DYNAMICS OF OXIDATIVE STRESS AND APOPTOSIS INDICATORS IN PATIENTS WITH SEVERE CONCOMITANT INJURY

DYNAMICS OF OXIDATIVE STRESS AND APOPTOSIS INDICATORS IN PATIENTS WITH SEVERE CONCOMITANT INJURY

Shabanov A. K., Evseev A. K., Goroncharovskaya I. V., Badygov S. A., Cherpakov R. A., Kulabukhov V. V., Klychnikova E. V., Borovkova N. V., Grebenchikov O. A., Petrikov S. S.

N.V. Sklifosovsky Research institute for Emergency Medicine, Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitation, Moscow, Russia

Among causes of death, trauma occupies the third place after cardiovascular diseases and oncology, which makes it an equally significant socio-economic problem [1, 2]. Trauma is the leading cause of loss of working capacity among people of the most able-bodied age (up to 45 years) [2]. The greatest participation in the treatment of this category of patients, in addition to surgical specialists, is taken by anesthesiologists-resuscitators [3].
Unlike a number of diseases, which today are of more historical interest due to the development of scientific and technological progress, the frequency of occurrence of concomitant injury is steadily increasing against the background of the escalation of armed conflicts, man-made disasters and the general technical vector of the development of civilization [4].
In response to a severe injury and its consequences, the body of the affected person urgently implements a genetically formed protective program aimed at maintaining the functional activity of vital organs [3]. Clinically, this manifests itself in the form of shock and is accompanied by centralization of blood circulation.
The severity of the patient's condition is mainly determined by trauma-associated afferent impulsation, and in particular its intensity, which is directly related to the severity of the injury. Against the background of the development and progression of this process, a number of factors are triggered that determine the severity and intensity of the emerging inflammatory response. These include an increase in mast cell secretion products as a result of their activation, namely histamine and potassium and hydrogen ions, an increase in the level of serotonin and adenosine diphosphate (ADP) as a result of platelet involvement in the process, an escalation of interleukins produced by macrophages, as well as TNF-α, interferon-γ and serotonin. The specific event of involvement of the vascular endothelium is a change in the level of tumor necrosis factor (TNF). However, there is also a change in the plasma concentration of specific interleukins, endothelins and prostaglandins. The greatest influence on such an important parameter of the homeostasis system as vascular tone is exerted by the level of catecholamines and glucocorticoids, which level also directly depends on the severity of the injury. In concomitant injury, many of these substances are considered as deregulating and pathogenic. However, their true role remains unclear. Moreover, in the absence of a critical state, it fits within the framework of physiological regulatory manifestations [5].
In order to expand the understanding of the functioning of certain body systems in a critical state, a constant search for new markers is underway. In the early post-traumatic period, the deficiency of the enzymatic and non-enzymatic antioxidant systems of the body against the background of an increase in the production of active radicals (oxygen, nitrogen, chlorine, etc.) leads to the development of oxidative stress [6], which, along with hypoxia, triggers apoptosis [7-9]. An imbalance in the oxidant-antioxidant system of the body, impaired functioning of the immune system, damage to cells of various organs under the influence of endogenous and exogenous factors, and other processes lead to such severe consequences as acute respiratory distress syndrome, multiple organ failure, sepsis, and death.
The interest in the study of oxidative stress and apoptosis in trauma patients is indicated by the almost threefold increase in publications in this area over the past decade, according to PubMed. In this regard, it seems relevant to study the markers of oxidative stress and apoptosis in the early post-traumatic period in patients who have undergone severe concomitant injury.

The objective of the study - to research the markers of oxidative stress and apoptosis depending on the outcome in patients who have undergone a severe concomitant injury in the early post-traumatic period.

