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UDC 577.1
B. Gutyj1, I. Khariv1, V. Khalak2 M. Khariv1, R. Vasiv1, Kh. Leskiv1, T. Martyshuk1, Z. Guta1
Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv,
Pekarska Str., 50, Lviv, 79010, Ukraine 2State Institution Institute of grain crops of NAAS, V. Vernadsky Str., 14, Dnipro, 49027, Ukraine DOI: 10.24412/2520-6990-2021-22109-14-18 THE EFFECT OF LIPOSOMAL DRUG "BUTAINTERVIT" ON THE ACTIVITY OF THE T-SYSTEM OF CELLULAR IMMUNITY OF RATS UNDER OXIDATIVE STRESS
Abstract
The results of investigations on the effect of the liposomal drug "Butaintervit" on rats' T-cell immune system activity under carbon tetrachloride poisoning are presented. We detected that administrating to rats of the experimental groups of 50% carbon tetrachloride at a dose of 0.25 ml per 100 g of body weight causes oxidative stress. It negatively affects cellular immunity and functional activity performance immunocompetent blood cells. The immunosuppressive impact of oxidative stress led to a decrease in the rats' blood of the first and second experimental groups, the T-lymphocytes number and their immunoregulatory subpopulations, mainly on the 2nd and 5 th days of the trial. At the equal background, the obtained data indicate a positive effect of butaphosphane, interferon, injectable milk thistle and vitamins A, D3, E in the liposomal drug "Butaintervit" on the relative number of T-lymphocytes in the rats' blood and on the redistribution of avidity in strengthening receptor membranes cells. It was found that the normalization on the 2nd day of studies of the level in the blood of rats of the second experimental group of total T-lymphocytes occurred due to medium-avid forms and active T-lymphocytes by changing the number of low-avid forms.
Key words: T-lymphocytes, blood, rats, oxidative stress, tetrachloroethane, butaphosphane, interferon, milk thistle, vitamins.
It is known that oxidative stress causes significant disorders of physiological functions and metabolic processes in animals and adversely affects the immune system [2, 7, 15-18]. Accordingly, the action of various chemical factors that contribute to oxidative pressure is relevant to study the immune system activity because adrenal cortex hormones play the key role in the development of stress syndrome - glucocorticoids, the level of which in the blood under these conditions increases significantly [1, 3, 10]. Under the glucocorticoids influence, peroxide processes are intensified, that leads to a significant supply in the formation of lipid peroxidation products (LPO), which have a destructive effect on the cellular immune system of animals [6, 8, 11, 26]. In this regard, the search for effective means would develop animals' adaptability and have a regulatory effect on various parts of the body's immunobiological reactivity to free radicals.
To date, scientists have found that among the many drugs that have a positive influence on the immune system due to stress on the body play an essential role, butaphosphane, interferon, milk thistle, as well as fat-soluble vitamins A, D3, E [12-14, 18, 2025]. In this regard, this work aimed to elucidate the impact of a complex immunotropic drug-containing butaphosphane, interferon, milk thistle, and vitamins A, D3, E in the form of a liposomal emulsion, on the activity of the cellular immune system of rats under conditions effects of carbon tetrachloride oxidative stress.
Materials and methods of research
The experimental part of the work was performed on young white male Wistar rats, weighing 180-200 g, kept in the vivarium of the State Research Control Institute of Veterinary Drugs and Feed Additives.
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During the studies, all groups of rats were provided with a balanced diet. In addition, the animals received drinking water without restrictions with free access to drinking bowls.
For the analyses, three groups of rats of 20 animals each were formed. Animals of the Ei and E2 groups were administered intramuscularly 50% carbon tetrachloride oil solution, dosage - 0.25 ml per 100 g of body weight. That was determined by daily weighing of animals for modeling oxidative stress on the first and third days of studies according to the method of O. V. Stefa-nov (2002). Rats of the C group were injected with saline in the appropriate volume as in the experimental groups. Rats of the E2 group on the first and third days of the study one hour after using tetrachloroethane were administered a liposomal drug at a dose of 2 ml/kg body weight. The studied liposomal drug was developed at the Institute of Animal Biology of NAAS and included the following components: butaphosphane, interferon, milk thistle, and vitamins A, D3, E.
Blood for immunological tests was taken from rats by decapitation under weak ether anesthesia on the second, fifth, and tenth days. In the rats' blood, the relative T lymphocytes number and their subpopulations in the reaction of spontaneous rosette formation with sheep erythrocytes were determined [24].
All animal manipulations were performed following the requirements of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Scientific Purposes (European Commission, 20i0).
The digital results were statistically processed using the computer program Origin Pro 8 using Student's t-test, differences at p<0.05-0.001 were considered probable.
Results and discussion
An essential role in investigating the cellular component of animal immunity in determining the Tand B-lymphocytes number as the leading immunocompetent blood cells that characterize the body's defenses and the state of specific immunity [4]. From the data in Table i we see that the simulation of oxidative stress in rats of the first and second experimental groups by intramuscular injection of 50% carbon tetrachloride oil solution significantly affects the T-lymphocytes number in the blood and their functional activity.
