Russian Journal of PhysiologyRussian Journal of PhysiologyРоссийский физиологический журнал им. И.М. Сеченова0869-81392658-655XThe Russian Academy of Sciences65175210.31857/S0869813924090134AJDIYLEXPERIMENTAL ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИResearch ArticleChanges in the expression of apoptosis-associated proteins in the temporal cortex and hippocampus of rats during long-term kindling and their correction with minolexinИзменения экспрессии апоптоз-ассоциированных белков в височной коре и гиппокампе крыс при длительном киндлинге и их коррекция с помощью минолексинаBazhanovaE. D.БажановаЕ. Д.
Russian Federation
bazhanovae@mail.ruKozlovA. A.КозловА. А.
Russian Federation
bazhanovae@mail.ruSokolovaYu. O.СоколоваЮ. О.
Russian Federation
bazhanovae@mail.ruSuponinA. A.СупонинА. А.
Russian Federation
bazhanovae@mail.ruDemidovaE. O.ДемидоваЕ. О.
Russian Federation
bazhanovae@mail.ruSechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of SciencesИнститут эволюционной физиологии и биохимии им. И.М. Сеченова Российской академии наукThe Federal State-Financed Institution Golikov Research Clinical Center of Toxicology under the Federal Medical Biological AgencyНаучно-клинический центр токсикологии им. академика С.Н. Голикова Федерального медико-биологического агентстваPavlov First St. Petersburg State Medical University of the Ministry of Healthcare of Russian FederationПервый Санкт-Петербургский государственный медицинский университет им. академика И.П. Павлова Министерства здравоохранения Российской Федерации1509202411091455147402022025Copyright ©; 2024, Russian Academy of SciencesCopyright ©; 2024, Российская академия наук2024Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0869-8139/article/view/651752

Epilepsy is one of the most common and serious diseases of the brain, affecting more than 70 million people worldwide. Available anticonvulsants are able to suppress seizures in two thirds of patients, and in the remaining third of patients, epilepsy is considered drug-resistant and other types of treatment are required, such as surgery, which also does not always lead to positive results. Overcoming resistance is a complex task that requires an understanding of the biochemical pathways and general pathological processes underlying epilepsy, primarily apoptosis. The purpose of this work was to study the effect of the antibiotic minolexin on the levels of apoptosis and the expression of apoptosis-associated molecules (p53, Bcl-2, caspase-3 and caspase-8) in the temporal cortex, underlying white matter and hippocampus of Krushinsky-Molodkina rats with hereditary audiogenic epilepsy with long-term kindling. Materials and methods. We used Krushinsky-Molodkina rats at the age of 11 months, which were subjected to audiogenic stimulation and administered intraperitoneally with 1 ml of saline solution or the second-generation tetracycline series minolexin at a dose of 45 mg/kg, dissolved in saline solution for 14 days. The temporal lobe cortex and underlying white matter, the hippocampus, were examined. Apoptosis levels (TUNEL) and expression of apoptosis-associated proteins (p53, Bcl-2, caspase-3 and -8) were assessed (immunohistochemistry, Western blotting). Results. In Krushinsky-Molodkina rats with hereditary audiogenic epilepsy, an increase in the apoptosis level was shown during long-term kindling. A p53-mediated, but caspase-independent mechanism of apoptosis activation has been identified. When minolexin was administered, an anti-apoptotic and neuroprotective effect was observed in the temporal lobe and hippocampus of rats.