MATERIALS AND METHODS

The study included 66 patients (44 men, 22 women, median age 39.5 (28.25; 46) years) with severe concomitant injury (SCI), who were treated in the intensive care unit of Sklifosovsky Research institute for Emergency Medicine in 2018-2021. Based on the outcome, two groups were formed: with a favorable outcome (49 patients) and a fatal outcome (17 patients).
To determine the severity of the condition, the following scales were used: ISS, APACHE-II, SOFA. The mean injury severity score was 30.0 ± 8.0 for ISS, 17.9 ± 8.4 for APACHE II, and 4.1 ± 2.8 for SOFA.
The inclusion criteria were: 1) age from 18 to 75 years; 2) the severity of damage on the ISS from 18 to 50 points. The exclusion criteria were: 1) victims transferred from other hospitals 24 or more hours after the concomitant injury; 2) morbid obesity with a body mass index of more than 35 kg/m2; 3) a history of renal failure; 4) aggravated allergic history; 5) oncological process.
The analysis of markers of oxidative stress and apoptosis was carried out in three time periods: 1-3, 4-7, and 8-14 days after injury.
Whole blood was obtained using the vacuum blood collection system. For further analysis, Vacutainer® SSTTM II Advance and Vacutainer® EDTA tubes (BD, UK) were used. Plasma and serum were obtained by centrifugation of whole blood at 1,500g for 15 minutes.
When determining the severity of oxidative stress in patients with severe concomitant injury, we assessed the level of malondialdehyde (MDA), the status of the body's antioxidant system, and the level of the platinum electrode potential at an open circuit (OCP).
Determination of the level of malonic dialdehyde in the serum of patients was performed using thiobarbituric acid. The state of the antioxidant system was assessed by the total antioxidant activity (TAA) of the blood serum, which was measured by spectrophotometry on an Olympus AU2700 biochemical analyzer (BeckmanCoulter, USA) using a TAS kit (Randox, UK), as well as by the total amount of electricity (Q) spent on the oxidation of all low molecular weight antioxidants [10]. Platinum electrode OCR was measured in blood plasma using the IPC Compact potentiostat (NTF Volta, Russia). Endogenous vascular regulation disorders were assessed by the content of stable nitric oxide metabolites nitrite/nitrate (NOx) in serum. Angiotensin-converting enzyme (ACE) concentration was assessed with photometric method on Olympus AU 2700 biochemical analyzer (Beckman Coulter, USA) using ACE test kit (Audit Diagnostics, Ireland).
The study of apoptosis and counting of dead blood leukocytes was performed using flow cytometry on CYTOMIC FC500 device (Beckman Coulter, USA). With the hematological analyzer Ac∙T diff2 (Beckman Coulter, USA), the total number of leukocytes in the blood (109/l) was calculated. The number of lymphocytes ready to enter into apoptosis was determined by Fas receptor expression using CD95+ monoclonal antibodies and was presented as a percentage of the total lymphocyte population. The eBioscience Hu AnnexinV-FITC Recomb Protein kit (Thermo Fisher Scientific, Invitrogen) was used to determine the relative number of lymphocytes in venous blood at different stages of apoptosis. At the early stage of apoptosis, the integrity of the cell membrane is preserved, but its phospholipid components are rearranged, and phosphatidylserine appears on the cell surface. Annexin V is able to bind to phosphatidylserine in the presence of calcium. Simultaneous staining of cells with the vital DNA-specific dye 7 amino-actinomycin D (7AAD) made it possible to differentiate cells at the early stages of apoptosis (Annexin V+/7AAD–, early apoptosis) from cells that had already died as a result of apoptosis (Annexin V+/7AAD+, late apoptosis) . The number of lymphocytes at different stages of apoptosis was presented as a percentage relative to the total population of lymphocytes. The number of antigen-presenting monocytes with the CD14+HLA-DR+ phenotype was assessed.
The reference values of the studied parameters were calculated on the basis of data from practically healthy people (n = 50).
Statistical data analysis was performed using the Statistica 10 software package (StatSoft, Inc., USA). Descriptive statistics of quantitative traits were presented as Me (Q25; Q75), where Me is the median, (Q25; Q75) are the lower (25 %) and upper (75 %) quartiles. The hypothesis about the correspondence of the distribution of quantitative traits to the normal distribution was evaluated using the Shapiro-Wilk test. Due to the rejection of this hypothesis for the studied parameters, the groups were compared using the non-parametric test of the Mann-Whitney U-test. Intragroup comparison was performed using the Wilcoxon test. Differences were considered statistically significant at p < 0.05. In the case of multiple comparisons, taking into account the Bonferroni correction, differences were considered statistically significant at p < 0.025.
The study complies with WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human (2013), and the Rules of clinical practice in the Russian Federation (June 19, 2003, No. 266). The study was approved by ethical committee of Sklifosovsky Research institute for Emergency Medicine.

