113 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 dOI 10.11603/IJMMR.2413-6077.2019.2.10698 IMpACT OF NITRIC OXIDE SYNTHESIS MODULATORS ON THE CYTOKINES pROFILE IN EXpERIMENTAL ANTIpHOSpHOLIpID SYNDROME *O.Z. Yaremchuk, K.A. Posokhova, I.P. Kuzmak, M.I. Kulitska, O.O. Shevchuk, A.S. Volska, P.H. Lykhatskyi I. HORBACHEVSKY TERNOPIL NATIONAL MEDICAL UNIVERSITY, TERNOPIL, UKRAINE Background. Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of specific antibodies. Objective. The aim of the study was to investigate the effect of combined use of L-arginine and aminoguanidine on cytokine profile (IL-1β, IL-6, TNF-α, IL-4, IL-10) in experimental APS. Methods. The study was performed on BALB/c female mice. L-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were used for correction. Serum cytokines concentrations were assessed using an ELISA test. Results. It was found that in APS the concentration of proinflammatory cytokines IL-1β, IL-6 and TNF-a increases in 3.2, 2.3 and 4.5 times respectively, compare to the control. At the same time a decrease of the IL-4 and IL-10 in 1.9 and 2.2 times was evidenced. Aminoguanidine, a selective iNOS inhibitor, caused a significant decrease of TNF-α by 57% (p<0.001), but there were no changes in IL-1β, IL-6, IL-4 and IL-10 compare to the APS-group. L-arginine combined with aminoguanidine caused a significant decrease in the concentration of IL-1β by 30% (p<0.01), IL-6 – by 16% (p<0.05), TNF-a – by 59% (p<0.001) compare to the control. At the same time, the concentration of IL-4 increased by 35% (p <0.01), IL-10 – by 25% (p<0.005). Conclusions. Combined use of the precursor of the NO synthesis L-arginine and aminoguanidine, a selective iNOS inhibitor, leads to a decrease in the concentrations of IL-1β, IL-6, TNF-a and an increase of IL-4 and IL-10 compare to the group of the BALB/c mice with APS and the group of animals administered with aminoguanidine. KEY WORDS: antiphospholipid syndrome; cytokines; nitric oxide; L-arginine; aminoguanidine. *Corresponding Author: Yaremchuk Olha, Ph.D., Associate Professor of the Department of Medical Biochemistry, I. Hor- bachevsky Ternopil National Medical University, Maidan Voli, 1, Ternopil, Ukraine. E-mail: yaremchuk@tdmu.edu.ua Introduction Antiphospholipid antibody syndrome (APS) is an autoimmune condition characterized by the presence of antiphospholipid antibodies (aPL) [1], encompassing primary APS, secondary APS, seronegative APS (SNAPS) and catastrophic APS (CAPS) [2]. Secondary APS can be found in association with other autoimmune conditions such as systemic lupus erythematosus, rheuma- toid arthritis, autoimmune thyroid disease, Crohn’s disease, Sjogren syndrome, systemic sclerosis, lymphoma or leukemia, malignancies of the ovary and cervix, drug induced as with oral contraceptive pills or in infectious disease such as HIV or syphilis [1]. In CAPS a systemic inflammatory response, systemic endothelial dysfunction and DIC develop. These processes are the pathogenetic basis for development of multiple organ failure [3, 4]. SNAPS is negative for lupus anticoagulant and antiphospholipid antibodies [2]. The diagnostic APS criteria are anticardiolipin (aCL), antiβ2­glycoprotein­I (aβ2GPI) and lupus anticoagulant (LA) [1, 5]. APS can be classified only in the presence of thrombotic (non­inflam­ matory arterial, venous or small vessel throm- bosis) obstetric complications (death of one or more morphologically normal fetus at or beyond the 10th week of gestation; one or more premature birth of normal fetus before the 34th week due to eclampsia, pre-eclampsia or pla- cental insufficiency), or increased aPL level [2]. The mechanisms of thrombosis in APS have not been fully studied yet [6]. aβ2GPI antibodies are central in pathogenic APS mechanisms and, although the full pa- thogenesis of APS is not clear yet, the binding of these aPL antibodies to the antigens on the cell surface of platelets, monocytes, endothelial cells and trophoblasts triggers intracellular sig- naling with subsequent activation and alteration International Journal of Medicine and Medical Research 