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16MEDICAL SCIENCES
NEW ABOUT THE MECHANISM OF DECAMETHOXINE ACTION ON PERIPHERAL BLOOD
LYMPHOCYTES
Kovalenko I.
PhD, Assistant Professor, Department of Microbiology, Danylo Halytsk yLviv National Medical University, Lviv, Ukraine
Melnyk O.
PhD, Assistant Professor, Department of Medical Biology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Vorobets Z.
D Sci, Professor, Department of Medical Biology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Abstract
It has been established that decamethoxine (DCM) and its dosage form decasan (DS) showed the same microbicidal activity against E. coli museum strains (15.62 ^g/ml), C. albicans (16 ^g/ml), E. faecalis (0,97 ^g/ml), Bac. subtilis (0.24 ^g/ml). DS was less microboccidally active against S. aureus (0,48 ^g/ml) compare to DMC (0.24 ^g/ml). DS was also less microbicidal active against P. aeruginosa (250 ^g/ml) than DCM (62.5 ^g/ml). Decamethoxine a concentration of 10-5-10-2M dose-dependently leads to an increase of arginase activity in blood lymphocytes. Furthermore, the activity of the constitutive isoform of NO synthase in control was (71.4±6.9) and the activity of the inducible isoform (1.58±0.18) nmol NADPH(H+)/min per 1 mg of protein. With the action of decamethoxine at a concentration of 10-5 M the activity of cNOS decreased in 1.25 times and the activity of iNOS increased in 10.8 times. Consequently, there are a slight inhibition of LP processes under the action of different concentrations of decamethoxine. Thus, the concentration of MDA in blood lymphocytes is (62.3±4.6) nmol/mg protein of the control group. This numeric value decreases under the influence of the different concentrations of decamethoxine (10-5-10-2 M) but it is non-significant.
Simultaneously, with a slight decrease in process LP appropriate changes in the activity of glutathione system enzymes were revealed. Thus, it was shown that lymphocyte glutathione peroxidase activity is (154.2±13.4) nmol GSH/min-mg protein in control. Under the action of decamethoxine, this activity increases dose-dependently. Thus, the results obtained regarding the action of decamethoxin and fluoroquinolones indicate significant changes in the arginase-NO-synthase and glutathione antioxidant systems of blood lymphocytes, which leads to their imbalance and impaired regulatory role.
Kaywords: decamethoxine, lymphocytes, arginase, NO-synthase, glutathione, lipid peroxidation.
Introduction. Antiseptics are an important place in the etiotropic therapy of purulent-inflammatory complications is occupied and active against the pathogenic microflora. One of the most common antiseptics is the drug Decamethoxin [7, 13, 14]. Other drugs are made on its basis - gorosten, decazan, auridexan, septefril, etc. Due to the potent antibacterial activity against a wide range of bacteria and low toxicity the decamethoxine is widely used in the treatment of bacterial infections of various localization. These drugs of ultra-wide spectrum of action are active against gram-positive and gram-negative, aerobic and anaerobic microorganisms [7, 13, 14]. The formation of decamethoxin-resistant forms of microorganisms is very slow.
Since decamethoxin has hydrophilic and lipo-philic properties, it probably can penetrate cells and blood and are transmitted by blood to different organs and tissues through wound surfaces, mucous membranes, skin, etc. and thus causing a variety of biochemical effects. In this regard, the biological effect of dec-amethoxine has not been studied as extensively.
In this aspect, the role of Nitrogen (II) oxide as a universal cellular and tissue metabolite in the regulation of cellular functions and paracrine regulator of intercellular and intersystem interactions is no doubt [2, 4, 6, 8, 10, 15]. NO synthesis is carried out with the
participation of NO synthase (EC 1.14.13.39) from L-arginine by oxidative metabolism.
L-arginine is also metabolized by arginase [12]. According to modern ideas, the effects on the body of xenobiotics, drugs and the development of pathological processes accompanied by a violation of the mechanisms of antioxidant protection of cells [1, 9, 11].