RESULTS

The distribution according to the mechanism of injury and anatomical regions in patients is presented in Table 1. In the general structure of injury mechanisms, falls from a height prevail (41.0 %), followed by traffic accidents (38.0 %) (Table 1). At the same time, the combination of head and chest injuries (70.0 %) is most common in the structure of injury distribution by anatomical regions (Table 1). 57.5 % of the victims had limb injuries. Less than 30 % of patients had a combination of injuries to the head and abdomen (28.8 %), pelvis (22.4 %), and spine (18.8 %). It should be noted that 21 patients had an injury in 3 anatomical regions (33.4 %), and 17 ones had an injury in 4 regions (25.75 %).

Table 1. Distribution according to the mechanism of injury and anatomical regions in patients

Mechanism of injury

Number of patients (%)

Falling from height

41.0

Traffic accident

38.0

Work injury

6.0

Other

15.0

Injuries to anatomic regions

Limbs

57.5

Pelvis

22.4

Spine

18.8

Head + chest

70.0

Head + abdomen

28.8


The main causes and timing of deaths in patients with severe concomitant injury are presented in Table 2. The most common causes of deaths in patients with severe concomitant injury were shock and massive blood loss on days 1-3 (37.5 %) and purulent-septic complications on days 8-14 (37.5 %) from the moment of injury (Table 2). In addition, deaths were associated with edema and dislocation of the brain in 11.8 % of patients.

Table 2. The main causes and timing of deaths in patients with severe concomitant injury

Causes of death

Timing of the development of a lethal outcome from moment of injury

Total

days 1-3

days 4-7

days 8-14

Massive blood loss and shock

n = 6

n = 0

n = 1

7

Brain edema and dislocation

n = 1

n = 1

n = 0

2

Purulent-septic complications

n = 0

n = 1

n = 6

7

Other

n = 0

n = 0

n = 1

1

Note: n – the number of victims, abs.

The results of the study of the dynamics of markers of oxidative stress and apoptosis are presented in tables 3, 4.

Table 3. Dynamics of oxidative stress markers in patients with severe concomitant trauma

Index

Reference

Favorable outcome

Lethal outcome

days 1-3

days 4-7

days 8-14

days 1-3

days 4-7

days 8-14

MDA, µmol/l

2.27 (2.11; 2.47)

4.12 (3.46; 4.69)

4.90 (4.42; 5.65)

5.14 (4.67; 5.98)

3.61 (3.20; 4.85)

4.57 (4.02; 5.10)

4.44 (4.21; 7.69)

p1 < 0.000000*

p1 < 0.000000*

p1 < 0.000000*

p1 = 0.000001*

p1 = 0.000001*

p1 < 0.000000*

p3 = 0.000164*

p4 = 0.015208*

p2 = 0.511781

p2 = 0.480882

p2 = 0.361358

p3 = 0.020796*

p4 = 0.040861

OAA, mmol/l

1.61 (1.56; 1.68)

1.33 (1.20; 1.54)

1.20 (1.13; 1.34)

1.23 (1.09; 1.34)

1.47 (1.34; 1.78)

