EFFECT OF ERYTHROPOIETIN ON T-LYMPHOCYTES OF THE THYMUS OF RATS IN VITRO AFTER EXPOSURE TO INHIBITORS OF DIFFERENT CLASSES

Introduction. Erythropoietin (EPO) – physiological stimulator of erythropoiesis. It activates the mitosis and maturation of red blood cells from progenitor cells erythroid series. One of the main effects of EPO is the slowdown in the rate of apoptosis of erythroid progenitor cells in the bone marrow. Protective properties of EPO demonstrated in various diseases in clinical and experimental conditions. Previously, it was found that activating EPO-effect on T-lymphocytes (TLC) may result not only to the increase of the number of fluorescent mitochondria in cell (nm/c) with proton potential (Δφm), but also external growth of growth of external membrane potential electric potential of plasma (Δφp). The objective of this work was to investigate the TLC response upon EPO after exposure to specific inhibitors of phosphorylation reactions in the respiratory chain. Material and methods. We studied EPO («Eprex», Cilag) effect on rat TLC in vitro after their deenergization by several inhibitors: dinitrophenol (DNP) - uncoupler of oxidative phosphorylation and ingibitor of respiratory chain), pentachlorphenol (PCP)- uncoupler of oxidative phosphorylation, N,N -dicyclohexylcarbodiimide (DCCD)- of Ca2+- inhibitor of the membranebound part of the mitochondrial membrane ATP-ase with the help of a potential-sensitive vital fluorescent probe-cation 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). Rat TLC were isolated from thymuses according to the standard method. The microfluorimetric studies of DSM-stained TLC were performed by means of fluorescent microscope («Lumam I- 2», «LOMO», Russia) with thermostatic table. Fluorescence of 50–70 single cells was measured in each specimen, mean fluorescence intensity of TLC (F ~ ) was calculated. In addition, n _m/c was calculated in each fluorescent cell. Statistical processing of the experimental data was performed by the Spearman’s rank correlation coefficient. Results and discussion. In experiments with TLC from different thymuses, we registered a decrease in F ~ and n m/c after incubation with all used inhibitors. It was found that the difference in decrease velocity of nm/c and of F ~ depended on the type of inhibitor and on the duration of incubation. Maximum reduction of energy of the TLC achieved by incubation with a DNF, after which the EPO does not recover F ~ and _m/c. After incubation with PCP, EPO restores ~ 20–23 % _m/c and F ~ . The reaction of TLC on the DCCD confirms the important role of the ATP-ase in the maintenance of mitochondrial membrane potential. After deenergization of TLC under the action of DCCD, EPO has the maximum effect: restored ~ 42 % n m/c and ~ 38 % F ~ . Conclusions. EPO is able to partially recover the polarization of the mitochondria membranes in TLC violated as a result of exposure to DCCD.

erythropoietin, T-lymphocytes, energy activity, inhibitors, potential-sensitive vital fluorescent probe-cation 4-(p-dimethylaminostyryl)-1-methylpyridinium

1. Jelkmann W., Gross A.J.(Eds.). Erythropoietin,Springer-Verlag Berlin Yeidelberg New York, 1989,180 p.

2. Osikov M.V., Grigoryev T.A., Fedosov A.A., Kozochkin D.A., Ilinykh M.A. Effect of erythropoietin on the functional activity of platelets. Modern problems of science and education. 2012. № 6. URL: https://elibrary.ru/download/elibrary_23220530_33460976.pdf.

3. Baksheyev V. I., Kolomoyets N. M. Erythropoietin in clinical practice. Clinical medicine. 2007; 85 (9):30-37.

4. Biggar P, Kim GH. Treatment of renal anemia: Erythropoiesis stimulating agents and beyond. Kidney Res Clin Pract. 2017; 36(3):209-223.

5. Bonomini M1, Del Vecchio L2, Sirolli V3, Locatelli F2. New Treatment Approaches for the Anemia of CKD. Am J Kidney Dis. 2016; 67(1):133-42.

6. Livnah O, Johnson DL, Stura EA, Farrell FX, Barbone FP, You Y, Liu KD, Goldsmith MA, He W, Krause CD, Pestka S, Jolliffe LK, Wilson IA. Anantagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation. Nature Structural & Molecular Biology.1998; 5 (11): 993–1004.

