Bumetanid’in HT-22 Hipokampal Hücre Hattında Pentilentetrazol Kaynaklı Hücresel Hasar ve Oksidatif Stres Üzerine Etkisinin Araştırılması

Muzaffer Türkoğlu; Ali Tokgöz; Songül Bavli; Sebahattin Karabulut; Ayşegül Öztürk

EN TR

The Investigation of the Effect of Bumetanide on Pentylenetetrazol-Induced Cellular Damage and Oxidative Stress in the HT-22 Hippocampal Cell Line

Abstract

The present study aimed to investigate the potential neuroprotective effect of bumetanide (BMT), a Na⁺-K⁺-2Cl⁻ cotransporter-1 (NKCC1) inhibitor, against pentylenetetrazol (PTZ)-induced epileptiform cellular damage and to evaluate its association with oxidative stress parameters in the HT-22 neuronal cell line. HT-22 cells were divided into four groups: control, PTZ (60 mM), BMT, and BMT + PTZ. Cells were pretreated with BMT at different concentrations (1, 2, 4, and 8 µM) prior to exposure to PTZ. Cell viability was assessed using the XTT assay. Total antioxidant status (TAS) and total oxidant status (TOS) were measured to evaluate cellular oxidative stress. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc test. PTZ exposure significantly reduced cell viability and induced oxidative stress in HT-22 cells. BMT pretreatment, particularly at the 2 µM concentration, significantly attenuated PTZ-induced cell death (p < 0.05). At this dose, BMT increased TAS levels while significantly limiting the PTZ-induced elevation in TOS. BMT administered alone did not significantly affect cell viability. These findings indicate that BMT exerts a dose-dependent protective effect against PTZ-induced neuronal damage. This study demonstrates that bumetanide pretreatment provides neuroprotection against PTZ-induced cellular injury in HT-22 neurons. The observed protective effect appears to be mediated not by direct antioxidant activity, but rather through modulation of chloride homeostasis, leading to reduced neuronal hyperexcitability and secondary suppression of oxidative stress responses. Targeting NKCC1-dependent upstream mechanisms may represent a complementary therapeutic strategy for epileptiform neuronal damage.

Keywords

Bumetanid’in HT-22 Hipokampal Hücre Hattında Pentilentetrazol Kaynaklı Hücresel Hasar ve Oksidatif Stres Üzerine Etkisinin Araştırılması

Öz

Bu çalışmanın amacı, Na⁺-K⁺-2Cl⁻ kotransporter-1 (NKCC1) inhibitörü olan bumetanidin (BMT), pentilentetrazol (PTZ) ile indüklenen epileptiform hücresel hasara karşı olası nöroprotektif etkisini ve bu etkinin oksidatif stres parametreleri ile ilişkisini HT-22 nöronal hücre hattında araştırmaktır. HT-22 hücreleri kontrol, PTZ (60 mM), BMT ve BMT + PTZ olmak üzere dört gruba ayrılmıştır. Hücreler, farklı konsantrasyonlarda (1, 2, 4 ve 8 µM) BMT ile ön işleme tabi tutulduktan sonra 60 mM PTZ’ye maruz bırakılmıştır. Hücre canlılığı XTT testi ile değerlendirilmiştir. Hücresel oksidatif durumun belirlenmesi amacıyla total antioksidan seviye (TAS) ve total oksidan seviye (TOS) ölçümleri gerçekleştirilmiştir. Veriler tek yönlü ANOVA ve Tukey post-hoc testi kullanılarak analiz edilmiştir. PTZ uygulaması, HT-22 hücrelerinde hücre canlılığını anlamlı düzeyde azaltmış ve oksidatif stres belirteçlerinde belirgin artışa neden olmuştur. BMT ön tedavisinin özellikle 2 µM dozunda, PTZ’ye bağlı hücre ölümünü anlamlı şekilde azalttığı saptanmıştır (p<0.05). Bu dozda BMT uygulaması, TAS düzeylerini artırırken PTZ’ye bağlı TOS artışını anlamlı olarak sınırlamıştır. Tek başına BMT uygulamasının hücre canlılığı üzerinde anlamlı bir etkisi gözlenmemiştir. Bu çalışma, bumetanid ön tedavisinin PTZ ile indüklenen HT-22 hücre hasarına karşı nöroprotektif etki gösterdiğini ortaya koymaktadır. Bu etkinin, doğrudan bir antioksidan mekanizmadan ziyade, klorür homeostazının düzenlenmesi yoluyla nöronal hipereksitabilitenin azaltılmasına ve buna bağlı sekonder oksidatif stres yanıtlarının baskılanmasına bağlı olduğu düşünülmektedir. Bulgularımız, NKCC1 inhibisyonunun epileptiform hasarın upstream patofizyolojik mekanizmalarını hedefleyen tamamlayıcı bir terapötik yaklaşım olabileceğini desteklemektedir.

