03. Науково-дослідний інститут експериментальної та клінічної медицини ХНМУ
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Item Human HMGB1 does not induce eryptosis in vitro(2022) Tkachenko, Maryna; Onishchenko, Anatolii; Butov, Dmytro; Butova, Tetyana; Tkachenko, AntonAim: To study the ability of human high mobility group box protein 1 (HMGB1) to induce eryptosis in vitro. Material and methods: Blood collected from six healthy volunteers was incubated with HMGB1 (0-0.2-1-5 ng per ml). Eryptosis of red blood cells was assessed by Annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining by flow cytometry. The forward scatter (FSC) fluorescence was used to evaluate the morphology of red blood cells. Results: Exposure of erythrocytes to HMGB1 did not affect the morphology of erythrocytes, evidenced by no changes in the percentage of cells with small volume, i.e. shrunken cells, and erythrocytes with large volume, i.e. enlarged cells. HMGB1 had no impact on phosphatidylserine externalization, which is confirmed by the absence of statistically significant changes in the amount of phosphatidylserine-displaying cells and the mean fluorescence intensity (MFI) values of Annexin V-FITC in cells exposed to different concentrations of HMGB1. Furthermore, H2DCFDA staining revealed that the HMGB1 did not induce oxidative stress. Conclusion: HMGB1 does not promote eryptosis of human erythrocytes at concentrations of up to 5 ng/ml.Item Semi-refined carrageenan induces eryptosis in a Ca2+-dependent manner(2022) Tkachenko, Anton; Prokopiuk, Volodymyr; Onishchenko, AnatoliiBackground: Semi-refined carrageenan (food additive E407a) is a widely used thickener, which has been reported to exert toxic and pro-inflammatory effects. In particular, there is accumulating evidence that it induces eryptosis, i.e. a programmed cell death of eryptocytes, via ROS-mediated pathways. However, the role of Ca2+-dependent mechanisms in E407a-induced eryptosis is not elucidated. Material and methods: Semi-refined carrageenan at concentrations of 0 mg/ml, 1 mg/ml, 5 mg/ml and 10 mg/ml was incubated with blood of intact female WAG rats (n=9) for 24 h in RPMI and fetal bovine serum. After 24 h, the samples were used to obtain erythrocyte suspensions. The obtained suspensions were stained with a Ca2+-sensitive FLUO4 AM probe (30 min, 2.5 µM). The fluorescence of FLUO4 in erythrocytes was detected by a BD FACSCanto II flow cytometer. Results: The intracellular Ca2+ levels are proportional to the fluorescence of FLUO4. The mean fluorescence intensities (MFI) were compared. Low levels (1 mg/ml) of E407a had no impact on Ca2+ concentrations in erythrocytes (p>0.05). On the contrary, high concentrations (5 mg/ml and 10 mg/ml) of this food additive promoted an increase in the intracellular Ca2+ levels. The MFI values were 2.3- and 2.5-fold higher, respectively (p<0.0001). In addition, the exposure to E407a at concentrations of 5 mg/ml and 10 mg/ml (p<0.0001) increased the percentage of cells with high FLUO4 fluorescence. Conclusion: Food additive E407a induces eryptosis in a Ca2+-dependent manner.Item Current issues of resistant tuberculosis and smoking (review)(2022) Butova, Tetiana; Tkachenko, Anton; Butov, DmytroTuberculosis and smoking are among the most pressing issues in the modern health care system in the world. Tuberculosis patients who smoke are confirmed to have reduced the effectiveness of anti-tuberculosis therapy, unsatisfactory prognosis of a specific disease, higher risk of treatment failure and mortality from this disease. Diseases such as HIV and alcoholism increase ineffective treatment in tobacco smokers. In addition, smoking leads to increased virulence of the causative agent of tuberculosis - Mycobacterium tuberculosis.Item Intake of semi-refined carrageenan causes low-grade colonic inflammation and alters expression of epithelial-mesenchymal transition markers(2022) Onishchenko, Anatolii; Gubina-Vakulyck, Galina; Knigavko, Oleksandr; Sharashydze, Ketino; Pionova, Olena; Butov, Dmytro; Polikarpova, Hanna; Tkachenko, AntonThe aim of this research was to evaluate the effects of semi-refined carrageenan consumed orally on the amount of CD3 and CD68 positive cells residing in the colon and the expression of endothelial-mesenchymal transition (EMT) markers. Materials and methods. To assess colonic expression of CD3+, CD68+, fascin, vimentin and E-cadherin, sections obtained from 8 rats orally exposed to semi-refined carrageenan (E407a) at a dose of 140 mg / kg of weight during 14 consecutive days and 8 control animals were immunostained with the corresponding