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М.А. ГАЗАЛИЕВА, Т.Т. НШ РПЕИСОВ, Б.К. ЖШ МАБЕКОВА, Д.С. КШ РМАНГАЛИЕВА, М.М. ЖАНАСОВА
РЕЗИНОТЕХНИКАЛЫК 9 НД1Р1С1 АЭРОЗОЛ1Н1Н ЖЕДЕЛ ЭСЕР1НЕН БОЛАТЫН ФИЗИОЛОГИЯЛЫК КЭ РСЕТК1ШТЕРГЕ АРНАЙЫ ТАМАКТАНУ ТИ1МД1Л1Г1Н БАГАЛАУ
Тн шн: Антиуытты acepi бар арнайы э Hii^i эксперимент жануарларда жедел тозацдану аёсында ^олдану метаболикалыщ процесстщ оптимизациёсы, бщ лшыщ кн шлнщ щ лгаяы, электротерЫк ттркенудщ сезiмталдылыFыны кн шеяiне экелед^ ёгни резинатехникалыщ э нфрютщ аэрозольмен эсерлесуЫе аFзаныl^ бейiмделу реакциёсыныц тн зтуше экeлeдi. Тн йiндi сэ здер: резинотехникалыщ э нфрютщ аэрозол^ арнайы таFам, жедел эксперимент, жиынтыщ-межелт кэ рсеткiш, бщ лшыщ ет кн шл.
M.A. GAZALIYEVA, T.T. NURPEISSOV, B.K. ZHUMABEKOVA, D.S. KURMANGALIYEVA, M.M. ZHANASSOVA
ESTIMATION OF EFFICIENCY OF A SPECIALIZED FOOD ON PHYSIOLOGICAL INDICATORS AT UNDER SHARP INFLUENCE OF AEROSOL OF RUBBER-TECHNICALMANUFACTURE
Resume: Application of the specialised product with antitoxic action at experimental animals at under sharp dust action leads to optimisation of metabolic processes, increase in muscular force, sensitivity to electroskin irritation, so to formation of adaptive reactions of an organism to influence by an aerosol of rubber-technical manufactures.
Keywords: an aerosol of rubber-technical manufactures, a specialized product, under sharp experiment, a sum-threshold indicator, muscular force.
UDC: 616-099+612.112.91+616.24+616.381-002
M.R. GERASYMCHUK, L.M. ZAYATS, V.V. CHERKASOVA
Pathophysiology department, Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine
THE ROLE OF ENDOGENOUS INTOXICATION AND NEUTROPHILS IN MECHANISMS OF ACUTE LUNG INJURY
IN CASE OF EXPERIMENTAL PERITONITIS
The study was carried out on 78 albino Wistar male rats (180-230 g) divided at random into 3 groups. Group 1 consisted of 58 rats with experimental acute diffusive peritonitis (ADP), 2 group - control group with sterile 0,9% NaCl at equlibrium (n=10) and 3 intact group (n=10). ADP was induced by intraperitoneal injection of 10% suspension of feces. We analyzed total WBC count, Wet/Dry lung indexes, data of the oxidative and antioxidative systems and endogenous intoxication indexes, light and electron microscopy. Blood samples and lung tissue were obtained in 1,12, 24 and 48 h after ADP induction. It has been established that after ADP induction was noticed progressive endogenous intoxication with lipid and protein peroxidation and inhibition of antioxidant protection for 24 hours with further exhaustion after the first day of experiment. The most informative and sensitive biomarker of acute lung injury is a coefficient of leukocyte lung regulation. The latter suggests enhanced leukocyte sequestration in respiratory system even on the background of leucopenia, which implies lung injury. Furthermore, lung injury has been confirmed by the analysis of morphological alterations of laboratory rats' lung tissue micro- and ultrastructure. Keywords: acute lung injury, peritonitis, endogenous intoxication, neutrophils.
INTRODUCTION. The pathophysiological events associated with acute diffusive peritonitis (ADP) and acute lung injury (ALI) formation are poorly understood. In humans remains a major problem of morbidity and mortality worldwide despite developments in monitoring devices, diagnostic tools, and new therapeutic options. In many cases, respiratory failure is a significant contributor to this mortality [8].