Эпилепсия – одно из наиболее распространенных и одновременно серьезных заболеваний головного мозга, от которого страдают более 70 миллионов человек во всем мире. Имеющиеся противосудорожные препараты способны подавить приступы у двух третей больных, у оставшейся трети пациентов эпилепсия признается фармакорезистентной и требует иных видов лечения, таких как хирургическое вмешательство, которое также не всегда приводит к положительным результатам. Преодоление резистентности – сложная комплексная задача, для решения которой требуется понимание биохимических путей и общих патологических процессов, лежащих в основе эпилепсии, в первую очередь апоптоза. Целью данной работы стало изучение влияния антибиотика минолексина на уровни апоптоза и экспрессию апоптоз-ассоциированных молекул (p53, Bcl-2, каспаза-3 и каспаза-8) в височной коре, подлежащем белом веществе и гиппокампе крыс линии Крушинского – Молодкиной (КМ) с наследственной аудиогенной эпилепсией при длительном киндлинге. Использованы крысы КМ в возрасте 11 месяцев, которых подвергали аудиогенной стимуляции и вводили внутрибрюшинно физраствор или антибиотик тетрациклинового ряда второго поколения минолексин в дозе 45 мг/кг, растворенный в физрастворе, в течение 14 дней, далее следовали 7 дней отдыха, после которых была проведена некропсия. Исследована кора височной доли и подлежащее белое вещество, гиппокамп. Оценивали уровни апоптоза (TUNEL) и экспрессию апоптоз-ассоциированных белков (p53, Bcl-2, каспаза-3 и -8) (иммуногистохимия, вестерн-блоттинг). У крыс линии КМ с наследственной аудиогенной эпилепсией показано повышение уровня апоптоза при длительном киндлинге во всех исследованных областях мозга. Выявлен р53-зависимый, но не зависящий от каспаз механизм активации апоптоза. При введении минолексина наблюдался антиапоптотический и нейропротективный эффект в височной доле и гиппокампе экспериментальных крыс.

epilepsyapoptosisbcl-2p53caspase-3caspase-8minolexinэпилепсияапоптозBcl-2p53каспаза-3каспаза-8минолексинкрысы линии Крушинского-МолодкинойПравительство РФGovernment of the Russian FederationПравительство РФGovernment of the Russian Federation075-00264-24-00Pong A, Xu K, Klein P (2023) Recent advances in pharmacotherapy for epilepsy. Curr Opin Neurol 36: 77–85. https://doi.org/10.1097/WCO.0000000000001144Bazhanova E, Kozlov A (2022) Mechanisms of apoptosis in drug-resistant epilepsy. Zh Nevrol Psikhiatr Im SS Korsakova 122: 43–50. https://doi.org/10.17116/jnevro202212205143Henshall D (2007) Apoptosis signalling pathways in seizure-induced neuronal death and epilepsy. Biochem Soc Trans 35: 421–423. https://doi.org/10.1042/BST0350421Sokolova T, Zabrodskaya Y, Litovchenko A, Paramonova N, Kasumov V, Kravtsova S, Skiteva E, Sitovskaya D, Bazhanova E (2022) Relationship between Neuroglial Apoptosis and Neuroinflammation in the Epileptic Focus of the Brain and in the Blood of Patients with Drug-Resistant Epilepsy. Int J Mol Sci 23: 12561. https://doi.org/10.3390/ijms232012561Henshall D, Simon R (2005) Epilepsy and apoptosis pathways. J Cereb Blood Flow Metab 25: 1557–1572. https://doi.org/10.1038/sj.jcbfm.9600149Li H, Zhang Z, Li H, Pan X, Wang Y (2023) New Insights into the Roles of p53 in Central Nervous System Diseases. Int J Neuropsychopharmacol 26: 465–473. https://doi.org/10.1093/ijnp/pyad030Tsujimoto Y, Finger L, Yunis J, Nowell P, Croce C (1984) Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 226: 1097–1099. https://doi.org/10.1126/science.6093263Hollville E, Romero S, Deshmukh M (2019) Apoptotic cell death regulation in neurons. FEBS J 286: 3276–3298. https://doi.org/10.1111/febs.14970Vega-García A, Orozco-Suárez S, Villa A, Rocha