1.54 (1.35; 1.76)

1.27 (1.23; 1.45)

p1 = 0.000022*

p1 < 0.000000*

p1 = 0.000003*

p1 = 0.176269

p1 = 0.266482

p1 = 0.000914*

p3 = 0.010944*

p4 = 0.622424

p2 = 0.069498

p2 = 0.007765*

p2 = 0.289624

p3 = 0.858955

p4 = 0.213525

NOx, µmol/l

18.61 (17.70; 23.62)

17.32 (13.68; 21.96)

15.33 (11.85; 20.85)

14.12 (11.15; 17.02)

26.50 (18.20; 36.85)

25.24 (20.71; 35.33)

15.19 (12.06; 50.50)

p1 = 0.027314*

p1 = 0.002808*

p1 = 0.000037*

p1 = 0.126792

p1 = 0.062111

p1 = 0.427279

p3 = 0.241491

p4 = 0.204399

p2 = 0.019319*

p2 = 0.013516*

p2 = 0.188899

p3 = 0.929153

p4 = 0.040861

ACE, mmol/l

45.00 (36.45; 55.15)

27.10 (20.97; 34.3)

30.10 (22.05; 36.81)

35.37 (26.20; 46.03)

26.65 (24.79; 32.63)

28.72 (23.55; 30.63)

41.09 (33.87; 47.75)

p1 = 0.000001*

p1 = 0.000002*

p1 = 0.00279*

p1 = 0.00052*

p1 = 0.00011*

p1 = 0.297383

p3 = 0.842078

p4 = 0.067347

p2 = 0.64359

p2 = 0.531857

p2 = 0.149126

p3 = 0.33288

p4 = 0.016605*

PCR, mV

-39.26 (-18.97; -49.04)

14.18 (-6.68; 28.67)

30.89 (10.32; 43.05)

35.90 (17.51; 52.91)

15.63 (-5.60; 33.15)

34.22 (24.97; 43.92)

44.12 (33.20; 54.07)

p1 < 0.000000*

p1 < 0.000000*

p1 < 0.000000*

p1 < 0.000000*

p1 < 0.000000*

p1 < 0.000000*

p3 = 0.000030*

p4 = 0.007790*

p2 = 0.818553

p2 = 0.317774

p2 = 0.039869*

p3 = 0.002401*

p4 = 0.026156

Q, µC

21.76 (18.97; 24.92)

14.43 (10.07; 19.27)

11.48 (8.81; 13.04)

11.13 (9.68; 13.49)

18.76 (13.35; 24.91)

13.00 (9.61; 15.12)

10.25 (8.02; 15.81)

p1 < 0.000000*

p1 < 0.000000*

p1 < 0.000000*

p1 = 0.108071

p1 < 0.000000*

p1 < 0.000000*

p3 = 0.000066*

p4 = 0.155472

p2 = 0.017968*

p2 = 0.146624

p2 = 0.400793

p3 = 0.008147*

p4 = 0.637818

Note: the level of significance of differences (p): 1 - between control values and values in patients (Mann-Whitney U test, p < 0.05), 2 - between values in patients with favorable and fatal outcome (Mann-Whitney U test, p < 0.05), 3 -between values on days 1-3 and 4-7 (Wilcoxon test, p < 0.025, adjusted for multiple comparisons), 4 - between values on days 4-7 and 8-14 (Wilcoxon test, p < 0.025, adjusted for multiple comparisons), * – differences are statistically significant.

