Analysis of the expression of key protein regulators of apoptosis and autophagy in T-lymphocytes of patients with bronchial asthma

Yu.V. Skibo ^a*, M.V. Tikhomirova ^a**, S.N. Abramov ^a***, E.M. Biktagirova ^a****, I.D. Reshetnikova ^a,b*****, N.I. Akberova ^a******, Z.I. Abramova ^a*******

^aKazan Federal University, Kazan, 420008 Russia

^bFederal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Kazan, 420015 Russia

E-mail: ^*yuliya_ksu@mail.ru, ^**mary20011994@mail.ru, ^***007Abramov@mail.ru, ^****EMBiktagirova@kpfu.ru, ^*****reshira@mail.ru, ^******nakberova@mail.ru, ^*******ziabramova@mail.ru

Received March 14, 2019

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DOI: 10.26907/2542-064X.2019.4.505-520

For citation: Skibo Yu.V., Tikhomirova M.V., Abramov S.N., Biktagirova E.M., Reshet­nikova I.D., Akberova N.I., Abramova Z.I. Analysis of the expression of key protein regulators of apoptosis and autophagy in T-lymphocytes of patients with bronchial asthma. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2019, vol. 161, no. 4, pp. 505–520. doi: 10.26907/2542-064X.2019.4.505-520. (In Russian)

Abstract

Bronchial asthma is a disease characterized by T-lymphocyte resistance to apoptosis. Recent studies have shown that there is an alternative form of cell death – autophagy. Since apoptosis in T-cells of patients with asthma is reduced, we suggested that autophagy could be initiated. The aim of the study is to perform a comparative analysis of the expression of apoptosis and autophagy key proteins in T-lymphocytes of patients with mild and severe bronchial asthma.

To achieve this aim, the Western blot method was applied. The results were processed with the help of R statistics. Boxsplots were used to present the data.

We showed that in the group with mild asthma the amount of caspase-3 increases during cultivation (spontaneous apoptosis is activated). In the group with the severe form, its content did not change.

Autophagy activation was assessed by the presence of LC3-II protein. The results showed that form II of LC3-protein is present only in the group with the severe form. Therefore, the analysis of the content of key proteins of autophagy – Rubicon and UVRAG – in the cells of this category of patients was carried out and a significantly increased expression of these proteins was detected. In addition, we found a significant increase of UVRAG compared with Rubicon.

Thus, inhibition of apoptosis is characteristic of T-lymphocytes of patients with bronchial asthma. However, in the mild group, a decrease of nutrients in the culture medium stimulated the launch of apoptosis, which was manifested in an increase in the content of caspase-3 and a decrease in Bcl-2. In the severe group, cell cultivation did not cause an increase in apoptotic activity, autophagy was activated instead, and we assume that for this group of patients it is an alternative way of cell death.

Keywords: bronchial asthma, T-lymphocytes, apoptosis, autophagy, caspase-3, Bcl-2, LC3B,   Rubicon, UVRAG

Acknowledgments. This study was supported by the Russian Foundation for Basic Research (project no. 18-34-00739).

Figure Captions

Fig. 1. Assessment of the content of procaspase-3 and of caspase-3, its active form, in T-lymphocytes lysates following three and six days of culturing. The immunograms show control lysates of the cells and lysate samples obtained from the cells of patients with the mild and severe forms of atopic asthma. β-actin was used as “loading” control. The content of caspase-3 and procaspase-3 in different cell lysates was normalized with regard to the β-actin level.

Fig. 2. Statistical analysis of the data on capase-3 and its inactive form expression in T-lymphocytes from healthy donors and patients with the mild and severe forms of asthma. The analysis was performed in freshly isolated cells, as well as in cells cultured for three and six days: a) intergroup analysis of the coefficient k in three groups using the Kruskal–Wallis test followed by paired comparisons with the Bonferroni and Benjamini–Hockberg correction; b) analysis of the coefficient k in the control group in dynamics (0, 3, and 6 days); c) analysis of the coefficient k in the group with the mild form of atopic asthma in dynamics (0, 3, and 6 days); d) analysis of the coefficient k in the group with severe atopic asthma in dynamics (0, 3, and 6 days).

Fig. 3. Analysis of the Bcl-2 content in T-lymphocytes lysates following three and six days of culturing by the method of Western blotting. The immunograms show control lysates of the cells and lysate samples obtained from the cells of patients with mild and severe forms of atopic asthma. GAPDH was used as “loading” control.

Fig. 4. Analysis of the content of LC3-I and -II proteins in T-lymphocytes lysates following three and six days of culturing by the method of Western blotting. The immunograms show control lysates of the cells and lysate samples obtained from the cells of patients with the mild and severe forms of atopic asthma. β-actin was used as “loading” control. The content of caspase-3 and procaspase-3 in different cell lysates was normalized to the β-actin level.

Fig. 5. Statistical analysis of the data on Rubicon (a) and UVRAG (b) in T-lymphocytes from healthy donors and patients with the severe form of asthma. The analysis was performed in freshly isolated cells. Boxplots were used to present the data. The comparison was carried out with the help of the Wilcoxon test.

Fig. 6. Comparative analysis of the content of Rubicon and UVRAG proteins in T-lymphocytes of patients with the severe form of asthma. The analysis was performed in freshly isolated cells. Boxplots were used to present the data. The comparison was carried out with the help of the Wilcoxon test.

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