7. Saraeva N. About. The use of recombinant erythropoietin in haematological practice. Siberian medical journal. 2006; 64(6): 5-10. ( in Russ.).

8. Zakharov Yu. M., Melnikov I. Y., Tasevska N. In. The sheviakov A. A. study of the role of intercellular interactions in the regulation of erythropoiesis in erythroblastosis islets of the bone marrow. Bulletin of the Ural medical academic science. 2015; 55(4): 59-62. ( in Russ.).

9. Osikov M.V., Simonyan E.V., Saedgalina O.T., Fe-do sov A.A. Mechanisms of erythropoietin influence on the quantitative composition of blood lymphocytes in experimental thermal injury. Modern problems of science and education. 2016. № 2. URL: https://elibrary.ru/download/ elibrary_25869790_84106576.pdf. ( in Russ.).

10. Rocha J, Eduardo-Figueira M, Barateiro A, Fernandes A, Brites D, Pinto R, Freitas M, Fernandes E Mota-Filipe H, Sepodes B. Erythropoietin reduces acute lung injury and multiple organ failure/dysfunction associated to a scald-burn inflammatory injury in the rat. Inflammation. 2015; 38(1):312-26.

11. Lifshitz L., Avneon M., Prutchi-Sagiv S., Katz O., Gassmann M., Mittelman M., Neuman D. Immunomodulatory functions of Erythropoietin. Focus uni-luebeck Supplement. 2009; 28.

12. Todosenko NM1, Shmarov VA1, Malashchenko VV1, Meniailo ME1, Melashchenko OB1, Gazatova ND, Goncharov AG1, Seledtsov VI2. Erythropoietin exerts direct immunomodulatory effects on the cytokine production by activated human T-lymphocytes. Int Immunopharmacol. 2016; 36:277-281.

13. Wang S1,2, Zhang C1,2, Li J1,2, Niyazi S1,2, Zheng L1,2, Xu M1,2, Rong R1,2,3, Yang C1,2, Zhu T1. Erythropoietin protects against rhabdomyolysis-induced acute kidney injury by modulating macrophage polarization. Cell Death Dis. 2017; 8(4):e2725.

14. Morozova G.I., Parkhomenko T.V., Klitsenko O.A., Tomson V.V. Stimulating effect of erythropoietin on thy mocyte energetics established in vitro with a potential-sensitive fluorescent probe in the mitochondria. Biochem. Suppl. Series A: Membr Cell Biology. 2007; 1 (4):325-330.

15. Parkhomenko T.V., Morozova G.I., Klytsenko O.A., Tomson V.V. Quantitative evaluation of erythropoietin (EPO) influence on rat T-lymphocytes. Annals of Hematology. 2000; 79 (5): B8.

16. Parkhomenko T.V., Morozova G.I., Klytsenko O.A., Tomson V.V. Evaluation of restoring erythropoietin (EPO) effect on rat T-lymphocytes after their treatment with some inhibitors in vitro. Annals of Hematology. 2003;82 (6):S114.

17. Artuhov VG, Putinzetva OV, Bragina VA, Pashkov MV, Vasilenko DV. Fluorescent methods in the research induced by UV radiation changes of structural and functional state of human blood lymphocytes. Bull Exp Biol Med 2012; 153(6):891-895.

18. Parkhomenko T.V., Morozova G.I., Klitsenko O.A.,Tom-son V.V. The erythropoietin (EPO) response of cells isolated from rat thymuses in vitro. Focus uni-luebeck Supplement. 2009:32.

19. Futai M., Noumi T., Maeda M. ATP - synthase (H+- ATP - ASE) – results by combined biochemical and molecular biological approaches. Annual Review of Biochemistry. 1989; 58:111-136.

20. Clejan L., Bosch C.G., Beattie D.S. Inhibition by dicyclohexylcarbodiimide of proton ejection but not electron-transfer in rat-liver mitochondria. Journal of Biological Chemistry. 1984; 259 (21):13017-13020.

21. Linnane A., Lukins H., Nagley P., Marzuki S., Ha-di kusumo R., Jean-Francois M., John U., Ooi B., Wat-kins L., Willson T., Wright J., Meltzer S. Assembly of the yeast mitochondrial H+ATP-ASE correlative studies involving gene sequencing and immunochemical probes of assembly. Achievements and perspectives of mitochondrial research.1985;1(Bioenergetics): 211-222.