03. Науково-дослідний інститут експериментальної та клінічної медицини ХНМУ

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    Human HMGB1 does not induce eryptosis in vitro
    (2022) Tkachenko, Maryna; Onishchenko, Anatolii; Butov, Dmytro; Butova, Tetyana; Tkachenko, Anton
    Aim: 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.
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    Semi-refined carrageenan induces eryptosis in a Ca2+-dependent manner
    (2022) Tkachenko, Anton; Prokopiuk, Volodymyr; Onishchenko, Anatolii
    Background: 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.