Journal of Multidisciplinary Applied Natural Science
Open Access Journal
Scopus CiteScore 2024
4.8
Calculated on 05 May, 2025
SJR 2024
0.31
Powered by scimagojr.com
Open Access Journal
4.8
Calculated on 05 May, 2025
0.31
Powered by scimagojr.com
https://doi.org/10.47352/jmans.2774-3047.365
Marsintauli Hasudungan Siregar
Nurdiana Nurdiana
Farhad Bal’afif
Susanthy Djajalaksana
Arif Setiawansyah
GABAA receptor dysfunction and altered chloride homeostasis significantly contribute to seizure pathophysiology, with the sodium-potassium-chloride cotransporter 1 (NKCC1) playing a crucial role in regulating neuronal excitability. This study investigated furosemide's capacity to enhance GABAA receptor activity through NKCC1 antagonism and evaluated its therapeutic profile in combination with diazepam for seizure management. Comprehensive molecular docking analyses were conducted to assess binding affinities of furosemide and diazepam to NKCC1, followed by in vivo experiments using pentylenetetrazol-induced seizure models to evaluate GABAA receptor expression, seizure duration, and multiple pathophysiological biomarkers. Molecular analysis revealed that furosemide demonstrated measurable NKCC1 binding capacity (binding energy: -7.09 kcal/mol; Ki: 6.34 µM), though significantly lower affinity compared to diazepam (binding energy: -7.83 kcal/mol; Ki: 1.81 µM). The furosemide-diazepam combination exhibited complex competitive binding interactions, with furosemide substantially reducing diazepam's NKCC1 binding affinity. NKCC1 antagonism by furosemide effectively enhanced GABAA receptor expression by 29.8 ± 1.60% when used alone and 37.60 ± 2.0% in combination with diazepam. However, combination therapies resulted in significantly longer seizure durations (80 ± 3.0 s) compared to diazepam monotherapy (42.5 ± 2.10 s), suggesting antagonistic interactions on acute seizure suppression that may reflect altered chloride gradients or competitive pharmacokinetic effects. Despite reduced efficacy in seizure termination, combination therapy demonstrated selective advantages in other pathophysiological domains, including superior blood-brain barrier protection (reduced albumin level to 90.90 ± 2.70 µg/mL) and reduced excitotoxic damage. These findings indicate that furosemide-diazepam combination therapy presents a complex therapeutic profile characterized by trade-offs between acute seizure control and neuroprotective mechanisms. The data suggest potential utility in maintenance therapy or prevention of seizure-related complications rather than acute seizure termination, warranting further investigation into temporal optimization strategies and dose modifications.
[1] S. Sivakumaran and J. Maguire. (2016). "Bumetanide Reduces Seizure Progression And The Development Of Pharmacoresistant Status Epilepticus". Epilepsia. 57 (2): 222-232. 10.1111/epi.13270.
DOI: https://doi.org/10.1111/epi.13270[2] E. Sallard, D. Letourneur, and P. Legendre. (2021). "Electrophysiology Of Ionotropic GABA Receptors". Cellular and Molecular Life Sciences. 78 (13): 5341-5370. 10.1007/s00018-021-03846-2.
DOI: https://doi.org/10.1007/s00018-021-03846-2[3] S. B. Sarasa, R. Mahendran, G. Muthusamy, B. Thankappan, D. R. F. Selta, and J. Angayarkanni. (2020). "A Brief Review On The Non-Protein Amino Acid, Gamma-Amino Butyric Acid (GABA): Its Production And Role In Microbes". Current Microbiology. 77 (4): 534-544. 10.1007/s00284-019-01839-w.
DOI: https://doi.org/10.1007/s00284-019-01839-w[4] S. E. Oriaifo, I. Otokiti, and E. K. Omogbai. (2012). "The Effect Of Furosemide On Experimentally-Induced Seizures In Mice". Journal of Clinical Medicine and Research. 4 (7): 89-93. 10.5897/JCMR12.003.
[5] D. E. Naylor. (2023). "In The Fast Lane: Receptor Trafficking During Status Epilepticus". Epilepsia Open. 8 (S1): S35-S65. 10.1002/epi4.12718.
DOI: https://doi.org/10.1002/epi4.12718[6] T. Z. Deeb, J. Maguire, and S. J. Moss. (2012). "Possible Alterations In GABAA Receptor Signaling That Underlie Benzodiazepine-Resistant Seizures". Epilepsia. 53 (S9): 79-88. 10.1111/epi.12037.