Table 4. Dynamics of markers of blood apoptosis in patients with severe concomitant trauma

Index

Reference

Favorable outcome

Lethal outcome

days 1-3

days 4-7

days 8-14

days 1-3

days 4-7

days 8-14

Leukocytes, ×109 cells/l

6.3 (5.1; 5.9)

6.8 (5.6; 13.3)

9.2 (6.7; 15.5)

9.4 (5.6; 13.9)

8.2 (7.0; 15.8)

17.2 (13.9; 18.5)

11.0 (9.3; 13.3)

p1 = 0.018534*

p1 = 0.000055*

p1 = 0.000023*

p1 = 0.002642*

p1 = 0.000501*

p1 = 0.000013*

p3 = 0.247777

p4 = 0.55312

p2 = 0.334161

p2 = 0.026457*

p2 = 0.627569

p3 = 0.500185

p4 = 0.079617

Dead cells (DC), %

0.75 (0.56; 1.14)

0.80 (0.70; 1.12)

1.10 (0.90; 1.32)

1.30 (1.08; 1.46)

1.05 (0.80; 1.27)

1.44 (1.15; 1.74)

1.44 (1.08; 1.65)

p1 = 0.343534

p1 = 0.22704

p1 = 0.024002*

p1 = 0.049459*

p1 = 0.014671*

p1 = 0.003016*

p3 = 0.57482

p4 = 0.186377

p2 = 0.09527

p2 = 0.035431*

p2 = 0.203419

p3 = 0.172956

p4 = 0.224917

Dead cells (DC), ×106 cells/l

45.4 (28.5; 72.0)

68.0 (35.8; 123.0)

138.0 (83.6; 174.4)

101.8 (72.3; 183.0)

84.2 (61.3; 147.0)

203.5 (197.6; 222.4)

136.2 (115.2; 171.7)

p1 = 0.046038*

p1 = 0.003554*

p1 = 0.000039*

p1 = 0.000993*

p1 = 0.000583*

p1 = 0.000043*

p3 = 0.235551

p4 = 0.411531

p2 = 0.147502

p2 = 0.013918*

p2 = 0.363082

p3 = 0.685831

p4 = 0.043115

Early apoptosis, %

2.50 (1.60; 3.73)

5.83 (3.40; 7.34)

4.77 (2.78; 6.76)

5.70 (4.90; 6.70)

4.13 (2.85; 8.08)

1.99 (1.77; 3.58)

1.65 (1.37; 3.90)

p1 = 0.000092*

p1 = 0.006677*

p1 < 0.000000*

p1 = 0.008265*

p1 = 0.788808

p1 = 0.529078

p3 = 0.076813

p4 = 0.052957

p2 = 0.72343

p2 = 0.03551*

p2 = 0.001631*

p3 = 0.248865

p4 = 0.753153

Late apoptosis, %

0.05 (0.04; 0.12)

0.08 (0.03; 0.14)

0.10 (0.06; 0.12)

0.06 (0.03; 0.10)

0.08 (0.04; 0.10)

0.05 (0.02; 0.24)

0.03 (0.01; 0.07)

p1 = 0.887086

p1 = 0.994745

p1 = 0.699641

p1 = 0.845586

p1 = 0.901196

p1 = 0.123035

p3 = 0.648657

p4 = 0.569163

p2 = 0.955291

p2 = 0.8195

p2 = 0.093868

p3 = 0.892738

p4 = 1.000000

CD95+, %

41.6 (39.05; 50.45)

51.13 (37.83; 59.40)

49.00 (45.80; 66.43)

65.63 (53.87; 71.03)

43.60 (28.15; 66.87)

33.70 (32.54; 34.42)

30.68 (27.39; 39.07)

p1 = 0.896026

p1 = 0.02048*

p1 = 0.056934

p1 = 0.891086

p1 = 0.006323*

p1 = 0.001288*

p3 = 0.120446

p4 = 0.340883

p2 = 0.735581

p2 = 0.001165*

p2 = 0.00189*

p3 = 0.027709

p4 = 0.753153

CD14+HLA-DR+, %

84.7 (73.15; 87.45)

53.50 (44.59; 65.45)

40.40 (36.67; 57.20)

83.44 (59.18; 90.35)

35.57 (23.13; 43.20)

22.91 (15.81; 32.39)

