Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity / 2021 / Article
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The Role of Oxidative Stress in the Pathophysiology of Chronic Inflammatory Diseases 2021

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Research Article | Open Access

Volume 2021 |Article ID 2105406 | https://doi.org/10.1155/2021/2105406

Svetlana Soodaeva, Nailya Kubysheva, Igor Klimanov, Alexey Shutov, Tatyana Eliseeva, Viktor Novikov, Klavdiya Kontorshchikova, Dmitry Novikov, Ildar Batyrshin, "The Differences in the Levels of Oxidative Status Marker and Soluble CD95 in Patients with Moderate to Severe COPD during an Exacerbation and a Stable Period", Oxidative Medicine and Cellular Longevity, vol. 2021, Article ID 2105406, 12 pages, 2021. https://doi.org/10.1155/2021/2105406

The Differences in the Levels of Oxidative Status Marker and Soluble CD95 in Patients with Moderate to Severe COPD during an Exacerbation and a Stable Period

Academic Editor: Daniela Ribeiro
Received07 Jun 2021
Revised13 Sep 2021
Accepted25 Nov 2021
Published09 Dec 2021

Abstract

Studying the features of changes in markers of oxidative stress (OS) and inflammation indicators in COPD patients depending on the degree of bronchial obstruction is one of the priority directions for improving the prognosis and monitoring of the course of this pathology. We conducted a comparative investigation of changes in markers of OS and apoptosis at the systemic and local levels in patients with moderate to severe COPD during exacerbation and stable phase. 105 patients with COPD aged 46-67 and 21 healthy nonsmoking volunteers comparable in age were examined. COPD patients were divided into four groups: moderate COPD (GOLDII) during the exacerbation (GOLDIIex, ) and in the stable phase (GOLDIIst, ), severe COPD (GOLDIII) during the exacerbation (GOLDIIIex, ), and in the stable phase (GOLDIIIst, ). We studied the levels of such lipid peroxidation (LPO) products as diene conjugates (DC) and Schiff bases (SB) and parameters of induced chemiluminescence (Imax, total light sum-S, Imax/S) in blood serum, as well as sCD95 concentration in blood and exhaled breath condensate (EBC). The relationship between the values of the OS system indicators with sCD95, as well as with the parameters of lung function, was investigated. Multidirectional changes in OS indicator levels in COPD patients depending on the severity of obstructive airway disorders have been established. The maximum values of DC (), Imax ( RLU), and Imax/S () were typical for patients with moderate COPD, while the highest SB level ( RU) was observed in severe COPD during an exacerbation. The exacerbation of the disease was characterized by an increase in DC concentration in both GOLDIIex ( RU) and GOLDIIIex ( RU) compared to the stable moderate and severe COPD ( RU and RU, respectively, ). The established decrease in high values of DC, Imax, Imax/S, and sCD95 and an increase in SB concentration in GOLD III can serve as quantitative indicators of the prognosis of the severity of the disease. The serum concentration of sCD95 in GOLDIIex ( U/ml) and GOLDIIst ( U/ml) did not differ from the control group ( U/ml, ). In patients with during the exacerbation and stable phase, the serum levels of Imax/S ( and ) and sCD95 ( U/ml and  U/ml) were lower than the values of healthy volunteers ( and  U/ml, respectively, ). A positive correlation between sCD95 concentration and airway obstruction degree in all examined COPD patients was established. The revealed numerous associations between sCD95 and OS marker levels in GOLDIII indicate a relationship between systemic radical stress and apoptosis processes both in the respiratory tract and the whole body under conditions of severe inflammation. The established correlations between the values of DC, Imax, and sCD95 in the blood serum and the lung function parameters in all studied patients allow us to consider these indicators as additional prognostic indicators of disease intensification. Our work results help clarify the participation and detail of FRO and apoptosis processes in developing pathophysiological features in moderate to severe COPD in different periods and, accordingly, improve the efficiency of diagnosis and treatment of the disease.