DOI: https://doi.org/10.1111/epi.12037[7] M. Charalambous, H. A. Volk, L. Van Ham, and S. F. M. Bhatti. (2021). "First-Line Management Of Canine Status Epilepticus At Home And In Hospital: Opportunities And Limitations Of The Various Administration Routes Of Benzodiazepines". BMC Veterinary Research. 17 (1): 103. 10.1186/s12917-021-02805-0.
DOI: https://doi.org/10.1186/s12917-021-02805-0[8] C. H. Vinkers and B. Olivier. (2012). "Mechanisms Underlying Tolerance After Long-Term Benzodiazepine Use: A Future For Subtype-Selective GABAA Receptor Modulators". Advances in Pharmacological and Pharmaceutical Sciences. 2012 : 416864. 10.1155/2012/416864.
DOI: https://doi.org/10.1155/2012/416864[9] P. M. Abruzzo, C. Panisi, and M. Marini. (2021). "The Alteration Of Chloride Homeostasis GABAergic Signaling In Brain Disorders: Could Oxidative Stress Play A Role". Antioxidants. 10 (8): 1316. 10.3390/antiox10081316.
DOI: https://doi.org/10.3390/antiox10081316[10] J. T. Schulte, C. J. Wierenga, and H. Bruining. (2018). "Chloride Transporters And GABA Polarity In Developmental Neurological And Psychiatric Conditions". Neuroscience and Biobehavioral Reviews. 90 : 260-271. 10.1016/j.neubiorev.2018.05.001.
DOI: https://doi.org/10.1016/j.neubiorev.2018.05.001[11] M. Hamze, C. Brier, E. Buhler, J. Zhang, I. Medina, and C. Porcher. (2024). "Regulation Of Neuronal Chloride Homeostasis By Pro And Mature Brain Derived Neurotrophic Factor BDNF Via KCC2 Cation Chloride Cotransporters In Rat Cortical Neurons". International Journal of Molecular Sciences. 25 (11): 6253. 10.3390/ijms25116253.
DOI: https://doi.org/10.3390/ijms25116253[12] R. F. Berman. (2009). In: " P. A. Schwartzkroin (Ed.) Encyclopedia of Basic Epilepsy Research". Academic Press. 10.1016/B978-012373961-2.00270-8.
DOI: https://doi.org/10.1016/B978-012373961-2.00270-8[13] R. J. Burman, J. S. Selfe, J. H. Lee, M. Van Den Berg, A. Calin, N. K. Codadu, R. Wright, S. E. Newey, R. R. Parrish, A. A. Katz, J. M. Wilmshurst, C. J. Akerman, A. J. Trevelyan, and J. V. Raimondo. (2019). "Excitatory GABAergic Signalling Is Associated With Benzodiazepine Resistance In Status Epilepticus". Brain. 142 (11): 3482-3501. 10.1093/brain/awz283.
DOI: https://doi.org/10.1093/brain/awz283[14] R. Liu, J. Wang, S. Liang, G. Zhang, and X. Yang. (2020). "Role Of NKCC1 And KCC2 In Epilepsy: From Expression To Function". Frontiers in Neurology. 10 : 1407. 10.3389/fneur.2019.01407.
DOI: https://doi.org/10.3389/fneur.2019.01407[15] Z. Talifu, Y. Pan, H. Gong, X. Xu, C. Zhang, D. Yang, F. Gao, Y. Yu, L. Du, and J. Li. (2022). "The Role Of KCC2 And NKCC1 In Spinal Cord Injury: From Physiology To Pathology". Frontiers in Physiology. 13 : 1045520. 10.3389/fphys.2022.1045520.
DOI: https://doi.org/10.3389/fphys.2022.1045520[16] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell, and A. J. Olson. (2009). "AutoDock4 And AutoDockTools4: Automated Docking With Selective Receptor Flexibility". Journal of Computational Chemistry. 30 (16): 2785-2791. 10.1002/jcc.21256.
DOI: https://doi.org/10.1002/jcc.21256[17] Y. Zhao, K. Roy, P. Vidossich, L. Cancedda, M. De Vivo, B. Forbush, and E. Cao. (2022). "Structural Basis For Inhibition Of The Cation-Chloride Cotransporter NKCC1 By The Diuretic Drug Bumetanide". Nature Communications. 13 (1): 2747. 10.1038/s41467-022-30407-3.