Anahtar Kelimeler

Supporting Institution

TÜBİTAK

Project Number

1919B012305670

Ethical Statement

As this study was conducted on a secondary cell line (HT-22 Mouse Hippocampal Neuronal Cell Line), no Ethics Committee approval certificate is required.

Thanks

This study was supported by the Scientific Research Projects Support Programme for University Students 2023, 1st period, under application number 1919B012305670, conducted by the Scientific and Technological Research Council of Turkey (TÜBİTAK) Scientific Research Support Programmes Presidency (BİDEB).

References

Abruzzo, P. M., Panisi, C., & Marini, M. (2021). The Alteration of Chloride Homeostasis/GABAergic Signaling in Brain Disorders: Could Oxidative Stress Play a Role?. Antioxidants (Basel, Switzerland), 10(8), 1316. [https://doi.org/10.3390/antiox10081316](https://doi.org/10.3390/antiox10081316)
Barnard, S. N., Chen, Z., Holmes, M., Kanner, A. M., Hegde, M., Kuzniecky, R., Lowenstein, D., French, J. A., & Human Epilepsy Project (1) Investigators (2025). Treatment Response to Antiseizure Medications in People With Newly Diagnosed Focal Epilepsy. JAMA Neurology, 82(10), 1022–1030. [https://doi.org/10.1001/jamaneurol.2025.2949](https://doi.org/10.1001/jamaneurol.2025.2949)
Blaesse, P., Airaksinen, M. S., Rivera, C., & Kaila, K. (2009). Cation-chloride cotransporters and neuronal function. Neuron, 61(6), 820–838. [https://doi.org/10.1016/j.neuron.2009.03.003](https://doi.org/10.1016/j.neuron.2009.03.003)
Dalic, L. J., Warren, A. E. L., Young, J. C., Thevathasan, W., Roten, A., Bulluss, K. J., & Archer, J. S. (2020). Cortex leads the thalamic centromedian nucleus in generalized epileptic discharges in Lennox-Gastaut syndrome. Epilepsia, 61(10), 2214–2223. [https://doi.org/10.1111/epi.16657](https://doi.org/10.1111/epi.16657)
Dzhala, V. I., Talos, D. M., Sdrulla, D. A., Brumback, A. C., Mathews, G. C., Benke, T. A., Delpire, E., Jensen, F. E., & Staley, K. J. (2005). NKCC1 transporter facilitates seizures in the developing brain. Nature Medicine, 11(11), 1205–1213. [https://doi.org/10.1038/nm1301](https://doi.org/10.1038/nm1301)
Erel, O. (2004). A novel automated method to measure total antioxidant response against potent free radical reactions. Clinical Biochemistry, 37(2), 112–119. [https://doi.org/10.1016/j.clinbiochem.2003.10.014](https://doi.org/10.1016/j.clinbiochem.2003.10.014)
Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry, 38(12), 1103–1111. [https://doi.org/10.1016/j.clinbiochem.2005.08.008](https://doi.org/10.1016/j.clinbiochem.2005.08.008)
Geronzi, U., Lotti, F., & Grosso, S. (2018). Oxidative stress in epilepsy. Expert Review of Neurotherapeutics, 18(5), 427–434. [https://doi.org/10.1080/14737175.2018.1465410](https://doi.org/10.1080/14737175.2018.1465410)

