: How Pandora’s box opens. Nat Rev Mol Cell Biol. 2001;2(1):67–71.
10. 11. Engel T, Henshall DC. Apoptosis, Bcl-2 family proteins and caspases: The ABCs of seizure-damage and epileptogenesis? Int J Physiol Pathophysiol Pharmacol. 2009;1(2):97–115.
11. 12. Yamada K, Inagaki N. Neuroprotection by KATP channels. J Mol Cell Cardiol. 2005;38(6):945–9.
12. 13. Zhou F, Yao HH, Wu JY, Ding JH, Sun T, Hu G. Opening of microglial KATP channels inhibits rotenone-induced neuroinflammation. J Cell Mol Med. 2008;12(5A):1559–70.
13. 14. Banerjee PN, Filippi D, Allen Hauser W. The descriptive epidemiology of epilepsy-A review. Epilepsy Res. 2009;85(1):31–45.
14. 15. Shafaroodi H, Asadi S, Sadeghipour H, Ghasemi M, Ebrahimi F, Tavakoli S, et al. Role of ATP-sensitive potassium channels in the biphasic effects of morphine on pentylenetetrazole-induced seizure threshold in mice. Epilepsy Res. 2007;75(1):63–9.
15. 16. Kobayashi K, Nishizawa Y, Sawada K, Ogura H, Miyabe M. K+-channel openers suppress epileptiform activities induced by 4-aminopyridine in cultured rat hippocampal neurons. J Pharmacol Sci. 2008;108(4):517–28.
16. 17. Freiman TM, Kukolja J, Heinemeyer J, Eckhardt K, Aranda H, Rominger A, et al. Modulation of K+-evoked [3H]-noradrenaline release from rat and human brain slices by gabapentin: Involvement of KATP channels. Naunyn Schmiedebergs Arch Pharmacol. 2001;363(5):537–42.
17. 18. Varughese JF, Baxley T, Chalovich JM, Li Y. A computational and experimental approach to investigate bepridil binding with cardiac troponin. J Phys Chem B. 2011;115(10):2392–400. 19. Soytürk H. , Demir S. BÖ. Investigation of Physiological Role of Mitochondrial KATP Channel’s on Penicillin G Induced Experimental Epilepsy Model in Rats. Med Sci. 2021;16(1):19–29.
18. 20. Prince , D.A., Farrell D. Centrencephalic spike-wave discharges following parenteral penicillin injection in the rat. Neurology. 1969;19:309-310.
19. 21. Fariello RG. Parenteral Penicillin in Rats: An Experimental Model of Multifocal Epilepsy. Epilepsia. 1976;17(2):217–22.
20. 22. Macdonald, R.L., Barker JL. Pentylenetetrazol and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurons. Nature. 1977;267:720–1.
21. 23. Kotloski R, Lynch M, Lauersdorf S, Sutula T. Repeated brief seizures induce progressive hippocampal neuron loss and memory deficits. Prog Brain Res. 2002;135:95–110.
22. 24. Brayden JE. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol. 2002;29(4):312–6.
23. 25. Katebi, Majid & Soleimani, Manoocher & Mehdizadeh M. Neuroprotective effects of a mitochondrial K+-ATP channel opener (diazoxide) are mediated by Bcl-2 expression upregulation. Neural Regen Res. 6th ed. 2011;6(12):956-960.