|Year : 2012 | Volume
| Issue : 6 | Page : 710-713
Influence of antioxidant complex on the adhesion of leukocytes in chronic venous insufficiency of lower limbs in rats
Mark Plotnikov, Ivan Ivanov, Anastasia Sidehmenova, Oleg Aliev, Nonna Tykavkina
Institute of Pharmacology, Siberian Branch, Russian Academy of Medical Sciences, Lenin Avenue 3, 634028, Tomsk, Russian Federation, Russia
|Date of Submission||24-Oct-2011|
|Date of Decision||31-Aug-2012|
|Date of Acceptance||31-Aug-2012|
|Date of Web Publication||8-Nov-2012|
Institute of Pharmacology, Siberian Branch, Russian Academy of Medical Sciences, Lenin Avenue 3, 634028, Tomsk, Russian Federation
Source of Support: None, Conflict of Interest: None
Objectives: In this study, we have evaluated the influence of antioxidant complex (AOC) (dihydroquercetin and lipoic acid) on pool of nonadhesive leukocytes and leukocytes with low and high adhesion in chronic venous insufficiency (CVI) of lower limbs in rats.
Materials and Methods: Model of CVI of lower limbs was created by blood flow restriction in caudal vena cava. AOC was administered at 100 mg/kg b.w./day for 14 days p.o. The venous pressure in vena cava and adhesion of leukocytes were estimated on the 14 th day of the experiment.
Results: On the 14 th day, the venous pressure in groups of animals (experimental and control) was in 2.7-2.9-fold higher. In the control group, the significant increase of leukocytes pool with high adhesion was detected. Intragastric administration of AOC reduced the number of leukocytes with high adhesion and increased the number of nonadhesive leukocytes and leukocytes with a low adhesion.
Conclusions: Model of CVI of lower limb is accompanied by increased venous pressure and raised adhesion activity of leukocytes. Administration of AOC for 14 days reduces the adhesive activity of leukocytes.
Keywords: Adhesive activity of leukocytes, antioxidants, chronic venous insufficiency, dihydroquercetin, lipoic acid
|How to cite this article:|
Plotnikov M, Ivanov I, Sidehmenova A, Aliev O, Tykavkina N. Influence of antioxidant complex on the adhesion of leukocytes in chronic venous insufficiency of lower limbs in rats. Indian J Pharmacol 2012;44:710-3
|How to cite this URL:|
Plotnikov M, Ivanov I, Sidehmenova A, Aliev O, Tykavkina N. Influence of antioxidant complex on the adhesion of leukocytes in chronic venous insufficiency of lower limbs in rats. Indian J Pharmacol [serial online] 2012 [cited 2021 Apr 14];44:710-3. Available from: https://www.ijp-online.com/text.asp?2012/44/6/710/103260
| » Introduction|| |
Chronic venous insufficiency (CVI) of lower limbs is widely prevalent with considerable socioeconomic expenses for rehabilitation of patients. ,, In countries with developed health-care service, treatment of patients with CVI of lower limbs demands about 1-3% of health service budget. ,, The number of patients with CVI of lower limbs is constantly growing. Thus, the annual increase in new cases of CVI of lower limbs in a population of industrially developed countries reaches up to 2.6% among women and 1.9% among men. ,
The pathogenesis of CVI of lower limbs involves venous hypertension and venous stasis, activation and adhesion of leukocytes, endothelial dysfunction, and inflammatory mediators expression that leads to microcirculatory disorder and reduction of tissue oxygenation. ,,,, Activation of leukocytes and their adhesion to the endothelium is the basic starting mechanism of inflammatory reactions and is reflected in the theory of 'White Cell Trapping'. , In the process of adhesion and subsequent migration into subendothelial layer, a fraction of leukocytes is destroyed, and proteases and free radicals are released resulting in the degradation of collagen fibers of the venous wall. ,, The aseptic inflammation of a vessel wall is developed, which in conditions of venous insufficiency has a chronic character. ,,,
Different aspects of pharmacology of chronic venous insufficiency pharmacotherapy have been discussed in literature. One of the directions in CVI of lower limbs treatment is the arrest of inflammatory processes at the level of leukocyte endothelium. It is known that some antioxidants are capable of limiting rolling, adhesion, and migration of leukocytes to a certain extent. ,,,,,
We previously showed that the composition of dihydroquercetin and lipoic acid exhibits lymphokinetic, capillary protective, and hemorheological activity.  Furthermore, this composition demonstrated antiexsudorific effect in the model of chronic venous insufficiency. 