Thus, the relative total T-lymphocytes number in the blood of rats of the Ei group on the 2nd, 5th, and
10th day of the test was respectively 1.12 (p<0.01), 1.09 (p<0.01 ), and 1.09 (p<0.05) times less than in control. At the same time, the number of undifferentiated forms of total T-lymphocytes in the blood of rats of this experimental group in these study periods was respectively 1.22 (p<0.01), 1.18 (p<0.01), and 1.17 (p<0.05) times greater than in control. The total T-lymphocytes number with low receptor density in the blood of rats of the first trial group was 1.08 times lower, relative to the control, respectively, on the 2nd (p<0.05) and 10th (p<0.01) days of research. Compared to the control, in the blood of rats of this group, there were probably a smaller number of medium-avid forms of total T-lymphocytes on the 2nd day and high-avid -on the 5th day.
Similar changes were recorded in the blood of rats when examining the active T-lymphocytes number. Thus, compared with the control, a probable decrease in the relative number of active T-lymphocytes against the background of increasing their undifferentiated forms in the first experimental group was observed in all study periods, especially (p<0.001) on the 2nd and 5th days. The reduction in the relative number of active T-lymphocytes in the blood of rats was mainly due to a decrease in the number of low-avid forms in all study periods (p<0.05-0.01), and on the 5th day - the number of cells with medium receptor density (p<0.05).
The theophylline-resistant T lymphocytes number in the blood of rats has changed slightly under oxidative stress. The probable decrease compared to the control, the relative number of theophylline-resistant T-lymphocytes and an increase of their undifferentiated forms in the blood of rats of the E1 group were recorded only on the 2nd and 5th days of the research. On the 2nd day of investigations in the blood of rats of this group, the number of theophylline-resistant T-lymphocytes with medium and high receptor density was 1.24 and 1.47 times less (p<0.05) than in the blood animals of the control group.
Compared with the control, the theophylline-sensitive T-lymphocytes reduced 1.14 times (p<0.01) in the E1 group on the 2nd day of the study. In the research dynamics, there were no significant changes in the immunoregulatory index in rats of the experimental groups compared to the control.
The test results indicate an inhibitory effect of carbon tetrachloride on the number of T-lymphocytes and their immunoregulatory populations. Thus, due to oxidative stress, the formation of T-lymphocytes is inhibited, and the functional activity of these cells is significantly reduced.
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Table 1.
The T-lymphocytes number and their functional activity in the blood of rats, % (M±m, n=4)
Indexes Animals Research Day
groups second fifth tenth
1 2 3 4 5
C 33.60±0.33 33.00±0.58 34.00±i.i5
T-general, Ei 4i.00±0.9i** 39.00±i.08** 39.75±i.25*
0 E2 37.50±0.29*** 36.50±0.65* 34.00±0.4i
low-avid C 40.00±0.60 40.70±0.88 39.30±0.33
(3-5) Ei 37.00±0.7i* 39.00±0.4i 36.25±0.48**
E2 38.50±0.65 38.25±0.48* 39.25±0.48
medium avid C 23.67±0.33 22.30±i.45 23.70±0.66
(6-10) Ei 20.00±i.i0* i9.50±i.04 2i.00±0.9i
E2 2i.00±0.7i* 22.75±0.63 24.00±0.4i
highly avid C 2.70±0.66 4.00±0.i0 3.00±0.58
M Ei 2.00±0.4i 2.50±0.29** 3.00±0.9i
E2 3.00±0.4i 2.50±0.29** 2.75±0.25
% C 66.30±0.33 67.00±0.58 66.00±i.i5
Ei 59.00±0.9i** 6i.00±0.08** 60.25±i.25*
E2 62.50±0.29*** 63,50±0,65* 66.00±0.4i
T-active, C 57.30±0.70 56.30±0.30 57.70±0.30
0 Ei 64.75±0.63*** 63.25±0.25*** 63.50±0.86**
E2 63.25±0.48*** 60.75±0.48*** 55.50±0.86
low-avid C 33.30±0.33 34.60±0.33 33.70±0.60
(3-5) Ei 29.25±0.85* 30.50±0.65** 29.75±0.94*
E2 30.00±0.4i** 3i,00±0,4i** 35.50±0.86
medium avid C 7.00±0.58 7.70±0.33 6.60±0.30
(6-10) Ei 5.25±0.63 5.75±0,48* 6.00±0.4i
E2 6.00±0.4i 7.00±0.4i 8.00±0.4i
highly avid C i.66±0.33 i.30±0.33 i.30±0.30
M Ei 0.75±0.25 0.50±0.29 0.75±0.25
E2 0.75±0.25 i.25±0.25 i.00±0.4i
% C 42.70±0.66 43.60±0.33 42.30±0.30
Ei 35.25±0.63*** 36.75±0.25*** 36.50±0.86*
E2 36.75±0.48*** 39.25±0.48*** 44.50±0.86
T-helpers, C 57.00±0.58 58.00±0.58 62.70±i.76
0, Ei 6i.50±0.65** 60,75±0,25** 66.75±0.85
E2 60.00±0.4i** 58.50±0.65 59.25±0.85
low-avid C 25.60±0.33 25.30±0.88 24.00±0.58
(3-5) Ei 25.00±0.4i 25.00±0.9i 22.75±0.95
E2 25.50±0.65 27,00±0,7i 26.25±0.25*
medium avid C i4.00±0.58 i3.60±0.88 ii.33±i.20
(6-10) Ei ii.25±0.63* ii.75±0.75 i0.00±0.7i
E2 i2.75±0.25 i3.00±0.4i ii.25±0.63
highly avid C 3.30±0.30 3.00±0.58 2.00±0.58
M Ei 2.25±0.25* 2.50±0.65 i.50±0.64
E2 i.75±0.25* i.50±0.65 2.25±0.48
C 43.00±0.58 42.00±0.58 37.33±i.76