2019, Volume 5, Issue 2, p. 113-121 copyright © 2019, TNMU, All Rights Reserved O.Z. yaremchuk et al. 114 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 of diverse cell functions. Cellular activation starts after the binding of the complex aβ2GPI antibody/β2GPI [5, 7]. β2GPI is the most important antigenic target [2]. Platelets acti- vation and the subsequent release of throm- bo xane favor their aggregation. Thrombosis at the fine vasculature of the target organ is thought to be more dependent from antibodies against the anticoagulant AnV. Endothelial cells and monocytes activation determine a pro- aggregation status due to up-regulated expres- sion of adhesion molecules, such as E-selectin, and release of tissue factor (TF) and proin- flammatory cytokines [5]. Many patients with aPL antibodies remain asymptomatic [2]. An important factor in APS immuno pa- thogenesis is dysregulation of cytokine balance with increased synthesis of proinflammatory cytokines [8, 9]. Cytokines are the most versatile system of regulation. Cytokines, being synthesized at the inflammation site, affect virtually all cells invol­ ved in the inflammation development, as well as granulocytes, macrophages, fibroblasts, endothelial cells, epithelium cells, T and B lymphocytes [10].The inflammatory processes are controlled by the proinflammatory (IL­1, IL­2, IL­6, IL­8, IL­12, TNF­α, IFN) and anti­ inflammatory (IL­4, IL­10, TGF) cytokines [11]. Therefore, the study of pathobiochemical mechanisms of APS development, particularly establishment of the role of the cytokine system in development of this pathology, and search for effective methods of its treatment is an urgent and social issue [1, 6, 11, 12]. One of the links that are involved in the mechanisms of APS development is the nitric oxide (NO) system. In obstetric APS, the syn- thesis and bioavailability of nitric oxide (NO), which is involved in the regulation of vascular tone and blood coagulation properties, are impaired in the endothelium [4]. According to Cella M [13], a decrease in NO levels causes abortion and premature birth. On the other hand, NO overproduction mediated with indu- cible NO synthase (iNOS) increases uterine contractions and the risk of miscarriage [13]. Contradictions of the existing information on the involvement of the NO system in APS development as well as on the efficacy of NO precursors in reducing the manifestations of this pathology necessitates further study of the role of this system in APS. The objective of research is to investigate the effect of combined use of L-arginine and aminoguanidine on cytokine profile (concent­ ration of IL­1β, IL­6, TNF­α, IL­4, IL­10) in experimental antiphospholipid syndrome. Methods Female BALB/с mice, which were kept on a standard vivarium diet, were used in the re- search. The experiments were carried out following the principles of bioethics according to the “General Ethical Principles of Animal Experiments”, approved by the First National Congress on Bioethics (Kyiv, 2001) and in accordance with the provisions of the “European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes” (Strasbourg, 1986) and EU Directive 2010/10/63 EU for animal experiments. APS was modeled using cardiolipin (Sigma, USA), which was injected intramuscularly four times (30 μg per 1 injection, the injection inter­ val was 14 days) [14]. To enhance the effec- tiveness of the immune response, cardiolipin was emulsified in 75 μl of complete Freund’s adjuvant (first injection); subsequent injections were performed with incomplete Freund’s adjuvant. APS was developing for 2 weeks after the last cardiolipin injection. The experimental animals were divided into 4 groups: the 1st – the intact; the 2nd – the BALB/c mice with APS; the 3rd – the animals with APS administered with aminoguanidine, the 4th – the animals with APS administered with L-arginine in combination with aminoguanidine. L-arginine (Sigma, USA, 25 mg/kg) and aminoguanidine (Khimlaboratorreaktiv, Ukraine, 10 mg/kg) were administered intraperitoneally once a day for 10 days after APS development. The animals of the