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10ВПЛИВ Д1ЮЧО1 РЕЧОВИНИ «ДИКЛОФЕНАК НАТР1Ю» НА КЛ1ТИНИ К1СТКОВОГО МОЗКУ
МИШЕЙ
Багмут 1.Ю.
Д. мед. н., професор, завiдуюча кафедрою клШчног патофЫологи, топографiчноi анатомИ та оперативно'1' хiрургii ХМАПО, Харюв, Укра'та
1ванов О.С.
Астрант, ХМАПО Срофеева В.В.
Викладач, Лисичанський медичний колледж
Абрамова Т.В.
Викладач, Лисичанський медичний колледж
KioH О.1.
Викладач, Лисичанський медичний колледж
Срмолаш Т.О.
Викладач, Лисичанський медичний колледж
ОдЫов Р.1.
Студент, Лисичанський медичний колледж EFFECT OF THE ACTIVE SUBSTANCE "DICLOFENAC SODIUM" ON BONE MARROW CELLS
Bagmut I.
doctor of medical sciences, professor, head of the department of clinical pathophysiology, topographic anatomy and operative surgery
Kharkiv medical academy of postgraduate education, Kharkiv, Ukraine
Ivanov O.
Postgraduate student,
Kharkiv medical academy of postgraduate education, Kharkiv, Ukraine
Erofeeva V.
Teacher, Lysychansk Medical College Abramova T.
Teacher, Lysychansk Medical College
Kion O.
Teacher, Lysychansk Medical College Ermolash T.
Teacher, Lysychansk Medical College
Odiliov R.
Student, Lysychansk Medical College
АНОТАЦ1Я
Щвдтримання систем гомеостазу людини та тварин е прюритетним завданням багатьох оргашв та систем, одшею з яких е кровотворення. На клгшни, що постшно даться негативно впливае багато факторiв, основним з яких е лшарсьш засоби, першють серед яких належить груш не стеро!дних протизапальних засобiв (НПЗЗ). Класичним представником дшчо! речовини групи НПЗЗ е Диклофенак натрш, який вщк-рито в другш половиш ХХ сторiччя та не втрачае свое! популярносп серед лiкарiв рiзноманiтних спеща-льностей. Експериментальна частина проведена на 44 особинах бших безпорщних лабораторних мишей , вжом 60 днiв та вагою 60 грамiв, як1 були подiленi не три групи. Перша отримувала дшчу речовини Диклофенак натрш в шлькосп 0,09 мг, друга 0,18 мг, а третя фiзiологiчний розчин натрш хлориду 2 рази на добу впродовж 96 годин. Шсля проведения експерименту тварини були забил, видшено кiстковий мозок, в якому вивчали показники загально! кiлькостi клгтин, недиференцiйованих бластiв, лiмфоцитiв з викори-станням методики iммуномагнiтно! сепарацп, за допомогою тест-систем встановлено концентрацiю лшо-полiсахаридiв та Антигену-1 (LFA-1). У тварин першо! групи знизився показник загально! кшькосп клiтин на 63,7%, у другш на 90,9%, тдвищився показник недиференцшованих бластiв на 145,4%, тодi як в другiй груш знизився на 63,6%. Рiвень лiпополiсахаридiв збiльшився в першiй групi на 113,5%, а в другш на 1872,9%. Загальна кшьшсть лiмфоцитiв у тварин першо! групи знизилась на 24,6%, в другш на 66%. Щд-вищився рiвень Антигену-1 (LFA-1) в першш групi на 49%, тодi як в другiй на 18,2%. Дослвдження показало негативний вплив Диклофенаку натрiю в якостi дшчо! речовини в дозi 0,18 мг, та б№ш сприят-ливий вплив в дозi 0,09 мг на дослвджуваш показники.
