The Effects of Asthma on the Stress Oxidative, Inammation, and Endothelial Dysfunction Characteristics in Children with Severe Community-Acquired Pneumonia

Background: Pulmonary vascular endothelial activation, inammation, and stress oxidative have been implicated in adverse clinical outcomes of community-acquired pneumonia (CAP). Although chronic lung problems such as asthma may affect the consequences of pneumonia, the exact mechanism of this effect remains unclear. The present study aimed to assess the effects of asthma on the oxidative stress, inammation, and endothelial dysfunction biomarkers in children pneumonia. Methods: This cross-sectional study was performed at Amir Kabir Hospital aliated to Arak University of Medical Sciences, Arak, Iran. Participants were 25 children with severe CAP and asthma (group I), 25 children with severe CAP (group II), and 25 healthy children (group III) with 2 to 6 years of age. Fasting blood samples were taken to the assay of serum malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor-alpha (TNF-α), soluble vascular cell adhesion molecule-1 (sVCAM-1), and Plasminogen activator inhibitor-1 (PAI-1). Results: We observed a signicant reduction in TAC in groups I and II (0.997±0.22 and 1.23±0.21 mmol/l, respectively) compared with group III (1.46±0.19 mmol/l). This reduction was signicantly higher in group I than in group II. Also, we observed a signicant increase in MDA and TNF-α in groups I (2.57±0.40 µmol/l, 6.94±1.61 pg/ml, respectively) and II (6.94±1.61µmol/l, 5.54±1.84 pg/ml, respectively) compared with group III (1.89 ±0.27µmol/l, 3.42±1.32 pg/ml, respectively). The increase in MDA was signicantly higher in group I than in group II. Data are presented as mean value and standard deviation (SD); ∗ p < 0 05. Malondialdehyde (MDA); Total antioxidant capacity (TAC); Vascular cell adhesion molecule 1 (VCAM-1); plasminogen activator inhibitor-1 (PAI-1). Tumor necrosis factor alpha (TNF-α). P- value: Differences in the percentage changes on mean value of initial reading between groups I, II, and III.

Results: We observed a signi cant reduction in TAC in groups I and II (0.997±0.22 and 1.23±0.21 mmol/l, respectively) compared with group III (1.46±0.19 mmol/l). This reduction was signi cantly higher in group I than in group II. Also, we observed a signi cant increase in MDA and TNF-α in groups I (2.57±0.40 µmol/l, 6.94±1.61 pg/ml, respectively) and II (6.94±1.61µmol/l, 5.54±1.84 pg/ml, respectively) compared with group III (1.89 ±0.27µmol/l, 3.42±1.32 pg/ml, respectively). The increase in MDA was signi cantly higher in group I than in group II.
Conclusions: Asthma can exacerbate the consequences of pneumonia in children by increasing oxidative stress, in ammation, and endothelial dysfunction.

Background
Pneumonia is an infection of the lungs caused by bacteria, viruses, fungi, and parasites that impose signi cant costs for the health care system and exhibit the most common reason for the death of infectious origin (1). In this disease, polymorphonuclear neutrophils and macrophages ght with microorganisms by using reactive oxygen species (ROSs) and lysosomal enzymes (2). As a consequence of pulmonary defense mechanism in in ammatory diseases such as pneumonia and asthma, Oxidative stress (OS) at the systemic level may have a central role with adverse clinical outcomes of these diseases, such as the endothelial dysfunction (ED), exacerbation of in ammation, and shortness of breath, and ultimately acute respiratory distress syndrome (ARDS), and death (3)(4)(5).
ED causes pulmonary edema due to an increase in endothelial permeability. The activated endothelium mediates leukocyte binding to express the adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) that mediate leukocyte binding. Upon leukocyte binding, these adhesion molecules activate endothelial cell signal transduction and then alter endothelial cell shape for the opening of passageways, through which leukocytes can migrate (6)(7).
Characterized by chronic in ammation in the airway wall (8), asthma is the most common chronic respiratory disease in children, which is prevalent in developing countries. Although it cannot be considered a direct cause of pneumonia, children with asthma are more prone to develop pneumonia due to previous lung damage. As a result, a child with asthma may have more severe symptoms and complications from pneumonia. Asthma may exacerbate the clinical consequences of pneumonia, such as ED (9-11).
We are not aware of any studies on assessing the changes in OS, in ammation, and ED biomarkers in children with asthma and pneumonia together compared with children with pneumonia only. Therefore, the current study assessed the alterations in OS, TNF-α, and ED biomarkers in children with asthma and pneumonia, children with pneumonia only, and healthy children.