Gong, Y., Wu, M., Shen, J., Tang, J., Li, J., Xu, J., Dang, B., & Chen, G. (2021). Inhibition of the NKCC1/NF-κB signaling pathway decreases inflammation and improves brain edema and nerve cell apoptosis in an SBI rat model. Frontiers in Molecular Neuroscience, 14, 641993. [https://doi.org/10.3389/fnmol.2021.641993](https://doi.org/10.3389/fnmol.2021.641993)
Joha, Z., Başgöz, N., Özgür, A., & Taşkıran, A. Ş. (2025). Bromelain protects against PTZ-induced glial damage and inflammation: An in vitro and in silico study. Cell Biochemistry and Biophysics, 83(3), 3215–3223. [https://doi.org/10.1007/s12013-025-01703-8](https://doi.org/10.1007/s12013-025-01703-8)
Joha, Z., & Taşkıran, A. Ş. (2023). Hidrojen peroksit ile indüklenen C6 hücrelerinde oksidatif stres kaynaklı apoptosis ve inflamatuar artışına karşı astaksantinin koruyucu etkisi. Turkish Journal of Agriculture-Food Science and Technology, 11(9), 1686–1692. [https://doi.org/10.24925/turjaf.v11i9.1686-1692.6281](https://doi.org/10.24925/turjaf.v11i9.1686-1692.6281)
Kwan, P., Arzimanoglou, A., Berg, A. T., Brodie, M. J., Allen Hauser, W., Mathern, G., Moshé, S. L., Perucca, E., Wiebe, S., & French, J. (2010). Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia, 51(6), 1069–1077. [https://doi.org/10.1111/j.1528-1167.2009.02397.x](https://doi.org/10.1111/j.1528-1167.2009.02397.x)
Lam, P., Newland, J., Faull, R. L. M., & Kwakowsky, A. (2023). Cation-chloride cotransporters KCC2 and NKCC1 as therapeutic targets in neurological and neuropsychiatric disorders. Molecules, 28(3), 1344. [https://doi.org/10.3390/molecules28031344](https://doi.org/10.3390/molecules28031344)
Li, T., Deng, J., Qin, J., & Chu, X. P. (2024). Editorial: Neuromodulation for pharmacoresistant epilepsy: from bench to bed. Frontiers in Neurology, 15, 1354897. [https://doi.org/10.3389/fneur.2024.1354897](https://doi.org/10.3389/fneur.2024.1354897)
Liu, R., Wang, J., Liang, S., Zhang, G., & Yang, X. (2020). Role of NKCC1 and KCC2 in epilepsy: from expression to function. Frontiers in Neurology, 10, 1407. [https://doi.org/10.3389/fneur.2019.01407](https://doi.org/10.3389/fneur.2019.01407)
Monteiro, Á. B., Alves, A. F., Ribeiro Portela, A. C., Oliveira Pires, H. F., Pessoa de Melo, M., Medeiros Vilar Barbosa, N. M., & Bezerra Felipe, C. F. (2024). Pentylenetetrazole: A review. Neurochemistry International, 180, 105841. [https://doi.org/10.1016/j.neuint.2024.105841](https://doi.org/10.1016/j.neuint.2024.105841)
Morimoto, K., Fahnestock, M., & Racine, R. J. (2004). Kindling and status epilepticus models of epilepsy: rewiring the brain. Progress in Neurobiology, 73(1), 1–60. [https://doi.org/10.1016/j.pneurobio.2004.03.009](https://doi.org/10.1016/j.pneurobio.2004.03.009)
Namulodi, S., Torun, I. E., Bayiroglu, F., Kaya, M. S., & Kilinc, E. (2025). Adropin ameliorates behavioral seizures and the relevant neuroinflammation, oxidative stress, and neural damage in a rat model of pentylenetetrazole-induced seizure potentially by reducing the activation of NF-κB/IkB-α signaling pathway. Metabolic Brain Disease, 40(5), 222. [https://doi.org/10.1007/s11011-025-01654-2](https://doi.org/10.1007/s11011-025-01654-2)
Rao, S., Farhat, A., Rakshasbhuvankar, A., Athikarisamy, S., Ghosh, S., & Nagarajan, L. (2023). Effects of bumetanide on neonatal seizures: A systematic review of animal and human studies. Seizure, 111, 206–214. [https://doi.org/10.1016/j.seizure.2023.09.007](https://doi.org/10.1016/j.seizure.2023.09.007)