1. Introduction

Local and systemic inflammation in COPD is closely related to the intensification of free radical oxidation (FRO) processes and the development of oxidative and nitrosative stress [15]. The leading role of oxidative (OS) and nitrosative stress (NS) in the damaging effect on almost all lung structures, especially in the formation of lung tissue remodeling, is well known [1, 5].

Several studies have shown an increase in OS and NS marker concentration in various biological environments in COPD patients [1, 2, 4]. An increase in H2O2 concentration and the total oxidative status (TOS) in the EBC was revealed in this disease [68]. In some works, an increase in Fe2+ level in the respiratory tract was noted [9]. The interaction of hydrogen peroxide and divalent iron in the Fenton reaction can lead to the overproduction of extremely reactive hydroxyl radicals, which initiate the processes of FRO and lipid peroxidation (LPO). An increase in lipid peroxidation products such as MDA, 4-Hydroxy2-nonenal, and 8-isoprostane has also been found in serum, EBC, and sputum in COPD [8, 1013].

Along with an increase in FRO indices, it was found that in patients with this disease, the activity of antioxidant enzymes, such as SOD, catalase, and glutathione peroxidase, as well as nonenzymatic antioxidant concentration (vitamins A and C, glutathione, etc.) decreases [14, 15]. However, several other studies have obtained opposite data on OS indicators in COPD. For example, an increase in enzymatic antioxidant activity, the absence of an increase in the level of lipid peroxidation products in patients with this disease has been shown [16, 17].

A significant proportion of OS and NS studies in COPD are devoted to investigating biomarkers reflecting the intensity of FRO directly in the respiratory tract: exhaled breath condensate (EBC), sputum, and BAL fluid [1012, 1820].

Currently, information is accumulating on circulating systemic OS markers associated with various pathophysiological disorders in COPD patients [21]. However, the feature changes in the systemic indicators of radical stress in COPD depending on the severity and period of the disease have not been sufficiently studied. The activity of OS processes is often researched by the lipid peroxidation indicators, such as MDA and isoprostanes. At the same time, it is important to study the intensification of lipoperoxidation reactions by the values of the initial and end products of LPO—diene conjugates (DC) and Schiff bases (SB). It is also relevant to determine the DC to SB (DC/SB) ratio, which allows you to define the direction and expressive of lipoperoxidation processes stages. There are few studies of these molecular products in patients with COPD, depending on the severity and period of the disease. There are few studies of these molecular product concentrations in COPD patients depending on the severity and period of the disease.

It should be noted that one of the most significant ways to investigate FRO reactions is to determine the potential ability of lipid substrates to form free radicals, which can be detected using chemiluminescence (CL) analysis. This method allows us to comprehensively evaluate both the prooxidant and antioxidant properties of the biosubstrate [2224]. Given that there has been a growing interest in a complex assessment of the oxidant/antioxidant system in COPD, using this integral method to determine OS markers in developing this disease is essential.

In addition to the study of FRO, to identify the mechanisms of pathophysiological processes in COPD, special attention is paid to investigating the relationship between OS indicators and inflammation markers, such as cytokines and soluble differentiation molecules (sCD). Earlier in our works, we showed the role of several soluble forms of membrane molecules in the mechanisms of development of systemic and local inflammation in patients with this disease [3, 4, 25, 26]. In particular, changes in soluble CD95 (sCD95) concentration in serum and EBC were detected in moderate and severe COPD patients during an exacerbation [3]. The sCD95 molecules are one of the apoptosis-specific markers and play an essential role in developing and regulating inflammatory processes in the airways and at the systemic level [3, 27]. For a more detailed study of the pathogenetic mechanisms of COPD progression, it is necessary to comparatively research changes in FRO markers and sCD95 levels in the blood and airways in patients with COPD, depending on the stage and period of the disease.

The study is aimed at investigating changes in the concentrations of DC, SB, and CL parameters in the blood serum and the level of sCD95 in the circulation and exhaled breath condensate in moderate to severe COPD patients during the exacerbation and stable phase.