ABSTRACT
Maintaining the homeostasis system of humans and animals is a priority task of many organs and systems, one of which is hematopoiesis. The constantly dividing blood cells are influenced by many factors, the main among which are drugs, the first among which belongs to the group of non-steroidal anti-inflammatory series (NSAIDs). The classic representative of the active substance of the NSAID group is Diclofenac sodium, discovered in the
second half of the twentieth century, it does not lose its relevance and popularity among specialists in various specialties. The experimental part was carried out on 44 individuals of white outbred laboratory mice, 60 days old and weighing 60 grams, which were divided into three groups. The first received the active ingredient Diclofenac sodium in an amount of 0,09 mg, the second 0,18 mg, and the third saline solution 2 times a day for 96 hours. After the experiment, the animals were sacrificed and the bone marrow was removed, in which the indicators of the total number of cells, undifferentiated blasts, and lymphocytes were studied using the method of immunomag-netic separation, the concentration of lipopolysaccharides and Antigen-1 (LFA-1) was determined using test systems. In animals of the first group, the indicator of the total number of cells decreased by 63,7%, the indicator of undifferentiated blasts increased by 145,4% and the level of lipopolysaccharides by 113,5%. The total number of lymphocytes in animals of the first group decreased by 24,6%, the level of Antigen-1 (LFA-1) increased by 49%. In the second group, the total number of cells decreased by 90,9%, the number of undifferentiated blasts by 63,6%. An increase in the level of lipopolysaccharides was observed, by 1872,9%, the lymphocyte count decreased by 66%, the level of Antigen-1 (LFA-1) increased by 18,2%. In the course of the study, it was found that the effect of the active substance "Diclofenac sodium" on the studied parameters is more favorable at a dose of 0,09 mg, a dose of 0,18 mg has a negative effect.
Ключовi слова: Диклофенак натрш, токсична дiя, шстковий мозок, лiмфоцити.
Keywords: Diclofenac sodium, toxic effect, bone marrow, lymphocytes.
The homeostasis system in humans and animals is supported by a large number of organs that are closely interconnected, and the bone marrow is directly involved in this [1,2]. Its development begins long before the birth of the organism into the world, however, the influence of negative factors on the cells of hematopoi-esis occurs constantly, due to factors of the external or internal environment, which cannot but leave its mark on the structure and function [3,4].
Non-steroidal anti-inflammatory drugs (NSAIDs) are especially popular among doctors around the world, in connection with which the search for a substance with minimal effect on the body, primarily the bone marrow, is constantly being carried out. This problem is of particular relevance in connection with long-term use of drugs of the NSAID group to achieve stable remission or relief of pain [5-7].
Diclofenac sodium remains the classic active ingredient, a representative of NSAIDs, which has not lost its relevance since the second half of the twentieth century [8,9]. The chemical formula of Diclofenac sodium is a derivative of phenylacetic acid and a phenyl ring, which contains two chlorine atoms and is mainly a sodium salt. As a chemical substance, Diclofenac sodium has a minimum hazard class and is actively used in animal models in an amount of 3 mg / kg, which is the maximum daily dose for the treatment of rheumatoid arthritis. The optimal period of action of the substance is 12 hours, after which the effectiveness decreases. By increasing the refractory period by 2-3 days after the start of use, the active substance has its effect on the state of the immune system [10-13].
Diclofenac sodium is widely used as an active ingredient in many drugs as an analgesic and refers to non-selective cyclooxygenase (COX) blockers, but more so in relation to COX-2. The result of this effect may be an increase in the level of thrombosis. Often in the bloodstream cells with a changed structure are detected, biological activity increases, which is clearly manifested in immunodeficiency [14-18].
The aim of the work was to study under experimental conditions the effect of Diclofenac sodium as an active substance on the total number of bone marrow cells, undifferentiated blasts, lymphocytes, the expression level of Antigen-1 (LFA-1) and the concentration
of lipopolysaccharides in laboratory mice, provided that it is used for 96 hours.
Materials and methods. The experiment and subsequent studies were performed under the conditions of AskHels Medical and Diagnostic Center LLC, license of the Ministry of Health of Ukraine № 376 dated 21.04.2016, Kharkiv, with strict observance of all the rules of humane treatment of experimental animals and strict observance of aseptic rules according to the European Convention for the Protection of Vertebrate Animals Used for Research or Other Scientific Purposes "(Strasbourg, 1986)," General Ethical Principles for Conducting Experiments on Animals "(Kyiv, 2001) and Law of Ukraine № 3447-IV" On the Protection of Animals from Cruelty " - dated February 21, 2006 [19].