antibodies. Levels of expression were assessed quantitatively. Results. Oral exposure to semi-refined carrageenan resulted in an increase in CD3 and CD68 positive cells in the colonic lamina propria. Quantitative analysis of fascin, vimentin and E-cadherin immunostaining revealed changes in expression of these EMT markers both in the colonic stroma and epithelial cells. Vimentin and fascin were overexpressed in stromal and epithelial cells, while E-cadherin was upregulated in the stroma and downregulated in epithelia. Conclusions. Our observations suggest that oral intake of semi-refined carrageenan results in the development of low-grade colonic inflammation accompanied by infiltration with CD3 and CD68 positive cells and changes in the expression of EMT markers.Item Cytotoxicity of Hybrid Noble Metal-Polymer Composites(2022-10-11) Tkachenko, Anton; Virych, Pavlo; Myasoedov, Valeriy; Prokopiuk, Volodymyr; Onishchenko, Anatolii; Butov, Dmytro; Kuziv, Yuliia; Yeshchenko, Oleg; Zhong, Sican; Nie, Guochao; Kutsevol, NataliyaThe aim of the present research was to assess the cytotoxicity of gold and silver nanoparticles synthesized into dextran-graft-polyacrylamide (D-PAA) polymer nanocarrier, which were used as a basis for further preparation of multicomponent nanocomposites revealed high efficacy for antitumor therapy. The evaluation of the influence of Me-polymer systems on the viability and metabolic activity of fibroblasts and eryptosis elucidating the mechanisms of the proeryptotic effects has been done in the current research. The nanocomposites investigated in this study did not reduce the survival of fibroblasts even at the highest used concentration. Our findings suggest that hybrid Ag/D-PAA composite activated eryptosis via ROS- and Ca2+-mediated pathways at the low concentration, in contrast to other studied materials. Thus, the cytotoxicity of Ag/D-PAA composite against erythrocytes was more pronounced compared with D-PAA and hybrid Au/polymer composite. Eryptosis is a more sensitive tool for assessing the biocompatibility of nanomaterials compared with fibroblast viability assays.Item Isolation and Cryopreservation of Placental Cells: Search for Optimal Biotechniques in Experimental and Regenerative Medicine(2021) Prokopiuk, O.; Shevchenko, M.; Prokopiuk, V.; Musatova, I.; Safonov, R.; Prokopiuk, O.Abstract: A high effi cacy of placental cells application necessitates their investigation. Preclinical studies require an improvement of the methods for obtaining, standardizing and storage of placental cells of experimental animals. Cells were isolated from rats and mice placentas by means of diff erent enzymatic methods and the one of explants. Cells were cryopreserved with DMSO in DMEM using two-stage freezing. The number, morphological, cultural, metabolic features of cells were studied after isolation and storage. The maximum number of viable cells from the placentas of mice and rats was found to be obtained using the explant method or trypsin with ETDA. Cell cultures from mice and rats placentas after the third passage had stable morphofunctional characteristics. Viability of warmed rat placental cells according to dye exclusion was (92.3 ± 1.6)%, according to the adhesive test this was (81.3 ± 5.8)%. For mice placental cells, these values were (86.7 ± 3.7)% and (79.2 ± 8.1)%, correspondingly. The research results enabled the determining of eff ective biotechniques for obtaining the cryopreserved placental cells of rats and mice to perform preclinical studies of their biological eff ect in models of allo- and autotransplantations.Item The study of phospholipid bilayer of cell membranes in leukocytes incubated with high concentrations of the food additive E407a(2021) Tkachenko, Anton; Onishchenko, Anatolii; Roshal, Alexander; Posokhov, YevgenAim: The aim of the study was to assess the impact of semi-refined carrageenan (E407a) on the hydrophobic region of phospholipid bilayer in cell membranes of leukocytes exposed to the solution with high concentrations of this food additive. Material and methods: Fluorescent probe (2-phenylphenanthro[ 9,10-d]-1,3-oxazole) was used to investigate the influence of E407a on the state of lipid bilayer in leukocytes extracted from rats and treated with a 5% solution of the food additive E407a during 4 hours. Results: The shapes of the probe fluorescence spectra did not differ in leukocytes of rats treated with the 5% solution of semi-refined carrageenan and white blood cells of control samples. Such findings suggest that exposure to the E407a solution causes no changes in the proton-donor ability