The purpose of this research was to estimate the influence of antioxidant complex (AOC), including dihydroquercetin and lipoic acid, on the adhesion of leukocytes in conditions of CVI of lower limbs model in rats.
| » Materials and Methods|| |
Permission for the project was received from the local ethics committee of the Research Institute of Pharmacology, Siberian Branch of RAMNs (Tomsk, Russia). Males Wistar rats (n=16) weighing 300-350 g were used in this study. The rats were kept in the vivarium (at temperature 22 ± 2°C, relative humidity of 55 ± 15%, 12/12 h light-dark cycle). Complete food for conventional laboratory small rodents and water were freely available. Food contains antioxidants : v0 itamins A (30,000 IU/kg), E (1, 30,000 IU/kg), C (78.7 mg/kg), and selenium (0.07 mg/kg). Twelve hours before operative intervention and blood sampling, the animals were restricted in feeding. All experimental procedures were performed in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (March 18, 1986; Strasburg; ETS 123) and the Requirement of Public Health Ministry of Russian Federation Order No. 267 (Moscow, 2003).
Diethyl ether ("Kuzbassorgchem" Ltd, Russia), gentamycin ("Belmedpreparatins" OJSC, Belarus), streptocide ("Anjumi" Ltd, Russia), heparin ("Belmedpreparations" OJSC, Belarus), ficoll ("Sigma" Co Ltd, the USA), verografin ("Ivax Corporation", Czechia), dihydroquercetin ("Flavir" Ltd, Russia), lipoic acid ("Marbiofarm" Ltd, Russia), CaCl 2 × 2H 2 O, MgCl 2 × 6H 2 O, KCl, NaH 2 PO 4 , Na 2 HPO 4 , NaCl, NaHCO 3 , KH 2 PO 4 , and glucose (all chemical reagents of "Reahim" Ltd, Russia) were used in this research.
Administration of Antioxidant Complex
AOC is a mixture of dihydroquercetin and lipoic acid in proportion of 1:1. Animals of experimental group received AOC daily for 14 days intragasrically in a dose of 100 mg/kg (50 mg/kg dihydroquercetin and 50 mg/kg lipoic acid) in the form of suspension in 1% solution of starched slime; animals of control group and sham-operated animals received 1% solution of starched slime in equivolume quantity.
Chronic Venous Insufficiency of Lower Limbs Modeling
Animals were anesthetized with inhalation administration of diethyl ether. Before the operative intervention, gentamycin was injected to rats (4 mg/kg) intramuscularly to reduce the risk of postoperative infections. An estimation of anesthesia level was carried out according to the intensity of animal's reaction on pricking of tail tip and to the absence of a corneal reflex.
CVI of lower limbs model was created by blood flow restriction in caudal vena cava. For this purpose, animals under ether anesthesia were exposed to laparotomy, the site of caudal vena cava proximal to the right renal vena was isolated, ligature was brought under it, and after administration of heparin in a femoral vena in a dose of 250 U/kg, the partial occlusion of caudal vena cava was carried out. To do this, a needle having a diameter of 0.8 mm was imposed on a site of vena cava, and vena cava was ligated. Then, the needle was removed, partially restoring a vessel lumen. Operative treatment was completely repeated in sham-operated animals except the stage of vessel occlusion.
Pressure Measurement in Vena Cava
The development of resistant venous hypertension was a sign of CVI of lower limbs model repeatability. Pressure measurement in vena cava was performed on the 14 th day of the experiment. Before measurement of pressure, the rats were anesthetized by diethyl ether, then heparin was intravenously injected in a dose 250 U/kg. Pressure in a caudal vena cava was measured by the direct method at the level of ileac vena confluence. The approach was carried out through a femoral vein.
Adhesive Activity of Leukocytes
Blood assays stabilized with heparin were taken away in rats from the common carotid artery after pressure measurement. Suspension of leukocytes was received in a density gradient by the process of centrifugation. For this purpose, 4 ml of blood was layered on 1 ml of fikoll-verografin (7:16) solution and was centrifuged for 30 min 800g at room temperature. The suspension of leukocytes was washed twice by Hanks' solution without Ca 2+ and Mg 2+ (CMF-HBSS) (0.8% NaCl, 0.04% KCl, 0.035% NaHCO 3 , 0.006% Na 2 HPO 4 , 0.006% KH 2 PO 4 , 0.1% of glucose, pH 7.4), during the process of centrifugation at 400g for 15 min. Precipitation was resuspended in 5 ml of CMF-HBSS. In the initial suspension, the number of leukocytes was 7650 ± 670/μl. Received suspension of leukocytes was placed in a glass capillary with inside diameter 1 mm, pretreated with a solution of Dulbecco's Phosphate Buffered Saline (DPBS) (0.8% NaCl, 0.013% CaCl 2 × 2H 2 O, 0.01% MgCl 2 × 6H 2 O, 0.02% KCl, 0.02% NaHPO 4 , 0.115% NaH 2 PO 4 , pH 7.4). The cells were incubated at 37°C for 60 min. Nonadhesive leukocytes and leukocytes with low adhesion were deleted from the capillary at shear stress created by pressure of 0.49×10 4 N/m 2 . The capillary was repeatedly filled with DPBS and leukocytes with medium adhesion were removed from the capillary at shear stress created by pressure of 2.2×10 4 N/m 2 . The number of cells remaining in the capillary (leukocytes with a high adhesion) was calculated as the difference between the initial amount of leukocytes and the number of nonadhesive leukocytes and leukocyte with low and medium adhesion. 