DOI: https://doi.org/10.1038/s41467-022-30407-3[18] A. Setiawansyah, G. Susanti, R. Alrayan, I. Hadi, M. I. Arsul, D. Luthfiana, L. Wismayani, and N. Hidayati. (2024). "Aaptamine Enhanced Doxorubicin Activity On B-Cell Lymphoma 2 (Bcl-2): A Multi-Structural Molecular Docking Study". Ad-Dawaa' Journal of Pharmaceutical Science. 7 (1): 1-10. 10.24252/djps.v7i1.46796.
DOI: https://doi.org/10.24252/djps.v7i1.46796[19] Y. Rosa, R. Riyanto, G. Susanti, A. Setiawansyah, Z. Zuhana, A. Fatriansari, and S. A. Pratama. (2024). "Persea Americana Leaf Extract Promotes Wound Healing By Inhibiting NF-KB1". Journal of Applied Pharmaceutical Science. 10.7324/JAPS.2025.218012.
DOI: https://doi.org/10.7324/JAPS.2025.218012[20] N. Nurhidayati, Y. Yueniwati, A. T. Endharti, H. K. Permatasari, and A. Setiawansyah. (2025). "Chemical Binding Free Energy And Anti-Free Radical Of Chemical Compounds From Sea Cucumber (Holothuria Scabra) Against Malondialdehyde And Akt". Advanced Journal of Chemistry Section A. 1709-1723. 10.48309/ajca.2025.518195.1827.
[21] N. Chaachouay. (2025). "Synergy, Additive Effects, And Antagonism Of Drugs With Plant Bioactive Compounds". Drugs and Drug Candidates. 4 (1): 4. 10.3390/ddc4010004.
DOI: https://doi.org/10.3390/ddc4010004[22] M. Lakshmanan. (2019). In: " G. M. Raj and R. Raveendran (Eds.) Introduction To Basics Of Pharmacology And Toxicology: General And Molecular Pharmacology". Springer Singapore. 10.1007/978-981-32-9779-1_13.
DOI: https://doi.org/10.1007/978-981-32-9779-1_13[23] A. Setiawansyah and B. M. Gemantari. (2022). "Potential Activity Of Caryophyllene Derivatives As Xanthine Oxidase Inhibitor: An In Silico Quantitative Structure Activity Relationship Analysis". Journal of Food and Pharmaceutical Sciences. 10 (3): 700-708. 10.22146/jfps.5485.
DOI: https://doi.org/10.22146/jfps.5485[24] Y. Ben-Ari. (2017). "NKCC1 Chloride Importer Antagonists Attenuate Many Neurological And Psychiatric Disorders". Trends in Neurosciences. 40 (9): 536-554. 10.1016/j.tins.2017.07.001.
DOI: https://doi.org/10.1016/j.tins.2017.07.001[25] Y. Zhao, P. Vidossich, B. Forbush, J. Ma, J. Rinehart, M. De Vivo, and E. Cao. (2025). "Structural Basis For Human NKCC1 Inhibition By Loop Diuretic Drugs". The EMBO Journal. 44 (5): 1540-1562. 10.1038/s44318-025-00368-6.
DOI: https://doi.org/10.1038/s44318-025-00368-6[26] L. Chen, J. Yu, L. Wan, Z. Wu, G. Wang, Z. Hu, L. Ren, J. Zhou, B. Qian, X. Zhao, J. Zhang, X. Liu, and Y. Wang. (2022). "Furosemide Prevents Membrane KCC2 Downregulation During Convulsant Stimulation In The Hippocampus". IBRO Neuroscience Reports. 12 : 355-365. 10.1016/j.ibneur.2022.04.010.
DOI: https://doi.org/10.1016/j.ibneur.2022.04.010[27] T. Z. Deeb, Y. Nakamura, G. D. Frost, P. A. Davies, and S. J. Moss. (2013). "Disrupted Chloride Homeostasis Contributes To Reductions In The Inhibitory Efficacy Of Diazepam During Hyperexcited States". European Journal of Neuroscience. 38 (3): 2453-2467. 10.1111/ejn.12241.
DOI: https://doi.org/10.1111/ejn.12241[28] Y. Chen, M. M. Nagib, N. Yasmen, M. N. Sluter, T. L. Littlejohn, Y. Yu, and J. Jiang. (2023). "Neuroinflammatory Mediators In Acquired Epilepsy: An Update". Inflammation Research. 72 (4): 683-701. 10.1007/s00011-023-01700-8.