control group were managed with the same volumes of the solvent intraperitoneally. In 10 days after confirmation of APS the animals were taken out of the experiment by thiopental sodium anesthesia (intraperitoneal admi ni- stration of 1% solution at a dose of 50 mg/kg of animal body weight). The concentration of cytokines IL­1β, IL­6, TNF­α, IL­10, IL­4 in the serum of BALB/c mice was determined by enzyme immunoassay using standard kits adapted for mice of Express Biotech International, USA (Mouse IL­1β ELISA Assay, Mouse IL­6 ELISA Assay, Mouse TNF­α ELISA Assay, Mouse IL-10 ELISA Assay, Mouse IL-4 ELISA Assay). The concentration of cytokines was expressed in pg/ml. Statistical processing of digital data was performed by means of Excel software (Micro- soft, USA) and STATISTICA 6.0 (Statsoft, USA) using non-parametric methods of estimation O.Z. yaremchuk et al. 115 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 O.Z. yaremchuk et al. for the attained data. The arithmetic mean (M), its variance and standard error of the mean (m) were assessed for all parameters. The significant difference between the independent quanti- tative values was determined using the Mann- Whitney test. The changes were statistically significant at p≤0.05. Results According to the attained results, an in- crease of the concentration of IL­1β in 3.2 times (p<0.001) was proved in the BALB/c mice with APS compare to the control (Fig. 1). An increase in the concentration of IL-6 in 2.3 times (p<0.001) in the serum of the animals with APS was evidenced compare to the intact animals (Fig. 2). TNF­α concentration increased in 4.5 times (p<0.001) in the serum of the BALB/c mice with APS compare to the control (Fig. 3). At the same time, the anti­inflammatory cytokine IL-4 concentration reduced in 1.9 times (p<0.001) and IL-10 – in 2.2 times (p<0.001) compare to the control (Fig. 4-5). The administration of aminoguanidine, a selective iNOS inhibitor, did not cause significant changes concentrations of IL­1β and IL­6 in the serum of the BALB/c mice with APS compare to the control (Fig. 1-2). In the case of amino gua- Fig. 1. IL­1β content in the blood serum of the BALB/c mice with APS in the case of administration of L-ar- ginine and aminoguanidine (M±m, n=10). Notes: Herein, and Figures 2-5. Conventional name of animal groups: 1 – Control; 2 – Antiphos- pholipid syndrome (APS); 3 – APS + aminoguanidine; 4 – APS+L-arginine+aminoguanidine. * – р<0.05 compare to the control group; ** – р<0.05 compare to the group of animals with APS. Fig. 2. IL-6 content in the blood serum of the BALB/c mice with APS in the case of administration of L-ar- ginine and aminoguanidine (M±m, n=10). Fig. 3. TNF­α content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). Fig. 4. IL-4 content in the blood serum of the BALB/c mice with APS in the case of administration of L-ar- ginine and aminoguanidine (M±m, n=10). Fig. 5. IL-10 content in the blood serum of the BALB/c mice with APS in combined administration of L-argi- nine and aminoguanidine (M±m, n=10). Statistical processing of digital data was performed by means of Excel software (Microsoft, USA) and STATISTICA 6.0 (Statsoft, USA) using non-parametric methods of estimation for the attained data. The arithmetic mean (M), its variance and standard error of the mean (m) were assessed for all parameters. The significant difference between the independent quantitative values was determined using the Mann-Whitney test. The changes were statistically significant at p≤0.05. Results According to the attained results, an increase of the concentration of IL-1β in 3.2 times (p <0.001) was proved in the BALB/c mice with APS compare to the control (Fig. 1). Fig. 1. IL-1β content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). Notes: Herein, and Figures 2-5. Conventional name of animal groups: 1 – Control; 2 – Antiphospholipid syndrome (APS); 3 – APS + aminoguanidine; 4 – APS+L-arginine+aminoguanidine. * – р<0.05 compare to the control group; ** – р<0.05 compare to the group of animals with APS. An increase in the concentration of IL-6 in 2.3 times (p <0.001) in the serum of the animals with APS was evidenced compare to the intact animals (Fig. 2). 