of the media in the lipid membranes of leukocytes in the region where the probe locates. Conclusion: Exposure of white blood cell suspensions to the semirefined carrageenan solution does not affect the membrane hydration of the hydrophobic region of leukocyte phospholipid bilayer.Item Исследование состояния фосфолипидного бислоя цитоплазматических мембран лейкоцитов крыс при пероральном употреблении пищевой добавки Е407а(2021) Ткаченко, Антон Сергеевич; Посохов, Евгений АлександровичЦелью работы было оценить влияние перорально вводимого полуочищенного каррагинана (E407a) на гидрофоб- ный участок фосфолипидного бислоя клеточных мембран лейкоцитов периферической крови. Материал и методы. Флуоресцентный зонд (2-фенилфенантро[9,10-d]-1,3-оксазол) был использован для оценки состо- яния липидного бислоя мембран лейкоцитов, полученных от 8 взрослых крыс-самок линии WAG, которые перорально получали полуочищенный каррагинан в дозировке 140 мг на кг веса в течение 2 недель, и 8 контрольных животных. Результаты. В ходе проведения работы установлено, что заметных изменений формы спектров флуоресценции зонда в ответ на пероральное введение полуочищенного каррагинана не наблюдалось. Это указывает на то, что пероральный прием E407a не вызывает изменений протонодонорной способности среды в липидных мембранах лейкоцитов в области расположения зонда. Уменьшение интенсивности флуоресценции зонда у крыс, которым перорально вводили пищевую добавку E407a, связано с уменьшением количества молекул зонда, которые связа- лись с мембранами в течение 1 ч инкубации. Заключение. Пероральное употребление полуочищенного каррагинана не вызывает изменений гидратации ги- дрофобного участка фосфолипидного бислоя мембран лейкоцитов.Item Влияние пищевой добавки Е407а на экспрессию Е-кадгерина в тонком кишечнике крыс(2021) Ткаченко, Антон Сергеевич; Губина-Вакулик, Галина Ивановна; Поликарпова, Анна Валериевна; Онищенко, Анатолий ИгоревичАннотация Цель исследования: изучить особенности экспрессии эпителиального маркера Е-кадгерина в эпителиальном слое и строме слизистой оболочки тонкого кишечника при употреблении пищевой добавки Е407а. Материалы и методы. Экспрессию Е-кадгерина изучали иммуногистохимическим методом у 8 крыс популяции WAG, получавших ежедневно в течение 2 недель пищевую добавку Е407а перорально в количестве 140 мг на кг веса, и 8 контрольных животных. Результаты. Установлено, что употребление Е407а приводит к снижению экспрессии Е-кадгерина в эпителиоцитах (0,037 ± 0,004 УЕОП против 0,129 ± 0,021 УЕОП у контроля, р < 0,0001) тонкого кишечника на фоне повышения количества кадгерин-положительных клеток в строме органа (5,03 ± 0,30 клеток по сравнению с 2,55 ± 0,20 клеток в контрольной группе, р < 0,0001) на единицу площади (250 × 250 мкм). Заключение. Пероральное употребление пищевой добавки Е407а приводит к изменению особенностей экспрессии эпителиального маркера Е-кадгерина, что может вносить вклад в нарушение эпителиального барьера кишечника.Item Влияние пищевой добавки E407а на метаболическую активность клеток различных клеточных культур(2021) Ткаченко, Антон Сергеевич; Прокопюк, Владимир Юрьевич; Онищенко, Анатолий ИгоревичРезюме. Целью работы было изучить влияние различных концентраций пищевой добавки Е407а (полуочищенный карра- гинан) на метаболическую активность клеток фетальной печени, спленоцитов и клеток костного мозга. Материал и методы. Клеточные культуры фетальной печени, спленоцитов и костного мозга инкубировали с пи- щевой добавкой Е407а в концентрациях от 0 мг/мл до 10 мг/мл в течение 24 часов (n=8). Для анализа влияния до- бавки на метаболическую активность клеток использовали колориметрический МТТ тест, основанный на способ- ности живых, метаболически активных клеток превращать 3-(4,5-диметилтиазол-2-ил)-2,5-дифенил-тетразолий бромид в формазан. Статистическую обработку данных проводили с помощью расчета критериев Краскела-Уол- лиса и Данна. Результаты. Установлено, что наиболее чувствительной к каррагинану клеточной культурой является костный мозг. Более чем двухкратное статистически достоверное (р<0,0001) повышение метаболической активности кле- ток костного мозга наблюдалось при использовании от 200 мкг/мл Е407а. Метаболическая активность сплено- цитов увеличивалась приблизительно в 1,5 раза и более (р<0,0001) при использовании каррагинанов в концен- трации 500 мкг/мл и выше. Клетки фетальной печени оказались наиболее устойчивыми к прямому токсическому действию пищевой добавки Е407а. Заключение. Пищевая добавка Е407а цитотоксична по отношению к клеткам костного мозга и спленоцитам в концентрациях соответственно от 200 мкг/мл и 500 мкг/мл.