The experiment involved 44 individuals of white laboratory mice, weighing 60 grams and age 60 days. The animals were divided into three groups: the first included 16 individuals who received Diclofenac sodium as an active ingredient in the quadriceps muscle of the thigh in an amount of 0,09 mg, the second group of 16 individuals received 0,18 mg of the active ingredient in the same way of administration, and the third, which included 12 individuals, received a saline solution of sodium chloride. The introduction was carried out twice a day with a difference of 12 hours for 96 hours.
After the experiment, the animals were sacrificed under general anesthesia with sodium thiopental solution, and after making sure of the absence of reflexes and vital activity, they were decapitated. In all animals, the femurs were dissected, neatly destroyed, and the bone marrow was isolated, which was studied histolog-ically [20]. The total number of cells was calculated, and using the method of immunomagnetic separation, in which the loss of cells is minimal, clean rows of un-differentiated blasts and lymphocytes were obtained [21]. Using test systems, the concentration of lipopolysaccharides in tissue homogenates and Antigen-1 (LFA-1) was determined. The one-dimensionality of the distribution of data was checked using swing diagrams, like a "box with a mustache" [22]. Statistical processing, construction of diagrams, graphs was carried out using the Statistica10 program.
Research results and discussion.
A study of the effect of Diclofenac sodium on the indicators of the total number of bone marrow cells showed a negative result in both groups of animals. However, there was a jump in the number of undifferentiated blasts in the animals of the first group in comparison with the control. The reason may be the activation of stem cell factor and interleukin-6 (IL), which are involved in the process of bone marrow stem cell division. Along with this, the concentration of lipopolysac-charides, which are the main component of the mem-
branes of gram-negative bacteria and trigger the appearance of a cascade of the inflammatory reaction in the body, has increased [23,24]. When studying the number of lymphocytes, a decrease in their total number was established against the background of an increase in the level of expression of Antigen-1 (LFA-1). In the complex, such changes may indicate the activation of the immune system in response to the administration of Diclofenac sodium and the appearance of younger forms of cells (table 1).
Table 1
Indicators of the number of bone marrow cells, undifferentiated blasts, lymphocytes, lipopolysaccharides and Antigen-1 (LFA-1) of laboratory mice of the first (n = 16) and control (n = 12) groups after using Diclofenac
sodium for 96 hours.
Index I group (n=16) III group (n=12)
X Ax X Ax
The total number of cells (cells/ml) x106 0,4 0,1 0,05 1,1 0,1 0,05
Undifferentiated blasts (%) 5,4 0,2 0,09 2,2 0,1 0,07
Lipoplisaccharides (ng/ml) 0,079 0 0,04 0,037 0 0,01
Lymphocytes (%) 16,6 0,1 0,07 22 0,2 0,1
Antigen-1 (LFA-1) (ng/ml) 16,4 0,2 0,1 11 0,2 0,1
Note: X - average value, Sx - standard deviation, Ax -
mean square error at the level of 0,05.
As can be seen from the data shown in Table 1, the animals of the first group showed a decrease in the total number of cells by 63,7% (p<0,05), while the indicator of undifferentiated blasts increased by 145,4% (p<0,05) and the level of lipopolysaccharides in tissue homoge-nates by 113,5% (p<0,05). The total number of lymphocytes in animals of the first group decreased by 24,6% (p<0,05), against the background of an increase in the expression level of Antigen-1 (LFA-1) by 49% (p<0,05). The data obtained against the background of an increased number of undifferentiated blasts may indicate the activation of the immune system in the body.
A completely different picture was observed in animals of the second group, which received Diclofenac
Table 2
The indicators of the number of bone marrow cells, undifferentiated blasts, lymphocytes, lipopolysaccharides and Antigen-1 (LFA-1) laboratory mice of the second (n = 16) and control (n = 12) groups after using Diclofenac sodium for 96 hours.
sodium in an amount of 0,18 mg. The dose of the active substance had a toxic effect on the total number of bone marrow cells, and also led to the blocking of stem cell factor and IL-6. The result was a decrease in the number of undifferentiated blasts. The lipopolysaccharide index has significantly increased, which may indicate the development of an inflammatory process in the tissue. The toxic effect affected the number of lymphocytes in the bone marrow. Against the background of an increased amount of lipoplisaccharides, the Antigen-1 (LFA-1) index increased. This can be interpreted as an immunodeficiency state against the background of an inflammatory process in the tissue (table 2).