DOI: https://doi.org/10.1007/s00011-023-01700-8[29] N. Dupuis and S. Auvin. (2015). "Inflammation And Epilepsy In The Developing Brain: Clinical And Experimental Evidence". CNS Neuroscience and Therapeutics. 21 (2): 141-151. 10.1111/cns.12371.
DOI: https://doi.org/10.1111/cns.12371[30] M. Maroso, S. Balosso, T. Ravizza, J. Liu, E. Aronica, A. M. Iyer, C. Rossetti, M. Molteni, M. Casalgrandi, A. A. Manfredi, M. E. Bianchi, and A. Vezzani. (2010). "Toll-Like Receptor 4 And High Mobility Group Box-1 Are Involved In Ictogenesis And Can Be Targeted To Reduce Seizures". Nature Medicine. 16 (4): 413-419. 10.1038/nm.2127.
DOI: https://doi.org/10.1038/nm.2127[31] K. Riazi, M. A. Galic, J. B. Kuzmiski, W. Ho, K. A. Sharkey, and Q. J. Pittman. (2008). "Microglial Activation And TNFα Production Mediate Altered CNS Excitability Following Peripheral Inflammation". Proceedings of the National Academy of Sciences of the United States of America. 105 (44): 17151-17156. 10.1073/pnas.0806682105.
DOI: https://doi.org/10.1073/pnas.0806682105[32] V. I. Dzhala, D. M. Talos, D. A. Sdrulla, A. C. Brumback, G. C. Mathews, T. A. Benke, E. Delpire, F. E. Jensen, and K. J. Staley. (2005). "NKCC1 Transporter Facilitates Seizures In The Developing Brain". Nature Medicine. 11 (11): 1205-1213. 10.1038/nm1301.
DOI: https://doi.org/10.1038/nm1301[33] C. Chretien, A. M. R. Lobo, A. D. La Rossa, and P. Maechler. (2023). "Glutamate Dehydrogenase Mutation Reveals The Role Of Mediobasal Hypothalamus In The Development Of Seizures". IBRO Neuroscience Reports. 15 : S251-S252. 10.1016/j.ibneur.2023.08.425.
DOI: https://doi.org/10.1016/j.ibneur.2023.08.425[34] S. N. Rakhade and F. E. Jensen. (2009). "Epileptogenesis In The Immature Brain: Emerging Mechanisms". Nature Reviews Neurology. 5 (7): 380-391. 10.1038/nrneurol.2009.80.
DOI: https://doi.org/10.1038/nrneurol.2009.80[35] M. Barker-Haliski and H. S. White. (2015). "Glutamatergic Mechanisms Associated With Seizures And Epilepsy". Cold Spring Harbor Perspectives in Medicine. 5 (8): a022863. 10.1101/cshperspect.a022863.
DOI: https://doi.org/10.1101/cshperspect.a022863[36] P. Ambrogini, P. Torquato, D. Bartolini, M. C. Albertini, D. Lattanzi, M. Di Palma, R. Marinelli, M. Betti, A. Minelli, R. Cuppini, and F. Galli. (2019). "Excitotoxicity, Neuroinflammation And Oxidant Stress As Molecular Bases Of Epileptogenesis And Epilepsy Derived Neurodegeneration: The Role Of Vitamin E". Biochimica et Biophysica Acta - Molecular Basis of Disease. 1865 (6): 1098-1112. 10.1016/j.bbadis.2019.01.026.
DOI: https://doi.org/10.1016/j.bbadis.2019.01.026[37] A. Friedman, D. Kaufer, and U. Heinemann. (2009). "Blood Brain Barrier Breakdown Inducing Astrocytic Transformation: Novel Targets For The Prevention Of Epilepsy". Epilepsy Research. 85 : 142-149. 10.1016/j.eplepsyres.2009.03.005.
DOI: https://doi.org/10.1016/j.eplepsyres.2009.03.005[38] N. Marchi, L. Angelov, T. Masaryk, V. Fazio, T. Granata, N. Hernandez, K. Hallene, T. Diglaw, L. Franic, I. Najm, and D. Janigro. (2007). "Seizure Promoting Effect Of Blood Brain Barrier Disruption". Epilepsia. 48 (4): 732-742. 10.1111/j.1528-1167.2007.00988.x.
DOI: https://doi.org/10.1111/j.1528-1167.2007.00988.x[39] L. Han. (2021). "Modulation Of The Blood Brain Barrier For Drug Delivery To Brain". Pharmaceutics. 13 (12): 2024. 10.3390/pharmaceutics13122024.