28.32 (19.29; 45.54)

p1 < 0.000000*

p1 < 0.000000*

p1 = 0.107695

p1 = 0.000105*

p1 = 0.000324*

p1 = 0.000015*

p3 = 0.289959

p4 = 0.052024

p2 = 0.015377*

p2 = 0.00614*

p2 = 0.002362*

p3 = 0.463072

p4 = 0.345448

Note: the level of significance of differences (p): 1 – between control values and values in patients (Mann-Whitney U test, p < 0.05), 2 – between values in patients with favorable and fatal outcome (Mann-Whitney U test, p < 0.05), 3 – between values on days 1-3 and 4-7 (Wilcoxon test, p < 0.025, adjusted for multiple comparisons), 4 – between values on days 4-7 and 8-14 (Wilcoxon test, p < 0.025, adjusted for multiple comparisons), * – differences are statistically significant.

DISCUSSION

Our data are generally consistent with other studies of the main causes of death in patients with severe concomitant injury [11], in which three main causes of mortality can be distinguished: massive blood loss and resulting shock, edema and dislocation of the brain, as well as development infectious complications. Massive blood loss and shock were the main causes of mortality on the first day after injury. The same was true of cerebral edema with its subsequent dislocation. At later stages, purulent-septic complications were the predominant cause of mortality. It should be noted that, despite all anti-epidemic measures and strict infection control, when patients are artificially ventilated for more than 3 days, at least 70 % of the victims develop ventilator-associated pneumonia [12].
In response to a traumatic impact, the body launches a number of systemic reactions aimed at preserving its vital activity. Often, the post-traumatic period is accompanied by the development of hypoxia and shock, leading to impaired microcirculation and reduced perfusion of organs and tissues.
Epinephrine-induced vasoconstriction maintains local hypoxia and limits tissue turnover during shock. Lack of oxygen is one of the factors in the development of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). The pathogenesis of this condition is based on the predominance of anaerobic glycolysis, which results in the accumulation of lactate and hydrogen ions in the cell, which, in turn, is an inducer of mitochondrial pore closure (mPTP). This process mediates the accumulation of Na+ and Ca2+ ions inside the mitochondria, which contributes to increased production of free radicals through hypoxia-induced factor 1-alpha (HIF-1α). This process, under conditions of disruption of the intake of components of the antioxidant defense system into mitochondria, causes the accumulation of active radicals, intensification of lipid peroxidation processes, and damage to functional structures in mitochondria [13]. At the same time, coagulopathy, inflammation, anaerobic metabolism and oxidation contribute to the development of endotheliopathy [14]. In this case, relaxation dysfunction develops in the endothelium of arterioles, which is associated with local hyperproduction of reactive oxygen species (ROS) by CD11/CD18+ cells. In capillaries, activated leukocytes adhere to damaged endothelial cells.
The endothelium of postcapillary venules plays a key role in the occurrence of secondary complications in severe concomitant injury. Firstly, ROS cause complement (C5) activation and the production of a number of factors, such as B4 leukotriene, which is able to induce adhesion and activation of leukocytes on the endothelium. ROS also induce the release of Weibel-Palade bodies, which are large endothelial vesicles containing von Willebrand factor (vWF) and P-selectin. These compounds provide adhesion and penetration of CD11/CD18+ activated cells such as neutrophils and platelets. The inflammatory response is enhanced by mast cells and macrophages, which release inflammatory mediators such as TNF-α, nitric oxide (NO) and histamine. In addition, ischemia and inflammation often lead to disruption of endothelial tight junctions, adhesions, and glycocalyx components. Activated neutrophils cause breakdown of the glycocalyx during injury as they release proteolytic enzymes such as neutrophil elastase, which promotes the synthesis of local inducible nitric oxide synthase (iNOS) and ROS. All this contributes to the violation of the integrity of the connection of endothelial cells and the permeability of the endothelial barrier, which leads to a change in the vasotonic and hemostatic function of the endothelium [15].
However, the reperfusion stage, which occurs in conditions of intensive care, is more dangerous. When switching from anaerobic glycolysis back to oxidative phosphorylation, the synthesis of adenosine triphosphate (ATP) by mitochondria is triggered, but at the same time, an even greater production of active radicals occurs as a result of damage to mitochondrial structures. This process causes irreversible damage, including such important structures as mitochondrial DNA (mtDNA) while slowing down ATP synthesis. Restoration of intracellular pH to a physiological level activates the opening of the mitochondrial pore, leading to oversaturation of the mitochondria with Ca2+ and activation of calpain. On the other hand, there is a release of active radicals, as well as other compounds, including cytochrome C (cyt C), which cause the activation of caspases [13].