L, Feria-Romero I, Alonso Vanegas M, Guevara-Guzmán R (2021) Cortical expression of IL1-β, Bcl-2, Caspase-3 and 9, SEMA-3a, NT-3 and P-glycoprotein as biological markers of intrinsic severity in drug-resistant temporal lobe epilepsy. Brain Res 1758: 147303. https://doi.org/10.1016/10.1016/j.brainres.2021.147303Eskandari E, Eaves C (2022) Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol 221: e202201159. https://doi.org/10.1083/jcb.202201159D'Amelio M, Cavallucci V, Cecconi F (2010) Neuronal caspase-3 signaling: not only cell death. Cell Death Differ 17: 1104–1114. https://doi.org/10.1007/s12264-012-1057-5Fritsch M, Günther S, Schwarzer R, Albert M-C, Schorn F, Werthenbach J, Schiffmann L, Stair N, Stocks H, Seeger J, Lamkanfi M, Krönke M, Pasparakis M, Kashkar H (2019) Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature 575: 683–687. https://doi.org/10.1038/s41586-019-1770-6Galvis-Alonso O, Cortes De Oliveira J, Garcia-Cairasco N (2004) Limbic epileptogenicity, cell loss and axonal reorganization induced by audiogenic and amygdala kindling in wistar audiogenic rats (WAR strain). Neuroscience 125: 787–802. https://doi.org/10.1016/j.neuroscience.2004.01.042Abarrategui B, Mai R, Sartori I, Francione S, Pelliccia V, Cossu M, Tassi L (2021) Temporal lobe epilepsy: A never-ending story. Epilepsy Behav 122: 108122. https://doi.org/10.1016/j.yebeh.2021.108122Henning O, Heuser K, Larsen V, Kyte E, Kostov H, Marthinsen P, Egge A, Alfstad K, Nakken K (2023) Temporal lobe epilepsy. Tidsskr Nor Laegeforen 143. https://doi.org/10.4045/tidsskr.22.0369Jonas M, Cunha B (1982) Minocycline. Ther Drug Monit 4: 137–145.Singh T, Thapliyal S, Bhatia S, Singh V, Singh M, Singh H, Kumar A, Mishra A (2022) Reconnoitering the transformative journey of minocycline from an antibiotic to an antiepileptic drug. Life Sci 15: 120346. https://doi.org/10.1016/j.lfs.2022.120346Костюкова АБ, Мосолов СН (2013) Нейровоспалительная гипотеза шизофрении и некоторые новые терапевтические подходы. Московский НИИ психиатрии Минздрава РФ 4: 8–17. [Kostyukova AB, Mosolov CH (2013) Neuroinflammatory hypothesis of schizophrenia and new therapeutical approaches. Moscow Res Institute of Psychiatry Minzdrava Rossii 4: 8–17. (In Russ)].Singh T, Thapliyal S, Bhatia S, Singh V, Singh M, Singh H, Kumar A, Mishra A (2022) Reconnoitering the transformative journey of minocycline from an antibiotic to an antiepileptic drug. Life Sci 15: 120346. https://doi.org/10.1016/j.lfs.2022.120346Wang N, Mi X, Gao B, Gu J, Wang W, Zhang Y, Wang X (2015) Minocycline inhibits brain inflammation and attenuates spontaneous recurrent seizures following pilocarpine-induced status epilepticus. Neuroscience 287: 144–156. https://doi.org/10.1016/j.neuroscience.2014.12.021Nagarakanti S, Bishburg E (2016) Is Minocycline an Antiviral Agent? A Review of Current Literature. Basic Clin Pharmacol Toxicol 118: 4–8. https://doi.org/10.1111/bcpt.12444Singh S, Khanna D, Kalra S (2021) Minocycline and Doxycycline: More Than Antibiotics. Curr Mol Pharmacol 14: 1046–1065. https://doi.org/10.2174/1874467214666210210122628.Бажанова Е, Козлов А, Соколова Ю (2023) Этиопатологические механизмы эпилепсии и сравнительная характеристика экспериментальных моделей аудиогенной эпилепсии. Эпилепсия и пароксизмальн состояния 15: 372–383. [Bazhanova E, Kozlov A, Sokolova J (2023) Etiopathological mechanisms of epilepsy and comparative characteristics of experimental models of audiogenic epilepsy. Epilepsiya i paroksizmal'nye sostoyaniya 15: 372–383. (In Russ)]. https://doi.org/10.17749/2077-8333/epi.par.con.2023.161Горбачева ЕЛ, Куликов АА, Черниговская ЕВ, Глазова МВ, Никитина ЛС (2019) Особенности