0 10 20 30 40 50 60 1 2 3 4 IL -1 β, p g/ m l * ** Fig. 2. IL-6 content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). TNF-α concentration increased in 4.5 times (p <0.001) in the serum of the BALB/c mice with APS compare to the control (Fig. 3). Fig. 3. TNF-α content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). At the same time, the anti-inflammatory cytokine IL-4 concentration reduced in 1.9 times (p <0.001) and IL-10 – in 2.2 times (p <0.001) compare to the control (Fig. 4-5). 0 5 10 15 20 25 1 2 3 4 IL -6 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 TN F- α, p g/ m l * ** ** Fig. 2. IL-6 content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). TNF-α concentration increased in 4.5 times (p <0.001) in the serum of the BALB/c mice with APS compare to the control (Fig. 3). Fig. 3. TNF-α content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). At the same time, the anti-inflammatory cytokine IL-4 concentration reduced in 1.9 times (p <0.001) and IL-10 – in 2.2 times (p <0.001) compare to the control (Fig. 4-5). 0 5 10 15 20 25 1 2 3 4 IL -6 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 TN F- α, p g/ m l * ** ** Fig. 4. IL-4 content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). Fig. 5. IL-10 content in the blood serum of the BALB/c mice with APS in combined administration of L-arginine and aminoguanidine (M±m, n=10). The administration of aminoguanidine, a selective iNOS inhibitor, did not cause significant changes concentrations of IL-1β and IL-6 in the serum of the BALB/c mice with APS compare to the control (Fig. 1-2). In the case of aminoguanidine use, a significant decrease in the concentration of TNF-α by 57% (p <0.001) was evidenced compare to the intact animals (Fig. 3). It was found out that, under the influence of aminoguanidine, the concentrations of IL-4 and IL-10 did not change significantly compare to the control group of animals (Fig. 4-5). 0 5 10 15 20 25 30 35 1 2 3 4 IL -4 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 IL -1 0, p g/ m l * ** Fig. 4. IL-4 content in the blood serum of the BALB/c mice with APS in the case of administration of L-arginine and aminoguanidine (M±m, n=10). Fig. 5. IL-10 content in the blood serum of the BALB/c mice with APS in combined administration of L-arginine and aminoguanidine (M±m, n=10). The administration of aminoguanidine, a selective iNOS inhibitor, did not cause significant changes concentrations of IL-1β and IL-6 in the serum of the BALB/c mice with APS compare to the control (Fig. 1-2). In the case of aminoguanidine use, a significant decrease in the concentration of TNF-α by 57% (p <0.001) was evidenced compare to the intact animals (Fig. 3). It was found out that, under the influence of aminoguanidine, the concentrations of IL-4 and IL-10 did not change significantly compare to the control group of animals (Fig. 4-5). 0 5 10 15 20 25 30 35 1 2 3 4 IL -4 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 IL -1 0, p g/ m l * ** 116 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 nidine use, a significant decrease in the con­ centration of TNF­α by 57% (p<0.001) was evi­ denced compare to the intact animals (Fig. 3). It was found out that, under the influence of aminoguanidine, the concentrations of IL-4 and IL­10 did not change significantly compare to the control group of animals (Fig. 4-5). In the case of administration of the prede- cessor of the synthesis of NO L-arginine in combination with aminoguanidine, a significant decrease in the concentration of IL­1β by 30% (p<0.01), IL-6 by 16% (p<0.05), TNF-a by 59% (p<0.001) was established compare to the control (Fig. 1-3). At the same time, the con- centration of anti­inflammatory cytokines IL­4 increased by 35% (p <0.01) and IL-10 – by 25% (p<0.005) compare to the control animals (Fig. 4-5). The results of the study proved that a significant decrease in the concentration of IL­1β by 22% (p<0.05), IL­6 by 23% (p<0.005) was evidenced in the case of combined adminis- tration