ОКИСЛИТЕЛЬНАЯ МОДИФИКАЦИЯ БЕЛКОВ И АКТИВНОСТЬ КАСПАЗЫ-3 В ЛИМФОЦИТАХ КРОВИ ПРИ ОКИСЛИТЕЛЬНОМ СТРЕССЕ IN VITRO

Носарева О.Л.¹, Степовая Е.А.², Рязанцева Н.В.³, Шахристова Е.В.⁴

¹Кандидат медицинских наук, ²Доктор медицинских наук, профессор, ³Доктор медицинских наук, профессор, ⁴Кандидат медицинских наук, Государственное бюджетное образовательное учреждение высшего профессионального образования «Сибирский государственный медицинский университет» Министерства здравоохранения Российской Федерации

Исследование выполнено при финансовой поддержке Российского гуманитарного научного фонда в рамках научного проекта №15-36-01289.

ОКИСЛИТЕЛЬНАЯ МОДИФИКАЦИЯ БЕЛКОВ И АКТИВНОСТЬ КАСПАЗЫ-3 В ЛИМФОЦИТАХ КРОВИ ПРИ ОКИСЛИТЕЛЬНОМ СТРЕССЕ IN VITRO

Аннотация

В статье рассмотрена взаимосвязь между изменением уровня карбонилирования, глутатионилирования белков и активностью каспазы-3 в лимфоцитах крови при окислительном стрессе in vitro. Полученные показатели изменения активности каспазы-3 и концентрации белково-связанного глутатиона в лимфоцитах крови при окислительном стрессе in vitro могут быть использованы при разработке подходов таргетной терапии заболеваний, сопровождающихся дизрегуляцией апоптоза.

Ключевые слова: лимфоциты крови, окислительный стресс, окислительная модификация белков, апоптоз, каспаза-3.

Nosareva O.L.¹, Stepovaya E.A.², Ryazantseva N.V.³, Shakhristova E.V.⁴

¹Candidate of Medicine Science, ²Doctor of Medicine Science, Professor, ³Doctor of Medicine Science, Professor, ⁴Candidate of Medicine Science, Siberian State Medical University

The research was carried out with financial support from Russian Humanitarian Scientific Fund within the framework of the project №15-36-01289.

OXIDATIVE MODIFICATION OF PROTEINS AND CASPASE-3 POTENCY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

Abstract

In this abstract we consider interaction between the changes of level of protein carbonylation, glutathionylation and caspase-3 potency in blood lymphocytes during oxidative stress in vitro. The received alteration indices of caspase-3 potency and protein-bound glutathione concentration in blood lymphocytes during oxidative stress in vitro can be used when elaborating target therapy approaches to diseases accompanied by apoptosis disregulation.

Key words: blood lymphocytes, oxidative stress, oxidative protein modification, apoptosis, caspase-3.

Blood lymphocytes are an important and essential component of human body homeostasis system. According to present knowledge a lot of diseases connected with the involvement of blood lymphocytes into pathologic processes are accompanied by production of active oxygen species (AOS), cell redox-imbalance with following oxidative stress development and apoptosis disregulation.

One of the fundamental mechanisms of homeostasis maintaining and cell activity regulation is programmed cellular death, which represents active form of death as physiological mechanism of abolition of functionally inadequate, receptor representation defective cells. Enzyme caspase cascade activation is reckoned in apoptosis performance key mechanisms [6]. Caspases are cysteine protease, potential targets for AOS attack.

The goal of research is to ascertain the interaction between carbonylation and protein glutathionylation levels and caspase-3 potency in blood lymphocytes during oxidative stress in vitro.

Materials and methods

In this work we used lymphocytes isolated from healthy donors’ blood mononuclear leucocyte fraction (10 men and 7 women aged 20 to 45).

Mononuclear leucocyte isolation with following lymphocyte isolation from vein blood was carried out by means of gradient centrifugation method [1, 9]. Cell cultures containing to a maximum of 5% of dead cells were used for the experiment.

To simulate an experimental oxidative stress the isolated blood lymphocytes were cultivated in sterile conditions in full culture medium RPMI-1640 («Vector-Best», Russia) during 18 hours in semi-open system under 37°С in 5% СО₂ atmosphere with H₂O₂ of final 0,5 Mm concentration [8].

Protein-bound glutathione level was estimated with help of spectrographic method [2].

Carbonyl derived protein content estimation was conducted with help of enzyme immunoassay (EIA) using «Carbonyl Proteine ELISA Kit» («Immundiagnostik AG», Germany) according to the instruction of manufacturing company.

Caspase-3 potency was estimated by means of spectrofluorometry method [3, 7].

Received results statistical processing was carried out with help of programme Statistica 6,0. Verification of normality of quantitative indices distribution was conducted using Shapiro-Wilk test. The accuracy of differences was estimated by means of Mann-Whitney nonparametric test. The differences were considered statistically significant under р<0,05 [5].

Results and discussion

The increase of AOS intracellular production in blood lymphocytes leads to imbalance between pro- and anti-oxidants. AOS can also induce oxidative affection of cell basic macromolecules, proteins in the first place. Proteins affected by oxidative destruction change their functional activity and have a longer degradation period as compared to lipid modification products; that makes them a perspective peroxidation intensity marker [4].

During oxidative stress simulation in vitro in blood lymphocytes there was established a credibly significant increase of content of protein-bound glutathione by 2.80 times (р<0,05), of non-repairable carbonyl derived protein by 2.40 times (р<0,05), which was accompanied by caspase-3 activity increase by 2.06 times (р<0,05) in comparison with intact cells.

The received data point at caspase-3 enzyme activity activation in blood lymphocytes during oxidative stress in vitro because of non-repairable carbonyl derived protein cumulation. On top of that, the presence of cysteine remnants in caspase-3 structure allows us to include it with potential molecular effectors, which play role of AOS sensors when blood lymphocytes redox-status changes. In connection therewith, the activity growth of enzyme under study may be connected with the increase of protein-bound glutathione content in blood lymphocytes during oxidative stress in vitro.

Thus, the search for molecular mechanisms of protein oxidative modification participation in apoptosis key enzyme (caspase-3) activity redox-regulation will allow raising the effectiveness of pathogenetic therapy existing methods of a large number of socially important maladies involving in the process blood lymphocytes and will open wider prospects for programmed cell death selective control molecular technologies.

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