Index II group (n=16) III group (n=12)
X 5x Ax X 5x Ax
The total number of cells (cells/ml) x106 0,1 0,1 0,07 1,1 0,1 0,05
Undifferentiated blasts (%) 0,8 0,1 0,04 2,2 0,1 0,07
Lipoplisaccharides (ng/ml) 0,73 0,2 0,1 0,037 0 0,01
Lymphocytes (%) 7,5 0,1 0,06 22 0,2 0,1
Antigen-1 (LFA-1) (ng/ml) 13 0,2 0,1 11 0,2 0,1
Note: X - average value, Sx - standard deviation, Ax - mean square error at the level of 0,05.
From table 2 it becomes clear that the indicator of the total number of bone marrow cells decreased in the second group by 90,9% (p<0,05), the number of undifferentiated blasts by 63,6% (p<0.05). Against the background of the above changes, there was a sharp jump in lipopolysaccharides, by 1872,9% (p<0.05) in comparison with the control group. The lymphocyte count significantly decreased by 66% (p<0,05), which against
the background of inflammatory changes indicates suppression of immunity in animals of the second group. A decrease in the number of cells, including immune defense, resulted in a slight increase in the level of An-tigen-1 (LFA-1) in animals of the second group, by 18,2% (p<0,05). These changes once again confirm the hypothesis of toxicity of Diclofenac sodium when the dose is increased. Fluctuations of the studied indicators are clearly presented in the graphs (graph 1,2)
Graph 1
Indicators of the total number of cells, undifferentiated blasts and lipopolysaccharides of the experimental group
of animals in comparison with the control using Diclofenac sodium for 96 hours
Note: Total number of cells - cells/ml, undifferentiated blasts - %, Lipopolysaccharides - ng/ml
Graph 2.
Indicators of Lymphocytes and Antigen-1 (LFA-1) of the experimental group of animals in comparison with the
control when using Diclofenac sodium for 96 hours.
Note: Lymphocytes - %, Antigen-1 (LFA-1) - ng / ml.
The use of swing diagrams makes it possible to place three dose values along the abscissa axis, located at a uniform distance, making it possible to get an idea of the trends in the change in the effect obtained during
the experimental part of the effect of Diclofenac sodium from the dose used in the range 0 (control), 50 (first group), 100 (second group) % (diagrams 1-5).
Diagram 1. Total number cells (СОХ 5).
Diagram 2. Undifferentiated blasts (My3).
Diagram 3. Lipopolysaccharides (COX2)
Diagram 4. Lymphocytes (My8)
Доза, DL
Diagram 5. Antigen-1 (LFA-1)
According to the presented diagrams, it can be concluded that there is no vertical intersection of the boxes, which confirms the dependence of the result on the dose used. By the type of trends, an increase in the dose led to the appearance of a nonlinear variable with a maximum value in the second group for the index of undifferentiated blasts and Antigen-1 (LFA-1) (diagram 2.5). Non-linear increase in the indicator with increasing dose for lipopolysaccharides (diagram 3). An almost linear increase for the lymphocyte count (diagram 4).
Conclusions
During the study, it was found that the effect of the active substance "Diclofenac sodium" on the studied parameters is more favorable at a dose of 0,09 mg. It is this dosage that leads to the emergence of new bone marrow cells, activates the stem cell factor and IL-6, which contribute to the emergence of new stem cells.
The total number of cells in the first group decreased by 63,7%, while in the second group by 90.9%. The index of undifferentiated blasts increased by 145,4% in the first group, while the kA in the second
decreased by 63,6%. The concentration of lipopolysac-charides in the blood of animals of the first group increased by 113,5%, while in the second by 1872,9%. The number of lymphocytes in both groups decreased, in the first by 24,6%, while in the second by 66%. The Antigen-1 (LFA-1) index increased in both groups, in the first by 49%, in the second by 18.2%. The above changes may indicate the activation of the immune system under the influence of a medium therapeutic dose of Diclofenac sodium, and its increase is toxic to bone marrow cells.