Under the action of chemicals, oxidative free radical processes are often initiated in phospholipids of cell membranes that containing the polyunsaturated fatty acids. Intensification of lipid peroxidation leads to the accumulation of toxic products, accompanied by a decrease in the body's resistance [1, 9, 11]. At the same time, the buffer capacity of the antioxidant system is quite large and is provided by various components. An important place among the antioxidant cell systems occupies a glutathione system, the components of which take part in both enzymatic such as glutathione peroxi-dase, glutathione reductase, glutathione-S transferase, and non-enzymatic (glutathione) reactions of the anti-oxidant system.
It is believed that peripheral blood lymphocytes are a "metabolic mirror" of the body and, therefore, it's can serve as a model for studying the effects of antiseptics on the metabolic and regulatory systems of the cells of the macroorganism [3].
To find out the features of the antiseptic "Deca-methoxin" on arginase/NO-synthase and antioxidant systems of blood lymphocytes.
Materials and methods. The drug Decameth-oxin® was registered in Ukraine indefinitely by the order of the Ministry of Health of Ukraine (March 29, 2017, № 341).
Experiments on the antimicrobial action of deca-methoxine were performed on museum strains of microorganisms S. aureus, S. epidermidis, E. coli, P. aeruginosa, C. albicans.
Lymphocytes from the peripheral blood of women were isolated by the method of Boyum A. in a density gradient of ficol-triumbrast (X = 1.08 g/cm3) [5].
Peripheral blood lymphocyte arginase activity was determined by urea formation.
NO synthase activity was determined by oxidation of NADPH(H+) (Ravaiva M., 2011). The activity of Ca2+-independent iNOS was evaluated by adding to the incubation medium a selective inhibitor of the induci-ble isoform aminoguanidine instead of CaCl2+. The activity of Ca2+-dependent isoform of NOS, which according to the literature corresponds to the constitutive isoform of NOS (cNOS), was calculated as the difference between the total activity of NOS and the activity of Ca2+-independent isoform of NOS.
Glutathione peroxidase activity was determined by the development of a color reaction with 5,5-dithio-bis (2-nitro-benzoic acid) with the formation of a colored product of thionitrophenyl anion the number of
Table 1
Microbicidal activity of generic decamethoxine (DCM) and its dosage form of dexane (DS) on museum strains
of microorganisms
which is directly proportional to the number of SH-groups (Moin V., 1986).
Glutathione reductase activity of peripheral blood lymphocytes was determined by the method Mannervic V. (1991).
Glutathione transferase activity of peripheral blood lymphocytes was determined by the method Bu-lavin D., (1996), and the content of reduced glutathione (Naher Z et al., 2011).
The principle of the method of determining the MDA is that at high temperature in an acidic environment it reacts with 2-thiobarbituric acid, creating a colored trimethylene complex with an absorption maximum at X = 532 nm (Timirbulatov S., 1988).
Results and discussion. Despite the significant number of studies in Ukraine of the antiseptic drug decamethoxine (DCM) study of its microbicidal, physico-chemical and especially biochemical properties remains a pressing issue. It should be noted that foreign studies of this drug are virtually non-existent.
As a result of the conducted researches, it is established, that both DCM and its dosage form decasan (DS) showed the same microbicidal activity against museum strains of E. coli (15.62 ^g/ml), C. albicans (16 ^g/ml), E. faecalis (0.97 ^g/ml) , Bas. subtilis (0.24 ^g/ml). DS was less microbicidal against S. aureus (0.48 ^g/ml) compared with DCM (0.24 ^g/ml) (Tabl. 1).
