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UDC 612.1:615.324-092.4
ADAPTOGENIC EFFECT OF PRODUCTS OF ANTLER STAG BREEDING ON THE STATE OF HEMOSTATIC SYSTEM IN RATS BY SUPRATHRESHOLD PHYSICAL LOAD
Altai State Medical University, Barnaul
A.A. Blazhko, I.I. Shakhmatov, V.I. Kiselev, A.Yu. Zharikov
The article presents the results of the study of the hemostatic system of rats by means of an integral method -thromboelastography by suprathreshold physical load without prior intake of the adaptogene and by preliminary course 30-day intake of the concentrate containing blood and histolysate of the Siberian stag reproductive organs. There was also studied the influence of the course intake of the concentrate and separate additives (glucose, ascorbic acid, fruit essence) contained in the studied concentrate on the hemostatic system parameters. It was shown, that the preliminary course 30-day intake of the concentrate decreases the risk of development of the state of thrombotic readiness in rats after 8-hour physical load due to the increased intake of the concentrate of blood plasm fibrinolytic activity. It was also proved, that the components increasing adaptive qualities of the hemostatic system in rats are active agents contained in blood and histolysate of the Siberian stag reproductive organs. Key words: thromboelastography, histolysate of the Siberian stag reproductive organs, hemostatic system.
Introduction
Physical load is one of the most common types of stress in the human body which by regular activity can increase its resistance through mechanisms of cross adaptation [1, 2]. At the same time, the su-prathreshold physical load can lead to the development of damage to organs and systems, causing a state of distress in the body [3]. It is known that distress from the hemostasis system is manifested by the development of the state of thrombotic readiness [3, 4]. There are many methods for recognizing the development of thrombotic readiness, but the overall picture of coagulation from the onset of coagulation to the fibrinolysis process with the definition of viscoelastic properties of the clot can be fixed by only an integral method - thromboelastography [5, 6].
Earlier in our laboratory, it was established that in order to avoid the development of thrombotic readiness state in case of suprathreshold stress, it is necessary to increase the resistance of the body and the hemostasis system in particular, by physical training or by taking plant adaptogens [7, 8]. Adaptogenes of animal origin include products of antler stag breeding, which increase the mental and physical performance of the organism [9]. It is known that in products of antler stag breeding there are no prohibited doping substances or similar analogs [10]. Moreover, it was noted that concentrates a histolysate from male genital organs containing in addition to stag blood have a more pronounced tonic effect due to an increase in bio-synthetic activity in rat skeletal muscle cells [11].
Earlier, we already considered the effect of the exchange of products of antler stag breeding on the state of the hemostasis system [12, 13]. However, the effect of the preliminary intake of the concentrate and its constituents on the indices
of the hemostatic system, estimated by the method of thromboelastography, was not studied by su-prathreshold physical load.
The research objective is to evaluate the reactions of the hemostasis system by the method of thromboelastography in rats after suprathresh-old load by a preliminary course intake of concentrate containing blood and histolysate from the reproductive organs of stag and also to exclude the possible adaptive effect on the hemostatic system of supplements (glucose, ascorbic acid, fruit essence) included in the composition of the concentrate used.
Materials and methods
The studies were performed in 50 male Wistar rats weighing 250 ± 30 g. The animals were divided into five groups (intact and four experimental groups).
The first two experimental groups of animals were subjected to an 8-hour physical load in the form of an imposed run in a treadmill with a rotation speed of 6-8 m/min. In this case, the animals of the second experimental group, in contrast to the first, previously received the concentrate for 30 days. Immediately after the end of the physical load, the blood from the hepatic sinus was taken for the study in a volume of 5 ml into a polystyrene syringe containing 3.8% sodium citrate solution (blood and citrate ratio 9:1) under anesthesia.
The third and fourth groups of experimental animals took a concentrate (the third group) and, separately, additives (glucose, ascorbic acid, fruit essence), contained in the concentrate used (the fourth group) for 30 days, and were not subjected to physical load. Blood was taken on the 31st day of the intake of concentrate and supplements.
Experimental animals took a concentrate containing blood and histolysate of the genital organs of the stag males produced under the trade name "Pantohematogen (Lubyangem)" (FSBRI "All-Russian Scientific Research Institute of Antler Stag Breeging" of FASO of Russia, Barnaul), 4.5 ml per day. Calculation of the dose of concentrate for rats was performed taking into account the coefficients of interspecific counting [14]. Experimental animals were kept in individual cells and an aqueous solution of the concentrate was taken orally from individual drinkers. The solution was prepared by adding 4.5 ml of concentrate to water, bringing the solution to a total volume of 40 ml (the daily rate of water intake for these rats detected by us prior to the beginning of the experiment).