Analysis of the relationship between OS and sCD95 markers, as well as these indicators and lung function parameters, will help clarify the involvement of FRO and apoptosis processes in the development of pathophysiological features of COPD and improve the diagnosis efficiency and therapy of the disease.

2. Materials and Methods

The study included 126 people: patients with COPD () and healthy nonsmoking volunteers ().

The diagnosis of COPD was defined and classified according to the criteria of the Global Initiative on Chronic Obstructive Pulmonary Disease (GOLD) [28]. The COPD diagnosis was established based on largely irreversible airway obstruction with an improvement in after inhalation of 400 μg salbutamol. Lung function was measured again 15-20 minutes after inhalation of the bronchodilator to assess bronchodilator-induced bronchospasm reversibility.

COPD patients () were divided into four groups: patients with moderate COPD (GOLDII) during the exacerbation (GOLDIIex, ), patients with moderate COPD in the stable phase (GOLDIIst, ), patients with severe COPD (GOLDIII) during the exacerbation (GOLDIIIex, ), and patients with severe COPD in the stable phase (GOLDIIIst, ).

The presented study is a pilot, so we did not use traditional approaches to calculating the sample size [29]. Currently, there are several practical rules according to which the group size in the pilot study is from 15 to 35 people [30, 31].

The study was implemented based on the principles of the Helsinki Declaration. Written informed consent was obtained from all participants. The study was approved by the Ethics Committee of the Pulmonology Research Institute, Moscow, Russia (the protocol number N 05-19 от 16.10.2019).

The study included COPD patients meeting the following inclusion criteria: age over 40 years, active or ex-smokers (smoking pack-years), exacerbation of COPD and stable period, and evidence of obstructed lung function (postbronchodilator  and FEV1/) according to the GOLD [28]. An exacerbation was defined as a change in the symptoms of a cough, expectoration, and dyspnea beyond the daily variation and required changes in therapy in COPD patients.

The exclusion criteria were the following: asthma and other allergic diseases, pneumonia, history of congestive heart failure, severe arterial hypertension, diabetes, and conditions requiring the long-term use of systemic corticosteroids.

The control group included healthy nonsmokers with similar gender and age indicators who did not take any medications. Healthy subjects underwent a medical examination at the clinic and were randomly selected as a control group. The participants in the healthy group had no diagnosed respiratory diseases, diabetes, coronary heart disease, malignancies, or connective tissue diseases.

A pulmonary function study was carried out on a computer Spirograph “SpiroLab III” (Italy) for the evaluation of the FEV1, FEV1/FVC, and the parameters of inspiratory capacity (IC).

2.1. Serum and Exhaled Breath Condensate Preparation

Blood samples were obtained in the morning on an empty stomach from the middle cubital vein, immediately centrifuged at 3000 rpm for 10 minutes, and then extracted. Serum samples were frozen at -40°C.

EBC was collected using the RTube and following the guidelines for EBC by the ERS/ATS Task Force [32]. All patients were asked to refrain from drinking any liquid (except water) for 2 hours before the collection of EBC. To avoid oral or nasal contamination, the patients were asked to rinse their mouths with freshwater before collection and to wear a nose clamp during collection. The donors were asked to use tidal breathing into the mouthpiece for 10 minutes. After the 10-minute period of breathing is over, the samples were immediately stored and cooled to -40°C.

2.2. Measurement of the Diene Conjugates and Schiff Base Concentrations

The concentrations of DC and SB were determined as described in [33, 34] spectrophotometrically on a PerkinElmer LS-50 spectrophotometer. The levels of these LPO products were expressed in relative units (RU).

2.3. Chemiluminescence Analysis

To determine the intensity of free radical processes in blood serum, we used the CL method induced by hydrogen peroxide with ferrous sulfate. The measurements were carried out on a Dynatech chemiluminometer (Germany).

The following CL indicators were analyzed:

Imax (relative light units (RLU)) is the maximum value of the CL outbreak intensity, reflecting the biological system’s potential ability to develop FRO processes.

S (total light sum) is the area under the CL response curve, which characterizes the FRO activity and is inversely proportional to the antioxidant activity (AOA).