Museum strain of the microorganism Decamethoxine (^g / ml), M ± m
DCM DS
S. aureus ATCC 25923 0,24±0,02 0,48±0,03*
E. coli M-17 15,62±0,09 15,62±0,09
E. coli ATCC 25922 15,62±0,09 15,62±0,09
P. aeruginosa ATCC 27853 62,5±4,3 250±15,6*
E. faecalis ATCC 29212 0,97±0,07 0,97±0,08
B. subtilis ATCC 6632 0,24±0,02 0,24±0,02
C. albicans CC M 855 0,24±0,03 0,24±0,03
Note: *p < 0.05
Decasan was also less microbicidal active against P. aeruginosa (250 ^g/ml) than DCM (62.5 ^g/ml). However, both substances, according to the instructions for use of DCM® dosage forms, are not recommended for the prevention and treatment of infectious complications caused by P. aeruginosa, therefore it does not affect the effectiveness of antimicrobial agents according to the instructions for use. Inconsistency in the bactericidal action of DCM and DS against S. aureus ATCC 25923 and P. aeruginosa and the fact that DCM is 2 - 4 times more active against these strains indicates that manufacturers will have to take appropriate measures to improve quality to achieve microbicidal activity of the drug at the level of the patented sample DCM®.
When determining the effect of decamethoxine on the functioning of arginase in peripheral blood lymphocytes and elucidation of a number of kinetic parameters for the enzymatic reaction, L-arginine was added to the incubation medium in the concentration range from 1 to 200 mm (at a constant optimal concentration of Mn2+ - 2 mM).
Decamethoxine in concentrations of 10-5 - 10-2 M dose-dependently leads to an increase in arginase activity. At 10-5 M concentration of the drug is 1.2, and at 10-2 M concentration up to (175.1 ± 13.8) nmol of urea/min • mg of protein, ie 1.4 times relative to control (p < 0.05) (Tabl. 2).
Table 2
Effect of decamethoxine on arginase/NO-synthase and glutathione antioxidant systems (M±m, n=6-9)
Control Decamethoxine
10-5 M 10-4 M 10-3 M 10-2 M
Arginase, nmol urea/minmg 125,1±11,3 150,4±12,6 157,2±12,7 168,6±13,6 175,1±13,8*
cNOS, nmol NADPH/min • mg 71,4±6,9 57,1±5,8*
iNOS, nmol NADPH/min- mg 1,58±0,18 17,1±1,9***
MDA, ^molmg 62,3±4,6 60,2±4,5 59,7±4,9 57,6±4,7 55,1±4,3
GSH, nmol GSH/mg 17,2±1,6 17,9±1,8 18,1±1,8 18,3±2,1 18,4±1,8
GP, nmol GSH/min-mg 154,2±13,4 159,3±11,2 161,8±12,0 178,5±12,1 182,2±13,3*
GR, nmol NADPH/min- mg 51,7±4,2 53,7±5,0 58,6±5,1 60,5±5,2 62,3±5,4*
GT, nmol GSH/min-mg 120,2±9,2 128,3±11,4 136,6±12,1 148,4±12,4 156,6±12,4*
Note: *p < 0.05; ***p < 0.001
According to the literature, all NOS isoforms were identified in blood lymphocytes [5, 8]. nNOS (so-called type 1); iNOS (also known as type 2), which, unlike nNOS and eNOS, is not constantly expressed; the synthesis of this enzyme occurs only in pathological conditions and can be induced in cells of different types under the action of inflammatory stimuli. eNOS (also known as type 3), first identified in the blood vessel en-dothelial cells.
The activity of the constitutive isoform of NO synthase in the control was (71.4 ± 6.9), and the activity of the inducible isoform (1.58 ± 0.18) nmol NADPH(H+)/min per 1 mg of protein. Under the action of decamethoxine at a concentration of 10-5 M, the activity of cNOS decreased by 1.25 times (p < 0,05), and the activity of iNOS increased 10.8 times (p < 0,001) relative to control values.
Thus, it has been shown that the antiseptic deca-methoxine significantly affects the regulatory mechanisms of the cell, in particular blood lymphocytes. It stimulates arginase activity and inhibits cNOS activity with simultaneous iNOS activation.