The animals of the fourth experimental group took supplements (glucose, ascorbic acid, fruit essence) contained in the concentrate under study, in a volume of 1.7 ml per day for 30 days. The volume of 1.7 ml of the concentrate additives was diluted with water to 4.5 ml; thus, the putative active substance (blood and histolysate of the genital organs of the stag males) was replaced by water in appropriate proportions. Intact animals took water in the same volume as the experimental animals.
The thromboelastogram was recorded on a Ro-temGamma apparatus (Germany) in Natem mode for 35 minutes using the Star-tem activator. The following parameters of thromboelastogram were assessed:
- Coagulation time (CT) - the time from the moment of application of the activator to reaching a 2 mm amplitude by the thromboelastogram, this interval reflects the initiation phase of blood coagulation [15].
- Clot formation time (CFT) - time of amplitude change of thromboelastogram from 2 mm to 20 mm, this index characterizes the phase of strengthening of the process of thrombus formation [15].
Angle alpha (ALP) - the angle formed by the longitudinal axis of the thromboelasto-gram and the straight line drawn tangentially to the thromboelastogram from the point corresponding to the clot amplitude of 2 mm. This value reflects the kinetics of clot formation and characterizes the propagation phase [16].
- Maximum clot firmness (MCF) - an index corresponding to the maximum amplitude of the clot
and reflecting the function of platelets and fibrin-°gen [8].
- Maximum lysis (ML) - an indicator characterizing the level of maximum fibrinolysis recorded during the analysis. It is defined as finding the lowest amplitude after reaching the MCF.
The use of rats in experiments was carried out in accordance with the European Convention for the Protection of Vertebrate Animals used in the experiment, as well as the Directives -
86/609 /EEC. Anesthesia of animals was carried out in accordance with the "Rules of work with the use of experimental animals."
The data obtained during the study are presented in tables in the form (m [25-75%]), where m is the median in the sample multitude; [2575%] - the 25th and 75th percentiles. The statistical analysis was carried out on a personal computer using the Statistica 6.0 application package (StatSoft, USA). The significance of the differences was estimated using the nonparametric Mann-Whitney U-test, since the features examined did not obey the normal distribution. Differences were considered significant at a level of statistical significance p <0.05.
Results and discussion
As follows from the data presented in Table 1, an 8-hour physical load induced a shortening of coagulation time (CT) by 26% (p1 = 0.002) in rats (the first experimental group), an increase in the "alpha-alpha" index by 10% (p1 < 0.001) and a reduction in clot formation time (CFT) by 22% (p1 = 0.028) compared to that of intact animals, suggesting a shift in the hemostatic system towards hypercoagulability and consistent with the already described data obtained by coagulation tests. The maximum clot firmness (MCF) decreased by 11% (p1 = 0.018) at the end of the 8-hour physical load as compared to the intact animals, which indicated a decrease in the platelet count and a decrease in the fibrino-gen level in the blood of experimental animals due to their consumption for intravascular thrombus formation. The maximum lysis of the clot (ML) after an 8-hour load, unlike intact animals, was not determined, which indicated a decrease in the fibri-nolytic activity of the blood plasma. Thus, changes in the described indices of thromboelastogram indicate that an 8-hour physical load induces a state of thrombotic readiness in rats [3, 4].
In animals of the second experimental group which were exposed to 8-hour physical load after a 30-day course of the intake of concentrate containing blood and histolysate from the reproductive organs of the stag, the thromboelastogram indices did not differ from those in intact animals. Consequently, the preliminary administration of the adaptogen reduced the risk of thrombotic readiness in rats after an 8-hour physical load.
As follows from the data presented in Table 2, in rats, a 30-day intake of a concentrate containing blood and histolysate from stag reproductive organs caused a decrease in the maximum clot firmness by 12% (p1 = 0.014) and an increase in the maximum lysis (ML) by 125% (p1 = 0.021) in comparison with intact animals, which indicates an increase in the fibrinolytic activity of blood plasma. This adaptive effect of concentrate intake may be associated with activation of endothelial release of the tissue plasminogen activator (t-PA) and a de-
crease in the amount of plasminogen activation inhibitor (PAI-1) [17].