Imax/S is the ratio that characterizes antioxidant activity (AOA) of the reaction system.

2.4. Measurement of sCD95 Concentration

The levels of soluble CD95 molecules in the serum and the EBC were determined by enzyme-linked immunosorbent assay (ELISA) using an ELISA reader (Multiskan MS, Labsystems, Finland) wavelength of 405 nm. In determining the content of soluble CD95 molecules, we used goat polyclonal antibodies against PBMC superficial antigens and mouse monoclonal antibodies ICO-160 against the CD95 antigen conjugated with horseradish peroxidase. The results were expressed in conventional units (U/ml).

2.5. Statistical Analysis

The statistical analysis was carried out using the Statgraphics Centurion software package, v.9. The data were presented as the . To determine the distribution normality, the Shapiro-Wilk test was used. The student’s -test performed further analysis. To calculate the correlation coefficient (), the Pearson correlation test was used. The statistical significance level was considered to be .

3. Results

Figure 1 shows a block diagram of patient recruitment. A total of 258 people have successfully passed spirometry. Out of 258 patients, 153 people did not participate in this study according to the exclusion criteria. Therefore, 105 patients were recommended for further examination.

The demographic and clinical characteristics of individuals are shown in Table 1. There was no significant difference between the groups by age. In all groups, the majority of subjects were men. Spirometric values such as FEV1%, FEV1/FVC ratio, and IC were significantly lower in COPD patients compared to the control group (). Lung function parameters in GOLDII exceeded similar values in patients with severe COPD (). The exacerbation of the disease was characterized by a decrease in the values of the studied spirometric parameters compared to the stable phase in both GOLDII and GOLDIII ().


Healthy nonsmokersCOPD
ModerateSevere
12345

Subjects ()21Exacerbation ()Stable ()Exacerbation ()Stable ()
Sex, male/female15 (71%)/6 (29%)18 (72%)/7 (28%)22 (81%)/5 (19%)26 (90%)/3 (10%)20 (83%)/4 (17%)
Age (years)
Smoking pack-years0
FEV1 % pred









FEV1/FVC %









Inspiratory capacity IC (%)









COPD medication
 LAMA2 (8%)4 (14.8%)
 LAMA+LABA7 (28%)13 (48.1%)8 (27.6%)6 (25%)
 ICS+LABA+LAMA16 (64%)10 (37%)21 (77.8%)18 (75%)
 SCS9 (36%)29 (100%)

Data were presented as . COPD: chronic obstructive pulmonary disease; pack-years: number of cigarette packs per day multiplied by the number of smoking years; FEV1: forced expiratory volume in one second; % pred: % predicted; FVC: forced vital capacity; IC: inspiratory capacity (%); LAMA: long-acting muscarinic antagonists; LABA: long-acting β agonists; ICS: inhaled corticosteroids; SCS: systemic corticosteroids.
3.1. The Serum Concentration of Diene Conjugates in COPD Patients

The DC level was increased in all COPD patients compared to the control () (Table 2). The maximum value of these LPO products was observed in GOLDIIex relative to all the examined patients. The stable period of the disease was characterized by a decrease in DC level compared to during exacerbation in both moderate and severe COPD ().


Healthy nonsmoking volunteersGOLDIIexGOLDIIstGOLDIIIexGOLDIIIst
12345

Schiff bases (RU)









Diene conjugates (RU)









Diene conjugates/Schiff base









Imax (RLU)









S









Imax/S










Data were presented as .
3.2. Schiff Base Concentration in COPD Patients

The SB concentration in all the examined patients exceeded the values of the control () (Table 2). The highest SB level was found in GOLDIIIex relative to controls and patients with moderate COPD (). The concentration of these LPO end products in patients during the exacerbation did not differ from analogous values in the stable phase in both moderate and severe COPD ().

3.3. Index of DC/SB in COPD Patients

To identify the expressive of the initial or final stages of LPO, the DC/SB ratio was determined. The value of the DC/SB index in patients with moderate COPD was significantly higher compared to the control () (Table 2). At the same time, there was a decrease in DC/SB values in severe COPD both during exacerbation and stable periods relative to healthy volunteers and GOLDII patients (). There were no differences in DC/SB level depending on the periods of the disease in the examined individuals.