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VARIANT ANATOMY AND SYNTOPIC FEATURES OF THE ARTERIES OF THE SACRAL PART OF THE VERTEBRAL COLUMN DURING THE FETAL PERIOD AND IN HUMAN NEWBORNS
Riabyy Yu., Kryvetskyy V.
State higher education institution of Ukraine "Bukovinian State Medical University "
ABSTRACT
A set of morphological research methods studied the features of discharge and division of arterial vessels of the sacral part of the spine 35 fetuses and 30 newborns. Syntopia of arterial vessels of the sacral spine, as well as their variability and intervascular anastomoses, were determined. Keywords: spinal column, sacrum, arteries, ontogenesis, human.
Introduction. The study of the morphology of the spine and other related structures as a single organ complex is of great practical importance [1-4]. The study of the features of organogenesis and topography of organs in prenatal ontogenesis acquires significant clinical significance due to the introduction of ultrasound examination of its development, prenatal diagnosis of abnormalities of normal morphogenesis [5-7], surgical correction of some defects of the fetus in the uterus, prophylaxis and perinatal defects of internal organs [8-10].
According to the WHO, more than half a million women die each year during pregnancy, childbirth or the postpartum period. Every year, more than 3.3 million babies are born stillborn, more than 4 million die in the first month of life, and 6.6 million are under the age of five. Thus, the world community does not have almost 14 million children each year. These data were announced at a press conference dedicated to World Health Day [3].
Theoretical summary of the results of bone development allowed to show the dependence of vertebral formation on the adequacy of blood supply. According to this pattern, the development of the bone organ is in full accordance with the blood supply and load [4, 5].
The aim of the study. To study the formation and individual differences of the beginning and branching of blood vessels of the sacral spine, to investigate the symmetry and asymmetry of extra- and intraosseous vascular bed, their anatomical features of distribution in individual parts of the vertebra, intervascular anastomoses of this area.
Material and methods. The study was performed on 35 fetuses and 30 newborns. The study of the arteries of the sacral spine consisted of several stages:
1. Determination of injected extra organic arteries by conventional and fine preparation and radiography.
2. Detect intra organic arteries by preparing macroscopic preparations followed by radiography in the
frontal, sagittal and horizontal planes.
3. Describe the spatial distribution of arteries in the vertebrae by the method of enlightenment.
An aqueous suspension of lead red lead was used as a contrast agent.
Results and their discussion. Sources of arterial blood supply to the sacral spine are iliac-lumbar, middle sacral, lateral sacral and inferior sciatic arteries. The number of arteries that carry blood to the sacral vertebrae of the fetus ranges from 3 to 7, more often - 5 (50% of preparations). In newborns - respectively from 4 to 7, more often - 5(in 61% of preparations).
These data suggest that differences in the number of sacral arteries are present in both fetuses and newborns. The number of sources of nutrition of the sacrum in females and males is not constant.
It is determined that the middle sources of sacral vertebrae are the middle sacral and lateral sacral arteries. The iliac and lumbar arteries are slightly less likely to be involved in their blood supply.
The medial sacral artery as a source of blood supply to the sacral vertebrae was detected in 65 preparations. Its topographic and anatomical location on the anterior surface of the sacrum is subject to significant changes. Thus, on 18 preparations it is located closer to the right edge of the sacrum; at 21 - near the left edge, at 15 - occupies a middle position on the os sacrum. On 8 preparations the specified artery changed the direction twice: on I - II sacral vertebrae it was located in the middle of a sacrum, deviating then to its external edge, and on 3 - began at the level I of a sacral vertebra on the right, then went to the left edge and on level V of the sacral vertebra again returned to the midline of the sacrum.
On 5 preparations in fetuses and newborns, the trunk of the middle sacral artery was tortuous, on the other 60 - relatively straight. The medial sacral artery was traced from 2 to 5 vertebrae: 2 - on 8 preparations, 3 - on 29, 4 - on 19 and 5 - on 9. From this artery to the
