Ascorbic acid-containing compound efficacy in ischemic brain damage

Rita M. Termulaeva; Konstantin Y. Belanov; Natalya D. Bunyatyan; Aleksander S. Pirozhkov; Dmitrii E. Timoshkin; Ekaterina V. Blinova; Olga V. Vasilkina; Kirill D. Blinov; Elena V. Semeleva; Aleksander A. Dmitriev; Dmitrii S. Blinov

doi:10.18413/rrpharmacology.10.508

Authors

Rita M. Termulaeva Kadyrov Chechen State University https://orcid.org/0000-0001-5300-5700
Konstantin Y. Belanov Belgorod State National Research University https://orcid.org/0000-0003-0936-5551
Natalya D. Bunyatyan Belgorod State National Research University https://orcid.org/0000-0001-9466-1261
Aleksander S. Pirozhkov N.P. Ogarev National Research Mordovia State University https://orcid.org/0000-0003-1895-5342
Dmitrii E. Timoshkin Sechenov University https://orcid.org/0000-0002-1211-1096
Ekaterina V. Blinova Sechenov University https://orcid.org/0000-0003-0050-0251
Olga V. Vasilkina National Research Nuclear University MEPHI https://orcid.org/0000-0003-0881-8002
Kirill D. Blinov Sechenov University https://orcid.org/0009-0002-7195-2191
Elena V. Semeleva N.P. Ogarev National Research Mordovia State University https://orcid.org/0000-0001-6692-4968
Aleksander A. Dmitriev N.P. Ogarev National Research Mordovia State University, https://orcid.org/0009-0004-5106-6314
Dmitrii S. Blinov Dmitry Rogachev National Medical Research Center of Pediatric Hematology https://orcid.org/0000-0002-8385-4356

DOI:

https://doi.org/10.18413/rrpharmacology.10.508

Abstract

Introduction: Ischemic brain injury remains one of the main causes of disability and mortality worldwide. Protection of cellular population, depriving from oxygen supply and nutrients, is of extreme importance for further both clinical and health outcomes of timely implemented intravascular intervention. The aim: to assess anti-ischemic activity of 3-hydroxypyridine ascorbate in the in vitro and in vivo models of brain cell and tissue response to ischemia and reoxygenation.

Materials and Methods: 3-hydroxypyridine ascorbate (laboratory code 3-EA) was assessed as chemical substance (purity 99.8%) diluted in sterile phosphate-buffered saline. Intracellular Ca²⁺ response to glutamate excitotoxicity (GluTox), ischemia and reoxygenation as well as cellular viability was evaluated on NMRI murine fresh cortical neuro-glial cell culture incubated with 2-EA by registering intracellular Fura-2 and propidium iodide fluorescence respectively. Expression of apoptosis regulating genes BCL-2, STAT3, SOCS3, inflammation regulating genes TRAIL, MLKL, Cas-1, Cas-3, IL-1β и TNFα, and genes MAO-A and MAO-B was determined by real-time PCR. The substance neuroprotection was studied in male Sprague-Dawley rats with intraluminal middle cerebral artery (MCA) occlusion/reperfusion treated with 18 mg/kg of 2-EA along with neurological deficiency evaluation and morphological assessment of brain sections.

Results: Preincubation of cortical cells with 10-100 μM of 3-EA leads to inhibition of [Ca²⁺]i in cytosol of neurons and astrocytes under GluTox and oxygen-glucose deprivation (OGD) conditions. Reducing [Ca²⁺]i inhibits necrotic cell death in an acute experiment. Incubation of cerebral cortex cells with 3-EA leads to an overexpression of anti-apoptotic genes BCL-2, STAT3, SOCS3, along with downregulation of genes TRAIL, MLKL, Cas-1, Cas-3, IL-1β and TNFα. Intraperitoneal administration of 3-EA reduces the volume of necrotic areas, perinecrotic edema, cell damage, and neurological deficits in rats with MCA occlusion.

Conclusion: 3-EA dose-dependently suppresses the death of cerebral cortex cells under the excitotoxic effects of glutamate and ischemia/reoxygenation. Cell-protective effect of 3-EA involves changes in the basal and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and oxidative status proteins, which leads to inhibition of the late irreversible stages of apoptosis. A course administration of 3-hydroxypyridine ascorbate at a dose of 18 mg/kg per day reduces the severity of damage both by preserving the population of neurons in the penumbra zone and by limiting the local stress response.

