Modeling and dynamics of endogenous and exogenous oxidative stress  in vitro

Yu. V. Abalenikhina; Абаленихина Ю. В.; S. K. Pravkin; Правкин С. К.; A. V. Shchulkin; Щулькин А. В.; E. D. Rokunov; Рокунов Е. Д.; D. S. Nemtinov; Немтинов Д. С.; E. P. Vasilyeva; Васильева Е. П.; E. N. Yakusheva; Якушева Е. Н.

doi:10.29296/25877313-2022-12-02

Modeling and dynamics of endogenous and exogenous oxidative stress in vitro

作者: Abalenikhina Y.V.¹, Pravkin S.K.¹, Shchulkin A.V.¹, Rokunov E.D.¹, Nemtinov D.S.¹, Vasilyeva E.P.¹, Yakusheva E.N.¹
隶属关系:
1. Ryazan State Medical University
期: 卷 25, 编号 12 (2022)
页面: 10-17
栏目: Articles
URL: https://journals.eco-vector.com/1560-9596/article/view/321643
DOI: https://doi.org/10.29296/25877313-2022-12-02
ID: 321643

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Relevance. The effect of pro-oxidants on the cell can cause different effects depending on the dose and duration of exposure, therefore, adequate experimental models of oxidative stress (OS) in vitro are needed to study these processes.

The aim of the study was to study the dynamics of OS development in endogenous and exogenous in vitro models.

Material and methods. The study was carried out on a line of Caco-2 cells. Hydrogen peroxide (H₂O₂) and DL-butyonine sulfoximine (BSO) were added to cells at concentrations of 0.1–100 μM s and 1–500 microns, respectively, at the confluence of 3, 24 and 72 hours. At the end of the exposure, the percentage of viable cells was determined (MTT test), the level of reactive oxygen species (MitoTracker Red CM-H2 XRos), the amount of Nrf2 and glutathione peroxidase (ELISA), the concentration of carbonyl derivatives of proteins (photometric method.)

Results. H₂O₂ at concentrations of 5–50 μM and BSO – 10; 50; 100 μM cause an increase in the level of carbonyl derivatives of proteins, the level of transcription factor Nrf2 and antioxidant enzyme – glutathione peroxidase at exposure time of 24 and 72 hours. The concentration of H₂O₂ 100 μM and BSO 500 μM are toxic to the Caco-2 cell line. The incubation period of 3 hours does not cause the development of OS.

Conclusion. Hydrogen peroxide at concentrations of 5-50 μM, BSO – 10; 50; 100 μM and exposure time of 24 and 72 hours cause the development of compensated oxidative stress (eustress), and H₂O₂ at concentrations of 100 μM and BSO – 500 μM are toxic to cells of the Caco-2 line.

关键词

oxidative stress, hydrogen peroxide, D, L-butyonine sulfoximine, glutathione peroxidase, Nrf-2, carbonyl derivatives of proteins

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俄罗斯联邦, Ryazan

参考

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2. Figure 1. Viability of Caco-2 cells exposed to (a) hydrogen peroxide and (b) DL-butionine sulfoximine for 3, 24, and 72 h (M, n = 3).

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3. Figure 2. Change in the level of reactive oxygen species under the action of hydrogen peroxide (H2O2) and D,L-butionine sulfoximine (BSO) in Caco-2 cells: staining with MitoTracker Red CM-H2XRos, ×400

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4. Figure 3. The concentration of carbonyl derivatives of proteins in the cytoplasmic fraction of the homogenate upon exposure to hydrogen peroxide (a) at concentrations of 0.1–100 μM and upon exposure to D,L-butionine sulfoximine (b) at concentrations of 1–500 μM and exposure time of 3, 24 and 72 h (M, n = 3); p < 0.05 compared with *control; ^ incubation period 3 hours; + 24 h incubation period (Newman–Keuls test)

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5. Figure 4. The amount of Nrf2 in the cytoplasmic fraction of the Caco-2 cell lysate upon exposure to hydrogen peroxide at concentrations of 1–500 μM (left) and DL-butionine sulfoximine at concentrations of 1–500 μM for 3, 24, and 72 h (M ± SD, n = 3)

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