DOI: https://doi.org/10.3390/pharmaceutics13122024[40] S. Zhang, L. Gan, F. Cao, H. Wang, P. Gong, C. Ma, L. Ren, Y. Lin, and X. Lin. (2022). "The Barrier And Interface Mechanisms Of The Brain Barrier And Brain Drug Delivery". Brain Research Bulletin. 190 : 69-83. 10.1016/j.brainresbull.2022.09.017.
DOI: https://doi.org/10.1016/j.brainresbull.2022.09.017[41] L. A. Mendez-Cuesta, B. Marquez-Valadez, V. P. La Cruz, C. Escobar-Briones, S. Galvan-Arzate, Y. Alvarez-Ruiz, P. D. Maldonado, R. A. Santana, A. Santamaria, and P. Carrillo-Mora. (2011). "Diazepam Blocks Striatal Lipid Peroxidation And Improves Stereotyped Activity In A Rat Model Of Acute Stress". Basic and Clinical Pharmacology and Toxicology. 109 (5): 350-356. 10.1111/j.1742-7843.2011.00738.x.
DOI: https://doi.org/10.1111/j.1742-7843.2011.00738.x[42] S. Musavi and P. Kakkar. (2000). "Pro And Antioxidant Responses To Repeated Administration Of Diazepam In Rat Brain". Molecular and Cellular Biochemistry. 206 (1): 97-103. 10.1023/A:1007007630118.
DOI: https://doi.org/10.1023/A:1007007630118[43] B. L. S. Amaral, T. P. D. Santos, R. M. D. Santos, P. F. P. Andrade, A. Rocha-Gomes, V. J. Lages, K. B. Costa, D. A. Freitas, B. F. Mendes, G. E. B. A. Melo, and W. F. Pereira. (2023). "Furosemide Reduces TNF Levels And Increases Antioxidant Activity In Animal Models Of Nephrotic Syndrome". Journal of Advances in Medicine and Medical Research. 35 (21): 66-79. 10.9734/jammr/2023/v35i215213.
DOI: https://doi.org/10.9734/jammr/2023/v35i215213[44] S. Kovac, A. M. Domijan, M. C. Walker, and A. Y. Abramov. (2012). "Prolonged Seizure Activity Impairs Mitochondrial Bioenergetics And Induces Cell Death". Journal of Cell Science. 125 (7): 1796-1806. 10.1242/jcs.099176.
DOI: https://doi.org/10.1242/jcs.099176[45] N. N. Wang, F. Cao, L. H. Zhang, Y. F. Zheng, and D. Xu. (2025). "Multi Omics: A Bridge Connecting Genotype And Phenotype For Epilepsy". Biomarker Research. 10.1186/s40364-025-00798-8.
DOI: https://doi.org/10.1186/s40364-025-00798-8[46] J. A. French, A. M. Kanner, J. Bautista, B. Abou-Khalil, T. Browne, C. L. Harden, W. H. Theodore, C. Bazil, J. Stern, S. C. Schachter, D. Bergen, D. Hirtz, G. D. Montouris, M. Nespeca, B. Gidal, W. J. Marks, W. R. Turk, J. H. Fischer, B. Bourgeois, A. Wilner, R. E. Faught, R. C. Sachdeo, A. Beydoun, and T. A. Glauser. (2004). "Efficacy And Tolerability Of The New Antiepileptic Drugs I: Treatment Of New Onset Epilepsy". Epilepsia. 45 (5): 401-409. 10.1111/j.0013-9580.2004.06204.x.
DOI: https://doi.org/10.1111/j.0013-9580.2004.06204.x[47] T. Granata, L. Fusco, S. Matricardi, A. Tozzo, D. Janigro, and R. Nabbout. (2022). "Inflammation In Pediatric Epilepsies: Update On Clinical Features And Treatment Options". Epilepsy and Behavior. 131 : 107959. 10.1016/j.yebeh.2021.107959.
DOI: https://doi.org/10.1016/j.yebeh.2021.107959[48] E. Perucca, J. French, and M. Bialer. (2007). "Development Of New Antiepileptic Drugs: Challenges Incentives And Recent Advances". The Lancet Neurology. 6 (9): 793-804. 10.1016/S1474-4422(07)70215-6.
DOI: https://doi.org/10.1016/S1474-4422(07)70215-6