функционального состояния гипоталама-гипофизарно-адренокортикальной системы у крыс линии Крушинского – Молодкиной. Рос физиол журн им ИМ Сеченова 105: 150–164. [Gorbacheva EL, Kulikova АА, Chernigovskayaa EV, Glazovaa MV, Nikitina LS (2019) Functional State of the Hypothalamic-Pituitary-Adrenal Axis in Krushinsky–Molodkina Rats. Russ J Physiol 105: 150–164. (In Russ)]. https://doi.org/10.1134/S0869813919020043Paxinos G, Watson C (1998). The rat brain in stereotaxic coordinates. Academic Press 273.Dengler C, Coulter D (2016) Normal and epilepsy-associated pathologic function of the dentate gyrus. Prog Brain Res 226: 155–178. https://doi.org/10.1016/bs.pbr.2016.04.005Nasr S, Moghimi A, Mohammad-Zadeh M, Shamsizadeh A, Noorbakhsh S (2013) The effect of minocycline on seizures induced by amygdala kindling in rats. Seizure 22: 670–674. https://doi.org/10.1016/j.seizure.2013.05.005Nowak M, Strzelczyk A, Reif P, Schorlemmer K, Bauer S, Norwood B, Oertel W, Rosenow F, Strik H, Hamer H (2012). Minocycline as potent anticonvulsant in a patient with astrocytoma and drug resistant epilepsy. Seizure 21: 227–228. https://doi.org/10.1016/j.seizure.2011.12.009Querol Pascual M (2007) Temporal lobe epilepsy: clinical semiology and neurophysiological studies.b Semin Ultrasound CT MR28: 416–423. https://doi.org/10.1053/j.sult.2007.09.004Alhusaini S, Whelan C, Doherty C, Delanty N, Fitzsimons M, Cavalleri G (2016) Temporal Cortex Morphology in Mesial Temporal Lobe Epilepsy Patients and Their Asymptomatic Siblings. Cereb Cortex 26: 1234–1241. https://doi.org/10.1093/cercor/bhu315Mizutani M, Sone D, Sano T, Kimura Y, Maikusa N, Shigemoto Y, Goto Y, Takao M, Iwasaki M, Matsuda H, Sato N, Saito Y (2021) Histopathological validation and clinical correlates of hippocampal subfield volumetry based on T2-weighted MRI in temporal lobe epilepsy with hippocampal sclerosis. Epilepsy Res 177: 106759. https://doi.org/10.1016/j.eplepsyres.2021.106759Schmitt A, Tatsch L, Vollhardt A, Schneider-Axmann T, Raabe F, Roell L, Heinsen H, Hof P, Falkai P, Schmitz C (2022) Decreased Oligodendrocyte Number in Hippocampal Subfield CA4 in Schizophrenia: A Replication Study. Cells 11: 3242. https://doi.org/10.3390/cells11203242Wang Y, Tian Y, Long Z, Dong D, He Q, Qiu J, Feng T, Chen H, Tahmasian M, Lei X (2024) Volume of the Dentate Gyrus/CA4 Hippocampal subfield mediates the interplay between sleep quality and depressive symptoms. Int J Clin Health Psychol 24: 100432. https://doi.org/10.1016/j.ijchp.2023.100432Shahid S, Wen Q, Risacher S, Farlow M, Unverzagt F, Apostolova L, Foroud T, Zetterberg H, Blennow K, Saykin A, Wu Y (2022) Hippocampal-subfield microstructures and their relation to plasma biomarkers in Alzheimer's disease. Brain 145: 2149–2160. https://doi.org/10.1093/brain/awac138Stirling D, Koochesfahani K, Steeves J, Tetzlaff W (2005) Minocycline as a neuroprotective agent. Neuroscientist 11: 308–322. https://doi.org/10.1177/1073858405275175.Naderi Y, Panahi Y, Barreto G, Sahebkar A (2020) Neuroprotective effects of minocycline on focal cerebral ischemia injury: a systematic review. Neural Regen Res 15: 773–782. https://doi.org/10.4103/1673-5374.268898He J, Mao J, Hou L, Jin S, Wang X, Ding Z, Jin Z, Guo H, Dai R (2021) Minocycline attenuates neuronal apoptosis and improves motor function after traumatic brain injury in rats. Exp Anim 70: 563–569. https://doi.org/10.1538/expanim.21-0028Li J, Chen J, Mo H, Chen J, Qian C, Yan F, Chi G, Hu Q, Wang L, Chen G (2016) Minocycline Protects Against NLRP3 Inflammasome-Induced Inflammation and P53-Associated Apoptosis in Early Brain Injury After Subarachnoid Hemorrhage. Mol Neurobiol 53: 