ALI is characterized by the accumulation of large numbers of neutrophils in the lungs and a pulmonary inflammatory response in which there is increased production of immunoregulatory cytokines. Also, one of the earliest manifestations is activation of pulmonary endothelium and macrophages (alveolar and interstitial), overregulation of adhesion molecules, and production of cytokines and chemokines that induce a massive sequestration of neutrophils within the pulmonary microvasculature [5]. These cells transmigrate across the endothelium and epithelium into the alveolar space and release a variety of cytotoxic and proinflammatory compounds, including proteolytic enzymes, reactive oxygen species (ROS) i.e.,
H2O2, O2 , OH, HOCl, NO, singlet oxygen and cationic proteins, lipid mediators, and additional inflammatory cytokines, for the purpose of killing bacteria and other microorganisms - oxidative stress. Conversely, excessive production of ROS may lead to acute tissue injury and organ failure. The lung is also at potentially higher risk of injury mediated by ROS, lipid and proteins peroxidation as compared with other organs, due to high exposure to oxygen and the fact that lung tissue contains unsaturated fatty acids that are substrates of lipid peroxidation [3]. Macrophages, neutrophils, endothelial cells, and other pulmonary cell populations have all been demonstrated to express proinflammatory cytokines, but the relative importance of neutrophils amongst these cell populations in contributing to the development of acute lung injury has not been well defined completely.
METHODS. The study was carried out on 78 albino Wistar male rats (180-230 g) divided at random into 3 groups. Group 1 consisted of 58 rats with experimental ADP, 2nd group - control group with sterile 0,9% NaCl at equlibrium (n=10) and 3rd intact group (n=10). The animals were kept in polycarbonate rodent cages with stainless steel mesh lid.
Drinking water was supplied ad libitum and the animals were fed standard rodent pellet diet. ADP was induced by intraperitoneal injection of 10% suspension of feces. The development of ADP was verified by leukocytosis with left shift towards stab neutrophils, elevation of rectal temperature, and presence of purulent exudation in the abdominal cavity. Our study complied with the requirements of the European Scientific Foundation. In anesthetized rats [ketamin (40 mg/kg ip)], blood samples (0,5 ml) were simultaneously obtained from the venous (right ventricle) and arterial (left ventricle) at 1, 12, 24 and 48 h after ADP induction.
To reveal objective signs of endogenic intoxication (EI) and pulmonary pathology in rats with experimental ADP, the analysis of results of blood and lung tissue examination was performed using determining indicators of free radical oxidation of lipids and proteins - thiobarbituric acid reactive substances (TBARS), oxidizing modification of proteins (OMP) and antioxidant protection - ceruloplasmin (CP) [2]; the level of EI was studied by distinguishing middle molecular weight of peptides (MMWP) [2] and calculation of leucocyte index of intoxication (LII) by Kalf-Kalif [4]; light and electron microscopy of lung tissue; wet-to-dry lung weight coefficients and they were calculated as follows: wet lung-to-body weight ratio=(mass wet lung/mass body)*10,000, representing lung edema (LEd); dry lung-to-body weight ratio=(mass dry lung/mass body)*10,000, used to assess lung exudates (LEx); water content (LWC) in the lung=(mass wet lung - mass dry lung)/mass wet lung*100; and lung wet-to-dry weight ratio=mass wet lung/mass dry lung used to compare the lung content with exudates (LCE) [10]. For evaluation of ALI we also proposed coefficient of leukocyte lung regulation (CLLR) and gain patent of Ukraine №UA 71009 [1]. CLLR was calculated as (B-A) / B x 100%, where A - quantity of leukocytes in the arterial blood, B - quantity of leukocytes in the venous blood.
Statistical analyses were performed using statistical software program "Statistica 7". Data are expressed as mean ± standard error (SE). Criterion for significance was taken to be P<0,05.
RESULTS AND DISCUSSION. At the 1 h of the experiment the level of lipid peroxidation by TBARS amount exceeded the control animals by 1,54 times (p<0,05) and had become
5,60 0,14 nmol/l (p<0,05). After 12 h TBARS significantly dicreased to 30,71% and through 24h swiftly grew by 2,18 times higher than in control group (Fig. 1). While at the 48th
h of research - again dicreased on 16,98%. Such changes of TBARS may indicate their partial use of the body's energy substrates during shortage of energy.
Fig. 1. Dynamics of TBARS changes during ADP
Activity of one of the key antioxidants of plasma of blood -CP, was certainly higher by 19,13% in comparison to the level in control animals through 1h from the beginning of experiment and continued to grow till 24 h exceeding a value for control animals by 2,2 times (p<0,05) [6]. However, by 48 h decreased by 24,87% (p<0,05) comparatively with CP through 24 h from the beginning of research, remaining 1,6 times (p<0,05) higher than control. It is well known that the CP takes part in the storage, transport or removal of metal ions of variable valency. Our results may indicate disorder of the ability to recover products of free radical oxidation during chain elongation stage by failure of antioxidant system (AOS) defence with exponentiated formation of new chains of lipoperoxidation.