3.4. Chemiluminescent Parameters in the Blood Serum in COPD Patients

The Imax and S values were increased in all examined COPD patients in comparison with healthy volunteers () (Table 2). At the same time, the high Imax level gradually decreased as the severity of the disease increased. The maximum CL intensity in the serum was recorded in GOLDIIex and was higher than in severe COPD patients during exacerbation () and in the stable phase ().

The highest light sum (S) values were found in GOLDIIIex patients. This indicator’s value was lower in all examined COPD patients in the stable phase than during exacerbation ().

The ratio Imax/S (AOA) was higher in moderate COPD compared to control () (Table 2). The level of Imax/S (AOA) in severe COPD was lower than in healthy volunteers and all GOLDII patients (). There were no differences in Imax/S (AOA) value depending on the period of the disease.

3.5. sCD95 Concentration in Blood Serum and Exhaled Breath Condensate in COPD during Exacerbation and Stable Periods

The serum level of sCD95 in patients with moderate COPD did not differ from the level of healthy nonsmoking volunteers ( U/ml, ) (Figure 2). However, the concentration of these molecules in severe COPD during the exacerbation ( U/ml) and stable period ( U/ml) was statistically lower than in healthy nonsmokers (), GOLDIIex ( U/ml), and GOLDIIst ( U/ml, ).

The level of sCD95 in EBC was higher in patients with moderate COPD both during the exacerbation ( U/ml) and the stable period ( U/ml) than in healthy volunteers ( U/ml, ). The endobronchial concentrations of sCD95 in GOLDIIIex ( U/ml) and GOLDIIIst ( U/ml) were significantly lower compared to the GOLDII patients (). They did not differ from the levels in the control group ().

3.6. The Correlations between Oxidative Stress Indicators, sCD95 Levels, and Lung Function Parameters

We found correlations between the values of spirometric indicators and the studied OS markers in COPD patients (Table 3). A negative association between DC level and FEV1 and FEV1/FVC was revealed in all COPD patients. The inverse correlation was established between SB concentration and FEV1/FVC (%), IC in severe disease. The multidirectional nature of the relationship of the Imax values and lung function parameters was revealed. The negative correlation between these indicators was established in moderate COPD; a positive relationship was found in severe disease.


GOLDIIGOLDIII
FEV1 (%)FEV1/FVC (%)ICFEV1 (%)FEV1/FVC (%)IC

Diene conjugates





Schiff base





Imax





Imax/S





sCD95 (serum)





sCD95 (EBC)






: correlation coefficient; FEV1: forced expiratory volume in 1 second; % pred: % predicted; FVC: forced vital capacity; IC: inspiratory capacity (%); EBC: exhaled breath condensate.

A positive relationship between the AOA value and all the studied lung function parameters in patients with severe COPD was established. The increase in Imax/S (AOA) values occurred against the background of an increase in the IC level in GOLD II.

3.7. Analysis of Correlation between Oxidative Stress Indicators

The results of the association analysis between the concentration of LPO products and the studied CL were the following: (i)The negative correlations between the values of Imax/S (AOA) and levels of SB and DC ( and , respectively)(ii)The negative relationship between the Imax value and the SB level ( )(iii)The positive association of the DC concentration and the value of light sum ( )

3.8. Associations between sCD95 Levels and Lung Function Parameters

An analysis of associations showed a positive correlation between serum and endobronchial sCD95 levels and the studied lung function parameters in all examined patients (Table 3).

3.9. The Relationship between the Studied Markers of Oxidative Stress and the Level of sCD95 in COPD Patients

The relationship between the sCD95 level and the studied OS indicators was revealed only in patients with severe airway obstructive disorders (Table 4). In this group of patients, a decrease in the concentration of sCD95 both in the bloodstream and in the EBC occurs against the background of an increase in the SB level, as well as a d