Graphical Abstract

Keywords:

3-hydroxypyridine ascorbate (3-EA), ischemia, brain damage, cell culture, excitotoxicity, Ca2 , cerebral artery occlusion, neurological deficits

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Author Contribution

Rita M. Termulaeva, Kadyrov Chechen State University

M.D., Cand. Sc. (Med), Associate Professor of the Department of Pharmacology, Kadyrov Chechen State University, Grozny, Russia; e-mail: ritaterm@mail.ru; ORCID ID https://orcid.org/0000-0001-5300-5700. Individual contributions: carrying out experiments, data validation, and writing the manuscript.

Konstantin Y. Belanov, Belgorod State National Research University

M.D., postgraduate student of the Department of General and Clinical Pharmacology, Belgorod State National Research University, Belgorod, Russia; e-mail: belanov.konstantin@yandex.ru; ORCID ID https://orcid.org/0000-0003-0936-5551. Individual contributions: methodology, data processing, and writing the manuscript.

Natalya D. Bunyatyan, Belgorod State National Research University

D. Pharm. Sc, Professor, Department of General and Clinical Pharmacology, Belgorod State National Research University, Belgorod, Russia; Lead researcher of the Center for Clinical Pharmacology; Research Advisor of Scientific Centre for Expert Evaluation of Medicinal Products. Moscow, Russia; e-mail: ndbun@mail.ru; ORCID ID https://orcid.org/0000-0001-9466-1261. Individual contributions: conceptualization of the study and data validation.

Aleksander S. Pirozhkov, N.P. Ogarev National Research Mordovia State University

M.D., postgraduate student of the Department of Public Health and Healcare, N.P. Ogarev National Research Mordovia State University, Saransk, Russia; e-mail: pirozhkov1996@yandex.ru; ORCID ID https://orcid.org/0000-0003-1895-5342. Individual contributions: carrying out experiments and writing the manuscript.

Dmitrii E. Timoshkin, Sechenov University

M.D., Cand. Sc. (Med), Associate Professor of the Department of Operative Surgery and Topographical Anatomy, Sechenov University, Moscow, Russia; e-mail: Dmtimo@mail.ru; ORCID ID https://orcid.org/0000-0002-1211-1096. Individual contributions: visualisation and writing the manuscript.

Ekaterina V. Blinova, Sechenov University

M.D., D. Med. Sc, Professor, Department of Clinical Pharmacology and Internal Diseases Propaedeutic, Sechenov University, Head of the Department of Basic Medicine, National Research Nuclear University MEPHI, Moscow, Russia; e-mail: bev-sechenov@mail.ru; ORCID ID https://orcid.org/0000-0003-0050-0251. Individual contributions: conceptualization of the study and data validation.

Olga V. Vasilkina, National Research Nuclear University MEPHI

M.D., Cand. Sc. (Med), Associate Professor of the Department of Basic Medicine, National Research Nuclear University MEPHI, Moscow, Russia; e-mail: ms.vasilkina@bk.ru; ORCID ID https://orcid.org/0000-0003-0881-8002. Individual contributions: morphological part of experiments and writing the manuscript.

Kirill D. Blinov, Sechenov University

Graduate student, Sechenov University, Moscow, Russia; e-mail: pyrk2@yandex.ru; ORCID ID https://orcid.org/0009-0002-7195-2191. Individual contributions: carrying out experiments and writing the manuscript.

Elena V. Semeleva, N.P. Ogarev National Research Mordovia State University

M.D., D. Med. Sc, Associate Professor, Head of the Department of Public Health and Healcare, N.P. Ogarev National Research Mordovia State University, Saransk, Russia; e-mail: shtanina37@mail.ru; ORCID ID https://orcid.org/0000-0001-6692-4968. Individual contributions: carrying out experiments and writing the manuscript.

Aleksander A. Dmitriev, N.P. Ogarev National Research Mordovia State University,

M.D., postgraduate student of the Department of Public Health and Healcare, N.P. Ogarev National Research Mordovia State University, Saransk, Russia; e-mail: Xandr_dent@mail.ru; ORCID ID https://orcid.org/0009-0004-5106-6314. Individual contributions: carrying out experiments and writing the manuscript.

Dmitrii S. Blinov, Dmitry Rogachev National Medical Research Center of Pediatric Hematology

M.D., D. Med. Sc, Head of the Department of Molecular and Clinical Pharmacology, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia; e-mail: blinov-pharm@yandex.ru; ORCID ID https://orcid.org/0000-0002-8385-4356. Individual contributions: conceptualization of the study, data validation, statistical analysis, and manuscript draft review.