SIÇAN PRİMER KORTEKS NÖRON KÜLTÜRÜNDE GLUTAMAT TOKSİSİTESİNE KARŞI KLORZOKSAZONUN NÖROPROTEKTİF ETKİSİ

Öz

Glutamat (Glut) toksisitesi, nörolojik hastalıklara zemin hazırlayan ana sebeplerden biridir. Klorzoksazon (CZ), akut ve kronik morluklar ve burkulmalarla ilişkili ağrı ve iltihabı azaltmak için kullanılan kas gevşeticidir. Burada, yenidoğan serebral kortekste Glut'un neden olduğu nörodejenerasyonu tersine çevirmek için uygulanan CZ'nin nöroprotektif etkisini anti-inflamatuar ve antioksidan mekanizmalar yoluyla araştırmayı amaçladık. Yenidoğan kortikal nöronları Glut’a maruz bırakıldı ve CZ’nin Glut toksisitesi üzerindeki etkisini değerlendirmek için CZ çeşitli dozlarda (10, 20 ve 40 µM) uygulandı. Ardından hücre canlılığı, oksidatif stres ve inflamasyondaki değişiklikleri inceledik. Hücre canlılık analizimiz, CZ’nin Glut kaynaklı nöranal hasardan hücreleri koruduğunu göstermiştir. Ayrıca CZ’nin nöroprotektif özelliği MDA, MPO, CAT, GSH, GPx ve SOD gibi oksidatif ve antioksidan parametrelerin incelenmesi ile değerlendirildi. Elde edilen veriler doğrultusunda,hücre canlılık oranı Glut grubunda % 60'a kadar düştüğü gözlendi. Ancak CZ uygulaması ile birlikte hücre canlılığında en anlamlı artış 40 μM dozunda (%86) görülürken, en az artış 10 μM CZ (%77) görüldü. Ayrıca CZ'nin oksidatif parametreleri ve inflamasyonu azaltırken, antioksidan parametrelerin aktivitesini arttırdığını kanıtladı. Bu nedenle mevcut bulgular toplu olarak CZ'nin Glut kaynaklı hasarı yenidoğan kortikal nöronlarında güçlü bir şekilde önlediğini göstermiştir. Mevcut çalışma, Glut eksitotoksisitesine maruz kalan yenidoğan korteks nöronlarında CZ’nin koruyucu etkisini gösteren ilk çalışmadır ve CZ’nin terapötik bir ajan olarak kullanılabileceğini göstermektedir.

Anahtar Kelimeler

• Klorzoksazon
• korteks
• glutamat
• nöron

NEUROPROTECTIVE EFFECT OF CHLORZOXAZONE AGAINST GLUTAMATE TOXICITY IN RAT PRIMARY CORTEX NEURON CULTURE

Öz

Glutamate (Glut) toxicity is one of the main causes of neurological diseases. Chlorzoxazone (CZ) is a muscle relaxant used to decrease pain and inflammation associated with acute and chronic twists and bruises. Here, we objected to research the neuroprotective effect of CZ applied to reverse Glut-induced neurodegeneration in the neonatal cerebral cortex through anti-inflammatory and antioxidant mechanisms. Neonatal cortical neurons were exposed to Glut and different doses of CZ (10, 20, and 40 µM) were applied to assess the effect of CZ on Glut toxicity. We then examined changes in cell viability, inflammation, and oxidative stress. Our cell viability analysis showed that CZ protected cells from Glut-induced neuronal damage. In addition, the neuroprotective properties of CZ were evaluated by examining oxidative and antioxidant parameters such as MDA, MPO, CAT, GSH, GPx, and SOD. In line with the data obtained, it was observed that the cell viability rate decreased to 60% in the Glut group. However, with CZ application, the most significant increase in cell viability was seen at the 40 μM dose (86%), while the least increase was seen at 10 μM CZ (77%). It also proved that CZ increased the activity of antioxidant parameters while reducing oxidative parameters and inflammation. Therefore, the present findings collectively demonstrated that CZ potently inhibits Glut-induced injury in neonatal cortical neurons. The present work is the initial to show the protective effect of CZ in neonatal cortical neurons exposed to Glut excitotoxicity and suggesting that CZ may be used as a therapeutic agent.

Anahtar Kelimeler

• Chlorzoxazone
• cortex
• glutamate
• neuron

Kaynakça

1. Hartmann P, Ramseier A, Gudat F, Mihatsch MJ, Polasek W. Normal weight of the brain in adults in relation to age, sex, body height and weight. Pathologe. 1994;15(3):165-170. doi:10.1007/s002920050040.
2. Zhou Y, Danbolt NC. Glutamate as a neurotransmitter in the healthy brain. J Neural Transm (Vienna). 2014;121(8):799-817. doi:10.1007/s00702-014-1180-8.
3. De Pittà M, Brunel N. Modulation of synaptic plasticity by glutamatergic gliotransmission: a modeling study. Neural Plast. 2016;2016:7607924. doi:10.1155/2016/7607924.
4. Baj A, Moro E, Bistoletti M, Orlandi V, Crema F, Giaroni C. Glutamatergic signaling along the microbiota-gut-brain axis. Int J Mol Sci. 2019;20(6):1482. doi:10.3390/ijms20061482.
5. Duman RS, Sanacora G, Krystal JH. Altered connectivity in depression: GABA and glutamate neurotransmitter deficits and reversal by novel treatments. Neuron. 2019;102(1):75-90. doi:10.1016/j.neuron.2019.03.013.
6. Marvin JS, Scholl B, Wilson DE, et al. Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat Methods. 2018;15(11):936–939. doi:10.1038/s41592-018-0171-3.
7. Haroon E, Chen X, Li Z, et al. Increased inflammation and brain glutamate define a subtype of depression with decreased regional homogeneity, impaired network integrity, and anhedonia. Transl Psychiatry. 2018;8(1):189. doi:10.1038/s41398-018-0241-4.
8. Boyko M, Gruenbaum SE, Gruenbaum BF, Shapira Y, Zlotnik A. Brain to blood glutamate scavenging as a novel therapeutic modality: a review. J Neural Transm (Vienna). 2014;121(8):971-979. doi:10.1007/s00702-014-1181-7.