of L-arginine and aminoguanidine compare to the indicators of the 3rd group of animals, which were administered with ami- noguanidine (Fig. 1-2). An increase of the anti- inflammatory cytokine IL­4 concentration by 32% (p<0.05) and IL-10 by 19% (p<0.05) was proved compare to the 3rd group of the BALB/c mice administered with aminoguanidine (Fig. 4-5). Discussion Besides the pathogenic role of the aPL, pro- inflammatory cytokines and chemokines are significant in the pathogenesis of APS [12]. IL­1, TNF-a and endotoxins induce tissue factor (TF) expression in endothelial cells, monocytes, macrophages promoting blood clotting [10, 15]. The inhibitors of IL-1 production are IL-4, IL-10, IL­12, TNF­α [16]. IL­6 is involved in regulation of T and B cell interactions, macrophage, endo- theliocytes activity. IL-6 induces production of acute-phase proteins, stimulates hematopoiesis and platelet formation [16]. The attained results on increased concent- rations of proinflammatory cytokines (IL­1β, IL­6, TNF­α) in the serum of the experimental animals with APS conform with the literature [6, 17, 18]. According to N.V. Seredavkina [6], an increased concentration of IL­6 and TNF­α in the patients with APS compare to the control group was established. It is not clear whether aPL affect endothelial cells directly or through TNF­α. Regardless of the mechanism, the pro­ thrombotic condition, typical of APS, is asso- ciated with both significantly increased aPL levels as well as high TNF­α concentration [11]. According to J. Swadzba et al. [15] TNF­α is one of the main proinflammatory cytokines in APS; its level is increased and reflects pathological processes in endothelial cells. According to the literature, aPL and TNF-a can activate the endo- thelium and induce prothrombotic phenotype of endothelial cells, leading to increased thrombin production. Activation of endothelial cells causes upregulation of TF, which has been suggested to be a major potential mechanism of APS-related thrombosis. Once endothelial cells are activated, TF regulation can be more enhanced by a synergizing effect of TNF-a and factor Xa, thus expression of adhesion molecules (ICAM-1, VCAM-1, E and P selectins) and for ma- tion of endothelial microparticles take place [15]. According to A. Farzaneh-Far et al. [17], who investigated the levels of CRP IL 6, ISAM-1, pTNF α­P2, pTNF α­P2 in the patients with SLE, only increased pTNF α­P1 and pTNF α­P2 were asso­ ciated with aPL positivity. According to NV Sere- davkin a negative correlation between CRP and I g G β 2 G P 1 l e v e l s w a s e s t a b l i s h e d [ 6 ] . R.R. Forastiero et al. [18] established that IL 6 levels were greater in the patients with APS and aPA carriers than in the control group. TNF concentration was the same in the patients with APS and aPA carriers but higher than in the control group. In the patients with positive aPA, a direct correlation between IL 6 and TNF α was proved [18]. Under the experimental conditions it has been established that TNF-a may manifest antiplatelet and antithrombotic activity [15] According to the literature, IL­1β activates the synthesis of IL­6, S100B, α1­antihymotrypsin, inducible nitric oxide synthase (iNOS) causing increased NO synthesis [19, 20]. The iNOS is crucial in the primary proinflammatory response in macrophages [21]. AG is a nucleophilic hydra- zine compound, structurally similar to L-arginine in that these compounds contain two chemically equivalent guanidino nitrogen groups and to L-arginine analogues that com petitively inhibit NO synthase. AG completely prevents inflam­ matory stimuli induced for mation of NO, and it is a potent inhibitor of the cytokine-inducible isoform NOS [22]. The results of our studies proved that intro- duction of aminoguanidine, a selective iNOS inhibitor, did not cause significant changes in the concentration of proinflammatory cytokines (IL­1β and IL­6) and anti­inflammatory cytokines (IL-4 and IL-10) in the serum of the BALB/c mice O.Z. yaremchuk et al. 117 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 O.Z. yaremchuk et al. with APS compare