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ECPB 2019, 88(4): 53–62
https://doi.org/10.25040/ecpb2019.04.053
Research articles

Impact of Nitric Oxide Synthesis Modulators on the Mechanisms of Apoptosis Development and Production of Reactive Oxygen Species in the Blood Leukocytes in Experimental Antiphospholipid Syndrome

O.Z. YAREMCHUK1, K.A. POSOKHOVA1, H. H. HABOR1, M.M. GUZYK2
Abstract

Antiphospholipid Syndrome (APS) is an autoimmune disorder characterized by the presence of antiphospholipid antibodies, increased risk of arterial and venous thrombosis, and pregnancy pathology. The aim of the research was to investigate the effect of L-arginine and aminoguanidine on the degree of reactive oxygen species (ROS) development and processes of apoptosis and necrosis of blood leukocytes in antiphospholipid syndrome in BALB/c mice. The experimental animals were divided into 5 groups: the 1st – the control; the 2nd – the animals with experimental APS, the 3rd – the animals with APS administered with L-arginine (25 mg/kg), the 4th – the animals with APS administered with aminoguanidine (10 mg/kg), the 5th – the animals with APS administered with L-arginine in combination with aminoguanidine. L-arginine and aminoguanidine were administered intraperitoneally once a day for 10 days after APS formation. The content of living, apoptotic and necrotic cells in blood leukocytes was evaluated using Annexin V conjugated of GFP (green fluorescent protein) and propidium iodide (PI). The fluorescence signals of the samples were registered by the channel FL1 (515-535 nm) for GFP and FL3 (620-630 nm) for PI using a flow cytometer. The redistribution between the leukocytes populations was estimated by two parameters: direct (FS, cell size) and side scatter (SS, cell granularity). The ROS in blood leukocytes was determined using 2′,7′- Dichlorofluorescin diacetate. The radiation intensity of the test samples was registered by the channel FL1 (515-535 nm) with a flow cytometer. The results were processed by means of FCS Express V3. It was established that the viability of blood leukocytes of mice decreased in APS compare to the control. L-arginine and aminoguanidine only as well as their combined administration led to normalization of the blood leukocyte viability in the BALB/c mice relative to that of the APS animals. Redistribution between granulocytes and agranulocytes in APS was proved. An increase in the number of granulocytes in blood in APS was evidenced as well. In L-arginine administration to the animals with APS, a further increase in the number of granulocytes in blood was observed. Under the combined use of L-arginine and aminoguanidine, a rebalancing of the number of granulocytes and agranulocytes to the control level took place. In addition, it was established that the basal level of ROS production in granulocytes reduced by 27% and in agranulocytes – by 19% compare to the control group. It was also proved that in case of administration of L-arginine and aminoguanidine only, the animals with APS experienced a decrease in ROS content in granulocytes and agranulocytes. The combined administration of L-arginine and aminoguanidine into the APS mice led to normal ROS content in granulocytes and agranulocytes relative to those of the APS animals. Therefore, an enhanced activation of apoptosis and deficient production of ROS is significant in the pathobiochemical mechanisms of APS. Combined administration of L-arginine and aminoguanidine leads to normalization of blood leukocytes viability of the BALB/c mice, rebalancing of granulocyte and agranulocyte counts to norm, and normalization of the ROS content in granulocytes and agranulocytes relative to that of the APS animals largely than those administered alone.

Received: 16.12.2019

Keywords: Antiphospholipid Syndrome, nitric oxide, apoptosis, leukocytes, reactive oxygen species

Full text: PDF (Ukr) 0.95M

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