Data were expressed as mean ± SEM. A value of P < 0.05 was considered statistically significant. Comparisons between groups were performed using a nonparametric analysis followed by Mann-Whitney U test. Statistical analysis was performed using Statistica 6.0 (Stat Soft Inc., USA).
| » Results|| |
After partial occlusion of the vena cava, the persistent venous hypertension developed, being maintained for 14 days [Table 1]. On the 14 th day, the venous pressure in groups of animals with partial occlusion of the vena cava was greater, 2.7-2.9 times ( p0 <0.05) as compared with the values in sham-operated animals.
|Table 1: Venous pressure (P, mm H 2 O) in sham-operated rats and rats with model of lower limb chronic venous insufficiency on the 14 th day|
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In 2 weeks after partial occlusion of the vena cava, there was a significant increase in quota of leukocytes with a high adhesion by 2.5 times and the decline in nonadhesive leukocytes and leukocytes with a low adhesion by 1.6 times in comparison with that in sham-operated animals [Table 2].
|Table 2: The influence of course (14 days) intragastric administration of AOC (100 mg/kg) on the adhesive ability of leukocytes in rats with model of lower limb chronic venous insufficiency|
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Course intragastric administration (14 days) of AOC reduced the number of leukocytes with a high adhesion by 2.2 times and increased the number of nonadhesive leukocytes and leukocytes with a low adhesion by 1.2 times compared with the values in the control [Table 2].
| » Discussion|| |
Leukocyte adhesion to vascular endothelium is the main etiological factor of trophic damages in patients with CVI of lower limbs. ,,,, The interaction between leukocytes and endothelium is realized with the participation of adhesion molecules intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 (ICAM-1 and VCAM-1) on endothelial cells, and the ligands corresponding to them - antigens lymphocyte function-associated antigen-1 and very late antigen-4 (LFA-1 and VLA-4) on leukocytes. ,,, During the process of adhesion and further migration into a subendothelial layer, the leukocytes become active and release cytokines, leukotrienes, proteolytic enzymes, and reactive forms of oxygen, leading to the expansion of endothelial damage areas, the increase of adhesion molecules in the blood, and the area of intercellular interaction.
Extravasation of plasma components, including fibrinogen, occurs through a damaged endothelium. ,,,,,, These processes described above result in chronic inflammation with the subsequent development of trophic disorder and venous thrombosis. As several authors have noted, even in the conditions of short-term venous hypertension, the leukocytes delay in the vessels of the lower limbs and the increase in ICAM-1 content in patients' plasma with CVI of lower limbs are observed. , Similar changes were observed by Bergan, et al., but on the model of arteriovenous anastomosis and occlusion of the mesenteric venule in rats. They observed accumulation of leukocytes in the valvate structures and vein walls and the increased ICAM-1 expression of vascular endothelium in the conditions of raised venous pressure.  Our study also showed an increase in adhesive activity of leukocytes in venous hypertension.
Leukocytes activation and their interaction with vascular endothelium are assumed to play an important role in the pathogenesis of CVI of lower limbs, which results in inflammatory processes in the vascular wall, and the great importance is attached to the practicability of such drugs application that can counteract the inflammation.
In the light of recent studies about the role of blood cells and venous endothelium interactions, our data showing the decrease in leukocytes adhesive activity in rats with CVI of lower limbs by the influence of AOC course administration (14 days) are of great interest. Possible mechanisms of AOC influence on leukocytes adhesive activity are contributed by the properties of each component in this complex.
It has been established that lipoic acid decreases the oxidative stress and adhesion processes by reducing the expression of main adhesion molecules in the vascular wall. , It is also known that lipoic acid can inhibit the activity of cells which determines the immune response by inhibiting interleukins in them and stimulating the synthesis of cyclic AMP (cAMP), which possesses an immunosuppressive effect. ,
Flavonoids can inhibit many enzyme systems involved in the realization of inflammatory processes. Dihydroquercitin is an inhibitor of cAMP phosphodiesterases localized in different cells, including leukocytes.  Dihydroquercetin while affecting a signaling pathway Janus kinase/signal transducer and activator of transcription (JAK/STAT) reduces the expression of adhesion molecules ICAM-1 induced by interferon γ (IFN-γ) in human keratinocytes.  On the model of cerebral ischemia reperfusion, it reduces the migration of leukocytes in brain tissues due to the decrease in the expression of adhesion molecules ICAM-1. 
Thus, the model of CVI of lower limbs is accompanied by increased venous pressure and raised adhesion activity of leukocytes. Course administration (14 days) of AOC reduces the adhesive activity of leukocytes in rats with model of lower limb chronic venous insufficiency.
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[Table 1], [Table 2]