to the control group of ani- mals. At the same time, in the case of amino- guanidine administration a significant decrease in TNF­α concentration was proved compare to the intact animals. According to EI Ferreira et al. [23] aminoguanidine decreases TNFα levels, oxidative stress indicators, and NO metabolites. It is established that increased concent- rations of TNF­α are associated with pregnancy miscarriage in APS [6, 8], endotheliocytes acti- vation, and chemokine amplification that leads to subendothelial leukocyte accumulation, endothelial dysfunction, microcirculation dis- tur bances [16]. Early endothelial dysfunction was observed in APS [24]. Patients with APS displaying throm- bosis exhibited low plasma levels of nitrites and nitrates, which are the stable metabolites of NO breakdown. aPL can act as antagonists of endo- thelial nitric oxide synthase (eNOS) through β2GPI, and this interaction may impair NO synthesis. In particular, attenuation of eNOS activation by aPL was mediated by reduced phosphorylation of eNOS serine. This inhibition of eNOS phosphorylation was shown to be dependent upon protein phosphatase 2A, β2GPI, and apolipoprotein E receptor 2. aPL inhibition of eNOS activity contributes to throm- bus formation, increased leukocyte adhesion, and alterations in vascular tone [4]. It is established that violation of the bio- availability of NO may be one of the causes of endothelial dysfunction. This may be associated with both the lack of substrate for NO L-arginine synthesis as well as formation of superoxide anion which rapidly binds and inactivates NO [24]. NO synthesis is not dependent on L-arginine concentration in physiological states. In patho- logical conditions, the availability of L-arginine may determine production of NO. It is proved that L-arginine is necessary for adequate trans- lation of iNOS. When iNOS is being activated, superoxide anion is produced, which forms a highly reactive peroxynitrite, which in turn pro- duces nitrosylation of amino acid residues sensitive to it, especially tyrosine, that leads to conformational changes in the structure of pro tein molecules. With administration of L-arginine the functional characteristics of T-cells enhance, production of antibodies increases as well. NO-dependent effect of L-arginine on the immune system may be hormone-mediated [25]. The next objective of our study was to in- vestigate the effect of combined use of L-argi- nine and aminoguanidine on cytokine profile in APS. In the case of the use of the precursor of NO L-arginine synthesis in combination with aminoguanidine, a selective iNOS inhibitor, a significant decrease in the concentration of proinflammatory cytokines (IL­1β, IL­6, TNF­a) and an increase in the concentration of anti- inflammatory cytokines (IL­4, IL­10) was estab­ lished compare to the control group of animals. The attained results are consistent with the literature [25, 26]. These effects can be explained by the fact that glutamine formed from L-ar ginine is a conditionally essential amino acid and reduces the level of TNF­α soluble receptors [26]. According to VM Sheibak et al. [25] ad ministration of L-arginine decreases the level of IL-6. According to P. Soltesz et al. [12], besides the conventional Th1 pathway, Th2 cytokines are crucial in the mechanisms of APS develop- ment, i.e. IL-4 and IL-10. Various immunocom- petent cells regulate the proinflammatory cascade that leads to cytokine imbalance and activated circulating lymphocytic pool in APS. This proinflammatory process leads to endo­ thelial dysfunction, development of arterial and venous thrombosis [12]. As a result of the re- search, a decrease in anti­inflammatory cyto­ kines (IL­4, IL­10) in APS was established; the results are consistent with the literature [11, 12]. According to P. Soltesz et al. [12], the mar- kers of endothelial dysfunction positively corre- late with IL-4 levels in APS. It allows suggesting that by activation of the humoral and cellular immune responses, IL-4 is crucial in development of endothelial dysfunction, atherosclerosis, arterial and venous thrombosis. IL-4 