For confirmation of development of respiratory damage in animals with the model of ADP we had been estimating level of OMP in the blood serum (OMPs) and lung tissue homogenate (OMPh). In 1h after experiment concentration of OMPs and OMPh were significantly higher than in control
group.
In the blood serum of experimental animals with ADP the level of OMPs (356 nm) and OMPs (370 nm) were enhanced compared to the control values by 1,7 times (p<0,05) after 1 h of experiment was started, and OMPs (430 nm) and OPMs (530 nm) - by 2,8 and 4,2 times (p<0,05) accordingly. That is the early criterion of severity of process of metabolic intoxication, which is accompanied with depression of adaptation mechanisms *3+. Development of "metabolic intoxication" syndrome during ALI on a background ADP closely related to the functional state of biological membranes, by a metabolic disturbance, with the subsequent decline of functional activity of the regulatory systems and initiation of peroxidation processes which results in the accumulation of their derivatives and metabolites with destabilization of processes of the oxidizing phosphorylation and violation of functioning of cellular membranes [9]. The tendency of OMPs indexes growth was
saved to 24h of experiment with its insignificant decline that followed.
At lung homogenate through 1 h the level of OMPh (356 nm) by 2,4 times (p<0,05) exceeded control data, being here to 4,38% (p>0,05) lower than OMPh (370 nm), which for certain made progress in relation to healthy rats by 2,9 times, while OMPh (430 nm) and OMPh (530 nm) grew as well in blood by 3,7 and 2,7 times (p<0,05) accordingly. A similar tendency is marked at different critical conditions and chronic diseases [6]. Albumens' oxidation under the action of ROS for formation of aldehyde- or ketogroups is one of the adaptation systems and stimulates activation of polycatalytic proteases, that preferentially destroy oxidated proteins, in particular stroma of lungs.
Futher research the concentration of OMPh by 48 h of experiment acquired maximal values comparatively with control: OMPh (356 nm) grew by 7,2 times (p<0,05), OPMh (370 nm) - 8,2 times (p<0,05), OMPh (430 nm) - 11,3 times (p<0,05) and OMPh (530nm) - 18,3 times (p<0,05). High sensitiveness of pulmonary tissue to the action of ROS generation at the terms of development of ALI during ADP, per our opinion, predefined the high level of oxygen, and also considerable maintenance of the unsaturated fatty acids and low activity of particular enzymes of AOS. So, on a background of endotoxemia, modification of albumens is completed by formation of sour groups of albumens, as a result of blocking of key enzymes of the first link of AOS such as CP, and it specifies a deep disbalance between pro- and antioxidant systems [3].
One of predominating constituents which characterize progression of intoxicative and dismetabolic syndrome during ADP is expression of processes of EI [4]. Often pathogenic states are accompanied with development of EI and can be described as polyetiologic and polypathologic syndrome,which predefine an accumulation in tissues and biological liquids of endogenous toxic substances - surplus of products of normal or dysmetabolism.
From the first hour of the experiment starts the appearance of level of toxic products of hydrophilic nature in the animals of I group which significantly exceeded the control values of MMWP1 (254 nm) and MMWP2 (280 nm) by 14,04% and 15,92% respectively and made progress till 48h of experiment, attaining almost double advantage in relation to the indexes of control (Fig. 2). The basic components of fraction of MMWP are middle molecular weight peptides which last appear as a result of disintegration of albuminous molecules taking place at the proteinases action. We mark predomination of MMWP2 at a wave-length 280 nm, which
testifies to albuminolysis, which is also found in literature [3]. As level of MMWP depends on one side, on intensity of biopolymers disintegration, and on other - on speed of their getting lead out through the organs of desintoxication, we can think about violation of both the constituents of this process [4].