% Ei 38.50±0.65** 39.25±0.25** 34.25±0.48
E2 40.00±0.4i** 4i.50±0.65 39.75±0.85
T-suppressors, C 23.30±0.33 25.00±i.i5 28.66±i.33
% Ei 20.50±0.50** 2i.75±i,ii 26.00±i.63
E2 22.50±0.50 22.00±0.7i 26.35±0.75
Immunoregulatory index C i.80±0.i0 i.63±0.08 i.27±0.i2
Ei i.85±0.i0 i.77±0.i0 i.30±0.i0
E2 i.73±0.i0 i.85±0.09 i.50±0.i0
Note. In this and the following table, the differences are statistically significant in relation to the rats of the control group: * - p<0.05; ** - p<0.01; *** - p<0.001.
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The resals in the table shows that the liposomal drug administration has a regulatory effect on the number of T-lymphocytes in the blood. Thus, on the 2nd and 5th day of trial in the blood of mice of the E2 group, the relative number of total T-lymphocytes was 1.06 (p<0.001) and 1.05 (p<0.05) times less than in control, and respectively 1.05 and 1.04 times greater than in rats of the E1 group. In comparison with the control, in these research periods, the relative number of active T-lym-phocytes in the blood of rats of the E2 group decreases to a lesser extent than in animals of the first group. It should be noted that the normalization in the blood of rats of the E2 group the number of total T-lymphocytes on the 2nd day of the study was due to medium-avid forms and active T-lymphocytes - their low-avid forms. Less pronounced than in rats of the E1 group, in the blood of animals of E2 group, there was an increase compared to control. The number of undifferentiated forms of total and active T-lymphocytes on the 2nd and 5th day was (p<0.05-0.001).
Studies have shown differences in the abundance of active and theophylline-resistant T-lymphocytes in the control and second experimental groups on the 10th. However, the introduction of liposomal drugs tended to increase. At the same time, in the blood of rats of the E2 group in this research period, a probable rise in the low-avid forms of theophylline-resistant T-lymphocytes number was detected. Thus, on the 10th investigation day, the lymphocytes number of these forms in the E2 group, compared with the control, was more significant (p<0.05). On the 2nd day of the ana-lisys, the relative number of theophylline-resistant T lymphocytes in the blood of the E2 group rats was less (p<0.01) than in control. These changes occurred mainly due to a reduction in the blood of rats of the experimental group 1.9 times (p<0.05) the evident forms number against the background of an expansion in 1.05 times (p<0.01) the undifferentiated cells number, compared with control.
Thus, from the obtained results, it was established that on the 2nd day, the effect of drug components was expressed. Although not yet completely manifested, and a probable increase in the blood of rats on the 10th day, the theophylline-resistant T- lymphocytes with low receptor density indicate a prolonged effect of its liposomal form on the functional immunocompetent cells activity.
Regarding the T-lymphocytes-suppressors number in the blood of the second experimental group on the 10th test day, there was a tendency to raise them compared to the control. Thus, an increase in the im-munoregulatory index in this group was observed on the 10th day of the study. In general, studies have shown that under the action of oxidative stress caused by tetrachloroethane, there is a reduction in the functional activity of immunocompetent cells. The liposo-mal preparation administration has a normalizing effect on the cellular component of specific immunity. It leads to an improvement of receptors on the surface of T lymphocytes. Interact with more antigenic determinants, resulting in the body's immune response [20, 21].
Thus, under the action of liposomal drug components in rat's immunocompetent cells, there is a
reorganization of lymphocytes' plasma membranes to strengthen the growth of receptors of a plasmolemma that indicates an expansion of a receptor field of cells.
Conclusions
1. The intramuscular tetrachloroethane administration to rats has an immunosuppressive effect on the T-cell immunity activity, as evidenced by a decrease in the relative T-lymphocytes number (total, active, and theophylline-resistant) and a reduction in functional activity due to redistribution of receptor cells.
2. The utaphosphane, interferon, milk thistle, and fat-soluble vitamins A, D3, E in the form of liposomal drugs application under oxidative stress has a normalizing effect on immunocompetent blood cells, in particular, increase the number of T lymphocytes and restore their functional activity
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