It is this cascade, i.e. the activation of leukocytes and neutrophils, as well as the expression of cytokines and adhesion molecules, leads to cell death due to apoptosis. In addition, there is a high probability of developing thrombosis with further damage to the microvasculature as a result of activation of the complement system and the coagulation cascade.
In understanding the fundamental molecular mechanisms of many physiological and pathophysiological processes, an important role is assigned to the main factor of vasodilation, and NO, which can have a multidirectional effect under certain conditions. In essential hypertension, it has a normalizing effect on hemodynamics, but in hemorrhagic or traumatic shock, on the contrary, it destabilizes it, lowering blood pressure due to hyperactivation of inducible nitric oxide synthase. Since NO is an active form of oxygen that easily interacts with the superoxide anion radical (O2–) to form peroxynitrite (ONOO–) under conditions of suppression of superoxide dismutase activity, the study of NO generation during oxidative stress, which is characteristic of combined injury, seems to be very important [5].
The described mechanisms of development of oxidative stress and apoptosis generally correspond to our data. On the first day, against the background of the consequences of ischemia-reperfusion syndrome, there is a shift in almost all analyzed parameters from their normal values. Among markers of oxidative stress, a particularly noticeable difference with control values is observed for MDA, ACE, ORC (p < 0.0001) (Table 2). The state of the body's antioxidant defense system deserves special attention in this period. In contrast to patients with favorable outcomes, when a quite natural noticeable decrease in the activity of the antioxidant system was noted on the first day [16], in patients with lethal outcomes, the decrease in the activity of this system was not so pronounced. This circumstance may be associated both with an increase in the production of antioxidants in response to increasing oxidative stress [17] and with the inhibition of free radical processes against the background of hypoxia [18] on the first day after injury.
This pattern of changes in indicators persists for about 7 days and can characterize the stage of acute oxidative stress [19]. The totality of the processes occurring in this period determines the further dynamics of the patient's condition, since significant differences are observed in the future. In patients with a favorable outcome, after 7 days, a sharper decrease in the intensity of changes in oxidative stress indicators (OAA, OCR, Q) is noted, which may indicate the achievement of the so-called “quasi-stationary” level of active radicals [19], which characterizes the state of chronic oxidative stress. At the same time, in patients with a fatal outcome in this period, a further decrease in the activity of the antioxidant system is observed against the background of an increase in oxidative processes, i.e., the transition of oxidative stress to an uncontrolled stage.
The level of stable metabolites of nitric oxide was statistically significantly reduced relative to normal values up to the 14th day of observation in the group with a favorable outcome (Table 2). In the group with a lethal outcome, the NOx indicator did not statistically significantly differ from the normal values at all periods of the study and was statistically significantly higher on the 7th day compared to the group with a favorable outcome (p = 0.014) (Table 2). However, it should be noted that in the group with a fatal outcome, especially on the 14th day, a number of patients had a very high level of NOx. The increase in the level of NOx, apparently, is due to the septic status, since in the group with a lethal outcome, the level of leukocytes at these times was also statistically significantly different from the group with a favorable outcome. It is known that the level of NO increases in sepsis and septic shock, since iNOS is activated during infection, which generates hyperproduction of NO. The interaction of NO and ACE provides multidirectional regulation of blood pressure levels under both physiological and pathophysiological conditions. ACE has a vasopressor effect, converting angiotensin I to angiotensin II, the most powerful vasopressor, the effect of which is 50 times higher than that of adrenaline. The ACE level statistically significantly decreases in the group with a favorable outcome relative to the norm at all follow-up periods (p < 0.005) (Table 2), which indicates conjugation in vascular regulation by the endothelium. While this balance was not observed in the group with a lethal outcome, the ACE level was statistically significantly below the norm against the background of normal NOx values, and by the 14th day did not differ from the norm against the background of a decrease in NOx (Table 2).