stimulates B and T cell proliferation as well as differentiation of CD4 + T cells into Th2 cells [12]. According to A. Menachem et al. [11] cytosolic and secreted IL­10 and IFN­γ levels in eAPS mice were lower at 6 and 15 weeks and higher at 24 weeks after immunization com- pared to adjuvant mice. IL­10 is significant in autoimmune diseases. As a result of other stu- dies, IL-10 level was decreased in the serum of the patients with APS [12]. IL-10 inhibits secretion of IL-4, IL-5 and IFN­γ, growth factors and chemokines, and therefore acts as a key counter-regulator of autoimmune processes [12]. One of the func- tions of IL-10 is inhibition of the synthesis of proinflammatory cytokines: IL­1, IL­6, IL­12 and TNFα via a STAT3­dependent mechanism [27] and enhancement of IL-1 receptor antagonist 118 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 expression [19]. Decreased level of IL-10 in the serum in cases of APS compare to the control confirms the fact that IL10­mediated processes are impaired in APS that is why it leads to vascular damage [12]. Low IL­10 levels enable TNF­α unregulated production, resulting in procoagulant state. Decreased IL-10 levels can be associated with lymphocyte activation, which leads to the con- tinuation of the autoimmune response. During the B-cell activation, IL-10 delivers signals that promote the apoptosis of B cells [11]. Conclusions Thus, that in the serum of BALB/c mice with APS, an increase in the concentrations of proin- flammatory cytokines (IL­1β, IL­6, TNF­a) and a decrease in the concentrations of anti­inflam­ matory cytokines (IL-4 and IL-10) was estab- lished compare to the control parameters. With the introduction of aminoguanidine, a selective iNOS inhibitor, a decrease in the concentration of TNF­α was proved compare to that of the animals with APS. In the case of the use of the precursor of NO synthesis L-arginine in com- bination with aminoguanidine, a significant decrease in concentrations of IL­1β, IL­6, TNF­α and an increase of IL-4 and IL-10 was evidenced compare to the group of BALB/c mice with APS and the group of animals administered with aminoguanidine. Funding This research received no external funding. Conflict of Interests The authors declare no conflict of interest. Authors Contributions Yaremchuk O.Z. – writing – original draft, con- ceptualization, project administration, metho- dology, investigation, formal analysis, Posokho - va K.A. – supervision, conceptualization, writing – review & editing, Kuzmak I.P. – data curation, Kulitska M.I. – investigation, Shevchuk O.O. – investigation, writing – review & editing, Volska A.S. – investigation, Lykhatskyi P.H. – data curation. ВплиВ модуляторіВ синтезу оксиду азоту на показники цитокіноВого проФілю при експерименталЬному антиФосФоліпідному синдромі О.З. Яремчук, К.А. Посохова, І.П. Кузьмак, М.І. Куліцька, О. О. Шевчук, А.С. Вольська, П.Г. Лихацький ТЕРНОПІЛЬСЬКИЙ НАЦІОНАЛЬНИЙ МЕДИЧНИЙ УНІВЕРСИТЕТ ІМЕНІ І. Я. ГОРБАЧЕВСЬКОГО, ТЕРНОПІЛЬ, УКРАЇНА Вступ. Антифосфоліпідний синдром (АФС) – це автоімунне захворювання, яке характеризується наявність антифосфоліпідних антитіл, артеріальними та венозними тромбозами, тромбоцитопенією, невиношування вагітності. Мета дослідження. Дослідити вплив комбінованого застосування L-аргініну та аміногуанідину на показники цитокінового профілю (концентрацію IL-1β, IL-6, TNF-α, IL-4, IL-10) при експериментальному антифосфоліпідному синдромі. Методи дослідження. Дослідження виконано на мишах-самках лінії BALB/с, в яких моделювали АФС. Для корекції використовували L-аргінін (25 мг/кг) та аміногуанідин (10 мг/кг). Визначення концентрації цитокінів IL-1β, IL-6, TNF-α, IL-10, IL-4 у сироватці крові мишей BALB/c проводили методом імуноферментного аналізу з використанням стандартних наборів реактивів. Результати й обговорення. Отримані результати свідчать, що у сироватці крові мишей BALB/с за умов АФС відбувається зростання концентрації прозапальних цитокінів IL-1β у 3,2 раза, IL-6 у у 2,3 раза, TNF-а в 4,5 разів, відносно контролю. Спостерігалось зниження концентрації протизапальних цитокінів IL-4 в 1.9 раза та