Leukocyte response to the pathological process in the 1st experimental group was ambiguous. After an hour of the experiment LII increased by 2 times (p<0,05), and in 48h exceeded control by
12,8 times (p<0,05) (Fig. 3). This indicates excessive involvement of leukocytes, including neutrophils in inflammatory process, their great loss, through which they got the name of "kamikaze" of acute inflammation, in the same time potentiation of EI increase. And as a result of depreciation loss, hyperproduction of immature forms, which are characteristic for the acute inflammatory response of the critical state of the body due to increased excretion of cytokines (granulocyte colony-stimulating growth factor, etc.) at the action of such chemotactic factors such as endotoxins, complement components, interleukins,
lysosomal enzymes, etc. Similar factors have a stimulatory effect on the monocytic sprout, mainly at increasing the functional activity of macrophages in tissues [7, 9]. On a background of severe EI in animals with ADP, they had violation of pulmonary blood circulation, which was confirmed by the CLLR coefficient proposed by us [1]. As a result of determination of which it became known that one of the key, and perhaps a turning point in the development of ALI during ADP, there is a significant delay of leukocytes in the lungs with subsequent aggression against the "target organ", which is the lung microcirculatory bed [6]. It is
wellknown that the first phase of ALI associated with severe neutrophils adhesion to the surface of endothelial pulmonary circulation, and the second - due to increased permeability of capillary endothelial cells, so similar to the
our assertion are publications of some scientists, representing the main pathogenetic factor as neutrophil-dependent injury [8, 10].
We noticed that CLLR was very sensitive marker, as evident from the growth of this indicator compared to the control at 7,3 times (p<0,05) during ADP as early as one hour from the start of the study (Fig. 4). In addition, CLLR represented the leukocyte delay in the lungs and in a case of leukopenia, that was observed after 24 h of the experiment in the 1 sg roup of rats, exceeding control values by 17,9 times [6]. Consequently, our results of CLLR dynamics during the research, in particular in the 24 hour is prognostically unfavorable and indicate uncontrolled delay of white blood cells, as well as represent the progressive development of ALI. This simple coefficient can take a valuable place in clinical practice, because it clearly represents the real picture of the pathological process in the lungs even at preclinical stage.
Increased neutrophilic sequestration is one of the major pathogenetic mechanisms of progression towards destructive changes of aerohematic barrier components [5]. Last we found at the submicroscopic level already from the first hour of the experiment in animals with ADP. Early, preclinical manifestations of micro-and ultramicroscopic levels have been established by other authors also [4]. At the submicroscopic level already after one hour of experiment in animals with ADP was found hyperhydration of the I and II types of alveolocytes and endotheliocytes of hemocapillaries, leading to weakening of intercellular contacts and disruption of organelles [6]. A similar pattern also was noted by other authors in case of the multiple organ failure in the experimental polytrauma conditions [5]. Such tendency of damage of the components aerohematic barrier rapidly grew with the extension of the research duration and indicators of progression of EI. These changes were accompanied by the development of intracellular and interstitial edema, with violation of both respiratory and nonrespiratory lung functions. Similar results are confirmed in other studies by the scientists not only in terms of the influence of bacterial endotoxemia, but also in reperfusion syndrome and action of exogenous factors [7]. In addition to morphological data analysis, development of lung injury in peritonitis was confirmed as increasing by
calculated Wet/Dry indexes. We noticed progressive, significant increase in all Wet/Dry indexes from 12 h ofh studies with maximum values in 48 th. In particular, LEd was significantly higher than the control data by 1,9 times, LEx -1,5 times, LWC - at 10,78% and LCE - on 21,82% [6]. The well-known fact is that when the accumulation of fluid in the interstitial tissue is from 35% to 50% it starts to penetrate the surface of the alveoli, alveolar pulmonary edema is forced [10]. Therefore, these data indicate the formation of ALI, due to the increasing influence of neutrophils by enhanced EI, followed by progressive development of pulmonary edema and tissue accumulation of exudative fluid. As a result of such mechanisms potentiation can develop respiratory failure, that is confirm by the numeral literary sources [8, 10]. Thus, the inflammatory activity of neutrophils is generated by the complement system (C5a), activation of which is combined with the damaging effects of oxidants and proteases on the endothelium of the capillaries [6]. So, the central pathogenetic component in the development of edema in ALI is interreaction between activated neutrophils by EI and endothelium [7, 9], with acute respiratory failure manifestation that found in other studies, both in ALI and other destructive processes in the lungs [5].
CONCLUSION. In experimental acute diffuse peritonitis was observed a significant correlation of violations in the oxidative and antioxidative systems, which was represented by the activation of lipid peroxidation, oxidative modification of proteins and changes in the activity of antioxidant defense system, which was featured by the initial activation and subsequent inhibition.
During development of the experiment we identified elevated levels of leukocyte sequestration in the lungs by CLLR, which grew steadily throughout the study, despite significant fluctuations in the number of white blood cells, with a parallel progressive development ALI, which was confirmed by the changes in Wet/Dry indices and morphological analysis.