Raol, Y. H., & Brooks-Kayal, A. R. (2012). Experimental models of seizures and epilepsies. Progress in Molecular Biology and Translational Science, 105, 57–82. [https://doi.org/10.1016/B978-0-12-394596-9.00003-2](https://doi.org/10.1016/B978-0-12-394596-9.00003-2)
Samokhina, E., & Samokhin, A. (2018). Neuropathological profile of the pentylenetetrazol (PTZ) kindling model. International Journal of Neuroscience, 128(11), 1086–1096. [https://doi.org/10.1080/00207454.2018.1481064](https://doi.org/10.1080/00207454.2018.1481064)
Tan, S., Wood, M., & Maher, P. (1998). Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells. Journal of Neurochemistry, 71(1), 95–105. [https://doi.org/10.1046/j.1471-4159.1998.71010095.x](https://doi.org/10.1046/j.1471-4159.1998.71010095.x)
Taşkıran, A. S., Özdemir, E., Gümüş, E., & Ergül, M. (2020). The effects of salmon calcitonin on epileptic seizures, epileptogenesis, and postseizure hippocampal neuronal damage in pentylenetetrazole-induced epilepsy model in rats. Epilepsy & Behavior, 113, 107501. [https://doi.org/10.1016/j.yebeh.2020.107501](https://doi.org/10.1016/j.yebeh.2020.107501)
Taşkıran, A., & Yıldız Asdemir, T. (2023). Investigation of the effect of pumpkin (Cucurbita pepo L.) seed oil on pentylenetetrazole-induced neuronal damage in HT-22 cell line. Turkish Journal of Agriculture-Food Science and Technology, 11(s1), 2499–2504. [https://orcid.org/0000-0002-5810-8415](https://orcid.org/0000-0002-5810-8415)
Yang, N., Guan, Q. W., Chen, F. H., Xia, Q. X., Yin, X. X., Zhou, H. H., & Mao, X. Y. (2020). Antioxidants targeting mitochondrial oxidative stress: promising neuroprotectants for epilepsy. Oxidative Medicine and Cellular Longevity, 2020, 6687185. [https://doi.org/10.1155/2020/6687185](https://doi.org/10.1155/2020/6687185)
Yulak, F., Joha, Z., Öztürk, A., İnan, Z. D. Ş., & Taşkıran, A. Ş. (2025). Enoxaparin protects C6 glioma cells from glutamate-induced cytotoxicity by reducing oxidative stress and apoptosis. Molecular Neurobiology, 62(4), 4631–4640. [https://doi.org/10.1007/s12035-024-04587-6](https://doi.org/10.1007/s12035-024-04587-6)
Zhang, S., Meor Azlan, N. F., Josiah, S. S., Zhou, J., Zhou, X., Jie, L., Zhang, Y., Dai, C., Liang, D., Li, P., Li, Z., Wang, Z., Wang, Y., Ding, K., Wang, Y., & Zhang, J. (2023). The role of SLC12A family of cation-chloride cotransporters and drug discovery methodologies. Journal of Pharmaceutical Analysis, 13(12), 1471–1495. [https://doi.org/10.1016/j.jpha.2023.09.002](https://doi.org/10.1016/j.jpha.2023.09.002)

Details

Primary Language

Turkish

Subjects

Clinical Sciences (Other)

Journal Section

Research Article

Authors

Muzaffer Türkoğlu
0009-0000-0085-2332
Türkiye

Ali Tokgöz
0009-0000-3752-9483
Türkiye

Songül Bavli
0000-0002-1688-7828
Türkiye

Sebahattin Karabulut ^*
0000-0002-3261-4125
Türkiye

Ayşegül Öztürk
0000-0001-8130-7968
Türkiye

Publication Date

April 14, 2026

Submission Date

January 26, 2026

Acceptance Date

March 23, 2026

Published in Issue

Year 2026 Volume: 4

IZ

https://izlik.org/JA64DD28EP

Cite

RIS / Bibtex

APA

Türkoğlu, M., Tokgöz, A., Bavli, S., Karabulut, S., & Öztürk, A. (2026). Bumetanid’in HT-22 Hipokampal Hücre Hattında Pentilentetrazol Kaynaklı Hücresel Hasar ve Oksidatif Stres Üzerine Etkisinin Araştırılması. Health Services Research Journal, 4. https://izlik.org/JA64DD28EP