IL-10 в 2,2 раза у групі тварин з АФС, порівняно із показниками контролю. На фоні застосування селективного інгібітора iNOS аміногуанідину встановлено достовірне зниження концентрації TNF-а на 57 % (р<0.001), проте концентрація IL-1β, IL-6 IL-4 та IL-10 достовірно не змінювалася у сироватці крові мишей BALB/c з АФС, порівняно з показниками тварин з АФС. На фоні застосування попередника синтезу NO L-аргініну в комбінації з аміногуанідином встановлено достовірне зниження концентрації IL-1β на 30 % (р<0.01), IL-6 на 16 % (р<0.05), TNF-а на 59 % (р<0.001), відносно контролю. Водночас зростала концентрація протизапальних цитокінів IL-4 на 35 % (р<0.01) та IL-10 на 25% (р<0.005), порівняно з показниками групи мишей BALB/c з АФС. O.Z. yaremchuk et al. 119 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2019 Vol. 5 issue 2 O.Z. yaremchuk et al. Висновки. Встановлено, що комбіноване застосування попередника синтезу NO L-аргініну та селективного інгібітора іNOS аміногуанідину призводить до зниження концентрації IL-1β, IL-6, TNF-а та зростання IL-4 та IL-10, порівняно з показниками групи мишей BALB/c з АФС та групи тварин, яким вводили аміногуанідин. КЛЮЧОВІ СЛОВА: антифосфоліпідний синдром; цитокіни; оксид азоту; L-аргінін; аміногуанідин Відомості про авторів Яремчук Ольга Зеновіївна – кандидат біологічних наук, доцент кафедри медичної біохімії Тернопільського національного медичного університету імені І.Я. Горбачевського, Тернопіль, Україна. Посохова Катерина Андріївна – доктор медичних наук, професор кафедри фармакології з клінічною фармакологією Тернопільського національного медичного університету імені І.Я. Гор­ бачевського. Кузьмак Ірина Петрівна – кандидат біологічних наук, доцент кафедри медичної біохімії Тернопільського національного медичного університету імені І.Я. Горбачевського. Куліцька Марія Іванівна – кандидат біологічних наук, доцент кафедри медичної біохімії Тернопільського національного медичного університету імені І.Я. Горбачевського. Шевчук Оксана Олегівна – кандидат медичних наук, доцент кафедри фармакології з клінічною фармакологією Тернопільського національного медичного університету імені І.Я. Горбачевського. Вольська Аліна Станіславівна – кандидат біологічних наук, доцент кафедри фармакології з клінічною фармакологією Тернопільського національного медичного університету імені І.Я. Гор­ бачевського. Лихацький Петро Григорович – доктор біологічних наук, професор кафедри медичної біохімії Тернопільського національного медичного університету імені І.Я. Горбачевського. Information about the authors Yaremchuk O.Z. – Ph.D., Associate Professor of Medical Biochemistry Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000-0001-5951-1137, e­mail: yaremchuk@tdmu.edu.ua Posokhova K.A. – MD, Ph.D., DSc., Professor of Pharmacology and Clinical Pharmacology Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000-0002-2696-5738, e­mail: posokhova@tdmu.edu.ua Kuzmak I.P. – Ph.D., Associate Professor of Medical Biochemistry Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000­0002­5035­8815, e­mail: kuzmak@tdmu.edu.ua Kulitska M.I. – Ph.D., Associate Professor of Medical Biochemistry Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000­0001­7116­6655, e­mail: kulitskam@tdmu.edu.ua Shevchuk O.O. – MD, Ph.D., Associate Professor of Pharmacology and Clinical Pharmacology Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000-0003-2473-6381, e­mail: shevchukoo@tdmu.edu.ua Volska A.S. – Ph.D., Associate Professor of Pharmacology and Clinical Pharmacology Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000-0002-4985-9559, e­mail: volska@tdmu.edu.ua Lykhatskyi P.H. – Ph.D., DSc., Professor of Medical Biochemistry Department, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine. ORCID 0000-0002-0021-782X, e­mail: luhatsky@tdmu.edu.ua References 1. Khangura RK, Cooper S, Luo GY. Anti phos- pholipid Antibody Syndrome: Pathogenesis, Diag­ nosis, and Management in Pregnancy. Maternal- Fetal Medicine. 2019 Jul 1;1(1):38­42. doi: 10.1097/FM9.0000000000000007 2. Ahluwalia J, Sreedharanunni S. 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