The content of leukocytes in the venous blood of the victims increased already from the first day after the injury and reached its maximum values by the 4-7th day both in the survivors and in the dead (Table 3). At the same time, on days 4-7, the number of leukocytes in the blood of deceased patients was 1.72 times higher than in patients with a favorable outcome of a severe concomitant injury. Also, patients noted an increase in the concentration of dead blood leukocytes, most pronounced in patients with an unfavorable outcome of injury. Leukocytosis and an increase in dead leukocytes in venous blood are due to the development of endotoxemia in patients with severe concomitant trauma caused by hypoxia, an array of damaged tissues, and an associated infection. Infectious complications in victims with severe concomitant injury often develop against the background of immune system dysfunction. Thus, already from the first day after a severe concomitant injury, a decrease in the population of monocytes with the CD14+HLA-DR+ phenotype, which present the antigen to T-lymphocytes, was noted in patients. The minimum content of CD14+HLA-DR+ monocytes in the blood was noted on the 4th-7th day after the injury, while the concentration of antigen-presenting monocytes in the blood of deceased patients was significantly lower than in the group of survivors at all follow-up periods (Table 3).

The concentration of lymphocytes expressing the Fas-receptor (CD95+) on their surface in the blood of victims on the 1-3rd day after the injury did not differ significantly in the compared groups and was recorded within the upper limits of the physiological norm. On days 4-7, a significant decrease in CD95 positive lymphocytes in venous blood was noted in deceased patients. It is known that Fas also mediates the transmission of non-apoptotic signals in lymphocytes, including facilitating their differentiation during the development of the immune response [20]. Thus, the low content of CD95+ lymphocytes in the peripheral blood of patients with an unfavorable outcome of a severe concomitant injury also causes a violation of the processes of activation of the immune system. The low concentration of CD95+ lymphocytes in deceased patients was also accompanied by a significant decrease in apoptotic lymphocytes in the blood, observed in deceased patients on days 4-7 and 8-14 after injury. It should be noted that the concentration of apoptotic lymphocytes in patients with a favorable outcome of injury was slightly higher than normal throughout the entire observation period.

CONCLUSION

Thus, our data indicate a difference in the dynamics of changes in markers of oxidative stress and apoptosis. It is shown by a sharper decrease in the intensity of changes in oxidative stress indicators (TAA, OCR, Q) in patients with a favorable outcome by the 7th day after injury, which may indicate the achievement of the so-called "quasi-stationary" level of active radicals, which characterizes the state of chronic oxidative stress. At the same time, in patients with a fatal outcome, there is a constant decrease in the activity of the antioxidant system against the background of an increase in oxidative processes during the entire observation period, i.e., the transition of oxidative stress to an uncontrolled stage. In addition, data on the dynamics of NOx and ACE levels indicate the development of endotheliopathy in patients with a fatal outcome, which, in particular, is manifested by a violation of vascular regulation. Analysis of the dynamics of apoptosis markers (the number of leukocytes in venous blood, CD95+ lymphocytes, CD14+HLA-DR+ monocytes) of the blood revealed a significant violation of the processes of activation of the immune system, which leads to the development of infectious complications in patients with severe concomitant injury.

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