According to modern ideas, the effect on the body of xenobiotics, drugs, as well as the development of pathological processes are accompanied by a violation of the mechanisms of antioxidant protection of cells [1, 11]. Under the action of chemicals, oxidative free radical processes are often initiated in phospholipids of cell membranes containing polyunsaturated fatty acids. Intensification of lipid peroxidation leads to the accumulation of toxic products, accompanied by a decrease in the body's resistance. At the same time, the buffer capacity of the antioxidant system is quite large and is provided by various components. An important place among the AOS of the cell is the system of glutathione, components of which are involved in both enzymatic (glutathione peroxidase, glutathione reductase,glutathi-one-S transferase), and in non-enzymatic (glutathione) reactions of AOS [9, 11]. Although the glutathione system has been the subject of much research, in the literature, there is no consensus regarding its role in the body's response to the action of certain antiseptics, antibiotics or the development of pathological conditions. Therefore, the analysis of the functional state of the glutathione system under the action of antiseptics will reveal its role in the mechanism of action of these drugs.
We conducted a comparative study of the processes of LP and glutathione system in peripheral blood lymphocytes under the action of decamethoxine. Slight
but unreliable inhibition of LP processes by different concentrations of decamethoxine is shown, which was evaluated by determining the concentration of malonic dialdehyde, which is a secondary product of lipid pe-roxidation.
Thus, in the control, the concentration of MDA in blood lymphocytes is (62.3 ± 4.6) ^mol/mg protein. Under the action of different concentrations of deca-methoxine (10-5 -
10-2 M) this value is slightly reduced to (55.1 ± 4.3) ^mol/mg protein (p > 0.001).
Consequently, in blood lymphocytes significant changes in the processes of LP under the action of dec-amethoxin were not detected, although there is a tendency to decrease. Simultaneously with a slight decrease in the processes of LP, corresponding changes in the activity of enzymes of the glutathione system were revealed. Thus, it is shown that in the control of glutathione peroxidase activity of lymphocytes is (154.2 ± 13.4) nmol GSH/min •mg protein.
Under the action of decamethoxine, this activity increases dose-dependently and reaches (181.2 ± 13.3) nmol GSH/min • mg protein (p < 0.05). However, the concentration of reduced glutathione under the action of decamethoxine did not change and was in the range (17.2 - 18.4) nmol GSH/mg protein. We have previously demonstrated that fluoroquinolones also dose-de-pendently stimulate glutathione peroxidase activity. Regarding the activity of glutathione reductase, in the control it was (51.7 ± 4.2) nmol NADPH/min-mg protein. Under the action of decamethoxine in concentrations of 10-5 - 10-2 M this activity dose-dependently increased to (62.3 ± 5.4) nmol NADPH/min-mg protein, ie 1.2 times relative to the control group (p < 0.05).
Glutathione-S transferase activity in the control was (120.2 ± 9.2) nmol GSH/min- mg protein. Addition to the incubation medium of decamethoxine in concentrations of
10-5 - 10-2 M dose-dependently activated this enzyme, whose activity increased to (156.6 ± 12.4) nmol GSH/min-mg of protein, ie 1.4 times (p < 0.05).
Conclusion. This allows the conclusion that antiseptic decamethoxine has significant microbicidal activity against a number of strains of microorganisms, and affects the regulatory mechanisms of cells of the macroorganism, in particular blood lymphocytes. Activates enzymes of the glutathione antioxidant system and leads to an increase in arginase and inducible NO-synthase activities.
References
1. Ayala A, Munoz MF, Arguelles S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehide and 4-hydroxy-2-non-enal. Oxidative Medicine and Cellular Longevity. 2014;36:1-31.
2. Bogdan C. Regulation of lymphocytes by nitric oxide. Methods Mol. Biol. 2011;677:375-393.
3. Bolin A, Guerra B, Nascimento S, Otton R. Changes of lymphocyte oxidant/antioxidant parameters after carbonyl and antioxidant exposure. Immunophar-macology. 2012;14(4):690-697.