2668–2678. https://doi.org/10.1007/s12035-015-9318-8Kelly K, Sutton T, Weathered N, Ray N, Caldwell E, Plotkin Z, Dagher P (2004) Minocycline inhibits apoptosis and inflammation in a rat model of ischemic renal injury. Am J Physiol Renal Physiol 287: F760–F766. https://doi.org/10.1152/ajprenal.00050.2004Xiong G, Hu T, Yang Y, Zhang H, Han M, Wang J, Jing Y, Liu H, Liao X, Liu Y (2024) Minocycline attenuates the bilirubin-induced developmental neurotoxicity through the regulation of innate immunity and oxidative stress in zebrafish embryos. Toxicol Appl Pharmacol 484: 116859. https://doi.org/10.1016/j.taap.2024.116859Ataie-Kachoie P, Pourgholami M, Bahrami-B F, Badar S, Morris D (2015) Minocycline attenuates hypoxia-inducible factor-1α expression correlated with modulation of p53 and AKT/mTOR/p70S6K/4E-BP1 pathway in ovarian cancer: in vitro and in vivo studies. Am J Cancer Res 5: 575–588.Roshanaee M, Abtahi-Eivary S, Shokoohi M, Fani M, Mahmoudian A, Moghimian M (2022) Protective Effect of Minocycline on Bax and Bcl-2 Gene Expression, Histological Damages and Oxidative Stress Induced by Ovarian Torsion in Adult Rats. Int J Fertil Steril 16: 30–35. https://doi.org/10.22074/IJFS.2021.522550.1069Levkovitch-Verbin H, Waserzoog Y, Vander S, Makarovsky D, Ilia P (2014) Minocycline mechanism of neuroprotection involves the Bcl-2 gene family in optic nerve transection. Int J Neurosci 124: 755–761. https://doi.org/10.3109/00207454.2013.878340Shokoohi M, Khaki A, Abadi A, Boukani L, Khodaie S, Kalarestaghi H, Khaki A, Moghimian M, Niazkar H, Shoorei H (2022) Minocycline can reduce testicular apoptosis related to varicocele in male rats. Andrologia 54: e14375. https://doi.org/10.1111/and.14375Rezaei A, Moqadami A, Khalaj-Kondori M, Feizi M (2024) Minocycline induced apoptosis and suppressed expression of matrix metalloproteinases 2 and 9 in the breast cancer MCF-7 cells. Mol Biol Rep 51: 463. https://doi.org/10.1007/s11033-024-09380-1Candé C, Cohen I, Daugas E, Ravagnan L, Larochette N, Zamzami N, Kroemer G (2002) Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria. Biochimie 84: 215–222. https://doi.org/10.1016/s0300-9084(02)01374-3Luo Q, Wu X, Zhao P, Nan Y, Chang W, Zhu X, Su D, Liu Z (2021) OTUD1 Activates Caspase-Independent and Caspase-Dependent Apoptosis by Promoting AIF Nuclear Translocation and MCL1 Degradation. Adv Sci (Weinh) 8: 2002874. https://doi.org/10.1002/advs.202002874Chang C-J, Cherng C-H, Liou W-S, Liao C-L (2005) Minocycline partially inhibits caspase-3 activation and photoreceptor degeneration after photic injury. Ophthalmic 37: 202–213. https://doi.org/10.1159/000086610Krady J, Basu A, Allen C, Xu Y, LaNoue K, Gardner T, Levison S (2005) Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy. Diabetes 54: 1559–1565. https://doi.org/10.2337/diabetes.54.5.1559Chen M, Ona V, Li M, Ferrante R, Fink K, Zhu S, Bian J, Guo L, Farrell L, Hersch S, Hobbs W, Vonsattel J, Cha J, Friedlander R (2000) Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med 6: 797–801. https://doi.org/10.1038/77528Wei X, Zhao L, Liu J, Dodel R, Farlow M, Du Y (2005) Minocycline prevents gentamicin-induced ototoxicity by inhibiting p38 MAP kinase phosphorylation and caspase 3 activation. Neuroscience 131: 513–521. https://doi.org/10.1016/j.neuroscience.2004.11.014Mishra M, Basu A (2008) Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis. J Neurochem 105: 1582–1595. https://doi.org/10.1111/j.1471-4159.2008.05238.xHirsch E, Breidert T, Rousselet E, Hunot S, Hartmann A, Michel P (2003) The role of glial reaction and inflammation in Parkinson's disease. Ann NY Acad Sci 991: 214–228. https://doi.org/10.1111/j.1749-6632.2003.tb07478.x