Admission of additives (glucose, ascorbic acid, fruit essence) contained in the test concentrate for 30 days in rats did not cause changes in the throm-boelastogram score in comparison with intact animals. Therefore, it can be assumed that active components contained in the blood and histolysate from the reproductive organs of stags are the active components of the concentrate that increase the adaptability of the hemostasis system, manifested in the increase in the fibrinolytic activity of the blood plasma.
Conclusions
1. The ultra-threshold 8-hour physical load causes the development of thrombotic state in rats.
2. A preliminary 30-day intake of a concentrate containing blood and histolysate from the reproductive organs of stags reduces the risk of the development of thrombotic readiness in rats after a supra-threshold exercise.
3. Increased adaptability of the hemostasis system when receiving a concentrate containing blood and histolysate from the reproductive organs of stags consists in the increase the fibrinolytic activity of the blood plasma.
4. The active ingredients contained of the used Pantohematogen (Lubyangem) concentrate which enhance the adaptive properties of the hemostasis system in rats are the active substances contained in the blood and histolysate from the reproductive organs of stag males.
Table 1.
Indices of thromboelastogram: intact animals; experimental rats after an 8-hour physical load; experimental animals after 8-hour physical load with a preliminary 30-day intake of concentrate
Indices Intact rats (n=10) 8-hour physical load 8-hour physical load on the 31st 1 f i i • 1 1 / -1 A\
(n=10) day of concentrate intake(n=10)
Coagulation time (CT), sec 193,0 [167,3-206,3] 143,0 [114,0-163,0] p1=0,002 (A1 - 26 %) 176,0 [163,5-183,5] p1=0,349
Clot formation time (CFT), sec 76,0 [64,3-106,5] 59,5 [52,5-67,8] p1=0,028 (A1 - 22 %) 74,0 [70,0-80,0] p1=0,690
Angle of alpha (ALP), 0 73,0 [68,5-77,0] 80,0 [79,5-82,0] p1<0,001 (A1 + 10 %) 74,5 [68,0-77,0] p1=0,939
Maximum clot firmness (MCF), mm 68,0 [62,0-70,8] 60,5 [59,0-65,3] p1=0,018 (A1 - 11 %) 67,5 [66,8-70,3] p1=0,562
Maximum lysis (ML), % 4,0 [1,0-6,0] 0,0 [0,0-1,0] p1=0,013 (A1 - 100 %) 3,0 [0,8-6,5] p1=0,894
Note: the results are presented in tables in the form (m [25-75%]), where m is the median in the sample multitude; [25-75%] - the 25th and 75th percentiles. A1 - statistically significant difference in the experimental group with intact animals at p <0.05; pi - the level of significance of differences in the experimental group with intact animals. CT - the coagulation time, CFT - the clot formation time, MCF - the maximum clot firmness, ML - the maximum lysis.
References
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Table 2.
Indices of thromboelastogram: intact animals; experimental group of rats taking concentrate for 30 days; experimental
animals taking the concentrate additives for 30 days
Indices Intact rats (n=10) 30-day intake
concentrate (n=10) concentrate additives (n=10)
Coagulation time (CT), 193,0 209,0 [173,0-250,0] 189,5 [181,5-197,8]
sec [167,3-206,3] p1=0,270 p1=0,979
Clot formation time 76,0 101,0 [90,0-106,0] 81,0 [72,8-90,0]
(CFT), sec [64,3-106,5] p1=0,064 p1=0,655
Angle of alpha (ALP), 73,0 73,0 [71,0-76,0] 71,5 [70,3-74,8]
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Maximum clot firm- 68,0 60,0 [57,0-65,0] 67,0 [63,3-72,0]
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(A1 - 12 %)
Maximum lysis (ML), 4,0 9,0 [5,0-12,0] 5,5 [2,0-10,0]
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(A1 + 125 %)
Note: the results are presented in tables in the form (m [25-75%]), where m is the median in the sample multitude; [25-75%] - the 25th and 75th percentiles. A1 - statistically significant difference in the experimental group with intact animals at p <0.05; pi - the level of significance of differences in the experimental group with intact animals. CT - the coagulation time, CFT - the clot formation time, MCF - the maximum clot firmness, ML - the maximum lysis.
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Contacts
Corresponding author: Blazhko Aleksandr Alek-
sandrovich - Lecturer of the Department of normal
physiology of ASMU, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: 8-903-073-6667.
Email: blazhko_1990@mail.ru