4. Bryan NS, Bian K, Murad F. Discovery of the nitric oxide signaling pathway and targets for drug development. Frontiers in Bioscience. 2009;14:1-18.
5. Fafula R., Efremova U., Lychkovska N., Melnyk O., Vorobets Z., Kulachkovskiy O. Methodological Approaches to the Study of Enzymatic Spectrum of Lymphocytes at Patolog3cal States with the Application of Saponin (ultrastructure research). Bulletin of Problems in Biology and Medicine. 2012;4(96):163-166.
6. Forstermann U. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch. 2010;459(6):923-928.
7. Honchar OO, Nazarchuk OA, Paliy DV, Ko-valenko IV, Burkot VM. Antimicrobial qualities of generics of Decametoxin. Medical and Clinical Chemistry. 2015;17(4):43-46 (in Ukrainian).
8. Ignarro LJ. Nitric oxide as a unique signaling molecule in the vascular system: a historical overview. J. Physiol. Pharmacol. 2002;53:503-514.
9. Kaynar H, Meral M, Turhan H. Glutathione
peroxidase, glutathione-S-transferase, catalase, xanthine oxidase, Cu-Zn superoxide dismutase activities, total glutathione, nitric oxide, and malondialdehyde levels in erythrocytes of patients with small cell and non-small cell lung cancer. Cancer Lett. 2005;227(2):133-139.
10. Luiking YC. Engelen MP, Deutz NE. Regulation of nitric oxide production in health and disease. Curr. Opin. Clin. Nutr. Metab. Care. 2010;13(1):97-104.
11. Marushchak M, Markiv K, Kynytska I, Stechyshyn I. Glutathione antioxidant system of lymphocytes in the blood of patients in a setting of concomitant chronic obstructive pulmonare disease and arterial hypertension. Pol. Merkur. Lekarski. 2019;47(281):177-182.
12. Morris SM. Enzymes of arginine metabolism. J. Nutr. 2004;134(10):43-47.
13. Paliy GK, Nazarchuk OA, Bobyr OO et al. Estimation of antibacterial and antifungal qualities of modern antiseptics. Microbiology and Biotechnology. 2015;4(32):67-74 (in Ukrainian).
14. Paliy GK, Nazarchuk OA, Honchar OO, Ko-valenko SV, Yatsula OV. The research of physical and chemical, antimicrobial qualities of "Decamethoxin®" remedy. Medical and Clinical Chemistry. 2016;18(1):36-44 (in Ukrainian).
15. Saluja R, Jyoti A, Chaterjee M. Molecular and biochemical characterization nitric oxide sythase isoforms and thear intracellular distribution in human peripheral blood mononuclear cells. Biochim. Biophis. Acta. 2011;1813(10):1700-1707.
ЭВФЕМИЗМЫ В ИСПАНОЯЗЫЧНОМ МЕДИЦИНСКОМ ДИСКУРСЕ
Андреева П.И.
магистрант 2-го года обучения Первого Московского Государственного медицинского университета им. И.М. Сеченова (Сеченовского Университета)
Слышкин Г.Г.
доктор филологических наук, профессор, директор Центра лингвистики и профессиональной коммуникации Института права и национальной безопасности ФГБОУ ВО «Российская академия народного хозяйства и государственной службы при Президенте Российской Федерации»
EUPHEMISMS IN HISPANIC MEDICAL DISCOURSE
Andreeva P.
2nd year master's student of the I.M. Sechenov First Moscow State Medical University (Sechenov University) Slyshkin G.
PhD in Philology, Director of the Center for Linguistics and Professional Communication, Institute of Law
and National Security, Russian Presidential Academy of the National Economy and Public Administration (Moscow).
Аннотация
В статье освещается проблема эвфемизации в испаноязычном медицинском дискурсе, обозначены причины использования эвфемизмов, выделены номинативные сферы, в которых осуществляется употребление эвфемизмов в коммуникации «врач-пациент».