9. Pinky NF, Wilkie CM, Barnes JR, Parsons MP. Region- and activity-dependent regulation of extracellular glutamate. J Neurosci. 2018;38(23):5351-5366. doi:10.1523/JNEUROSCI.3213-17.2018.
10. Marcos JL, Galleguillos D, Pelissier T,et al. Role of the spinal TrkB-NMDA receptor link in the BDNF-ınduced long-lasting mechanical hyperalgesia in the rat: a behavioural study. Eur J Pain. 2017;21:1688-1696. doi:10.1002/ejp.1075.
11. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev. 2010;4(8):118-126. doi:10.4103/0973-7847.70902.
12. Lopez-Alarcona C, Denicola A. Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays. Anal Chim Acta. 2013;763:1e10. doi:10.1016/j.aca.2012.11. 051.
13. Pisoschi AM, Pop A. The role of antioxidants in the chemistry of oxidative stress: A review. Eur J Med Chem.2015;97:55-74. doi:10.1016/j.ejmech.2015. 04.040.
14. Hohmann N, Blank A, Burhenne J, Suzuki Y, Mikus G, Haefeli WE. Simultaneous phenotyping of CYP2E1 and CYP3A using oral chlorzoxazone and midazolam microdoses. Br J Clin Pharmacol. 2019;85(10):2310-2320. doi:10.1111/bcp.14040.
15. Ceccarelli SM, Jaeschke G, Buettelmann B,et al. Rational design, synthesis, and structure-activity relationship of benzoxazolones: new potent mglu5 receptor antagonists based on the fenobam structure. Bioorg Med Chem Lett. 2007;17:1302-1306. doi:10.1016/j.bmcl.2006.12.006.
16. Quesnot N, Bucher S, Gade C,et al. Production of chlorzoxazone glucuronides via cytochrome P4502E1 dependent and independent pathways in human hepatocytes. Arch Toxicol. 2018;92(10):3077-3091. doi:10.1007/s00204-018-2300-2.
17. Upadhya SC, Tirumalai PS, Boyd MR, Mori T, Ravindranath V. Cytochrome P4502E (CYP2E) in brain: constitutive expression, induction by ethanol and localization by fluorescence in situ hybridization. Arch Biochem Biophys. 2000;373(1):23-34. doi: 10.1006/abbi.1999.1477.
18. Bai Y, Ma X. Chlorzoxazone exhibits neuroprotection against Alzheimer's disease by attenuating neuroinflammation and neurodegeneration in vitro and in vivo. Int Immunopharmacol. 2020;88:106790. doi:10.1016/j.intimp.2020.106790.
19. Gündoğdu G, Tagh İzadehghalehjoughi A, Çiçek B, et al. Investigation of protective effect of parietin against glutamate excitotoxicity in primary cortical neuron culture. Atatürk Üniversitesi Vet Bil Derg. 2018;13(2):165-173. doi:10.17094/ataunivbd.363 858.
20. Kumar P, Nagarajan A, Uchil PD. Analysis of Cell Viability by the MTT Assay. Cold Spring Harb Protoc. 2018;2018(6). doi:10.1101/pdb.prot095505.
21. Kumari S, Mehta SL, Li PA. Glutamate ınduces mitochondrial dynamic ımbalance and autophagy activation: preventive effects of selenium. PLoS One. 2012;7(6):e39382. doi:10.1371/journal.pone.0039 382.
22. Tansey MG, McCoy MK, Frank-Cannon TC. Neuroinflammatory mechanisms in Parkinson's disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp Neurol. 2007;208(1):1–25. doi:10.1016/j.expneurol.2007.07.004
23. Hirsch EC, Breidert T, Rousselet E, Hunot S, Hartmann A, Michel PP. The role of glial reaction and inflammation in Parkinson's disease. Ann NY Acad Sci. 2003;991:214-228. doi:10.1111/j.1749-6632.2003.tb07478.x.
24. Sánchez-Pernaute R, Ferree A, Cooper O, Yu M, Brownell LA, Isacson O. Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease. J Neuroinflammation. 2004;17(1):6. doi:10.1186/ 1742-2094-1-6.
25. Jose LM, Olivenza M, Moro MA,et al. Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain. Neuropsychopharmacology. 2001;24(4):420-429. doi:10.1016/S0893-133X(00)00208-6.
26. Yeni Y, Taghizadehghalehjoughi A, Genc S, Hacimuftuoglu A, Yildirim S, Bolat I. Glioblastoma cell-derived exosomes induce cell death and oxidative stress in primary cultures of olfactory neurons. Role of redox stress. Mol Biol Rep. 2023;50(5):3999-4009. doi:10.1007/s11033-023-08256-0.
27. Genc S, Pennisi M, Yeni Y, et al. Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion.Antioxidants (Basel). 2022;11(7):1225. doi:10.3390/antiox110 71225.
28. Saravanan KS, Sindhu KM, Mohanakumar K. Acute ıntranigral ınfusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson's disease. Brain Res. 2005;1049:147-155. doi:10.1016/j.brainres.2005. 04.051.
29. Madathil SK, Karuppagounder SS, Mohanakumar KP. Sodium salicylate protects against rotenone-ınduced parkinsonism in rats. Synapse. 2013;67:502-514. doi:10.1002/syn.21658.