Study design and participants
A cross-sectional study was conducted at the Pediatric Clinic of Amirkabir Hospital in Arak, Iran, from January 2019 to September 2019. The present study aimed to assess the effects of asthma on the oxidative stress, in ammation, and endothelial dysfunction biomarkers in children pneumonia.
To estimate the sample size, we considered type 1 (α) and type 2 errors (β) of 0.05 and 0.20 (power = 80%), respectively, and serum MDA level as a key variable. Based on a previous study (12), SD (σ1) of control MDA was 0.1 µmol/L, SD (σ2) of case MDA was 0.07 µmol/L, and the difference in mean (d) of insulin levels was 0.15 µmol/L. We reached the sample size of 9 participants for each group. The sample size of our study consisted of 25 children diagnosed with severe community-acquired pneumonia (sCAP), 25 patients diagnosed with asthma and sCAP, and 25 healthy children.
Pneumonia was de ned as an acute pulmonary in ltrate evident on chest radiography with symptoms and signs of a lower respiratory tract infection: fever, cough, and purulent sputum. Pneumonia was con rmed with physical exams, microbiologic culture data, and Chest x-ray. CAP in children was de ned as a lower respiratory tract infection in a child who has not resided in a hospital or health care facility in the preceding 14 days. CAP in children is one of the most common acute infections that require going to the hospital. Children with sCAP, due to respiratory distress, are not able to eat, drink, and alert. They also have undesirable hydration status and oxygenation status (13)(14).
The asthma of children was con rmed by a physician via the symptoms of recurrent coughing, wheezing, and chest tightness.
Exclusion criteria included children with severely smoking parents and severe or multiple systemic diseases.

Ethics and consent
The present study was ethically approved by the Committee on Human Research, Publication and Ethics (CHRPE) at Arak University of Medical Sciences, Arak, Iran (IR.ARAKMU.REC.1397.3001). Informed consent to participate in the study was obtained from parent of the children.

Biochemical assessments:
Blood samples of all the subjects were taken, and aliquot samples of serums were saved after centrifugation (20 min, 3000 rpm) at -80°C.
According to the method of Benzie & Strain in 1996 (15), TAC was analyzed using uorescence recovery after photobleaching (FRAP) assay, which depends on the capacity of serum to reduce Fe 3+ to Fe 2+ .
Serum MDA levels were determined by the thiobarbituric acid reactive substances test (TBARS) spectrophotometric test, as described by Santos in 1980 (16).
Serum TNF-α was measured through the ELISA method according to the manufacture's instruction (Biovendor, Germany, Cat≠ RAF128R).

Statistical analysis
The Kolmogorov-Smirnov test was employed to assay the normal distribution of variables. The one-way ANOVA and Kruskal-Wallis test were employed to compare Anthropometric and Biochemical factors between groups. Post Hoc and Mann-Whitney tests were utilized to compare subgroups (I, II, III). All statistical analyses were performed using SPSS version 17 (SPSS, Chicago, IL, USA).

Discussion
In this study, we observed that in children with sCAP, biomarkers of OS, in ammation, and ED were signi cantly higher than healthy children, and it is also higher in asthmatic children with pneumonia than in non-asthmatic children. This is probably because asthma may exacerbate OS and in ammation in children with pneumonia.
Studies have shown the interaction between pneumonia and cardiovascular diseases (CVDs). According to the cohort study of Yeh et al. 2019, patients with CVDs had a higher risk of CAP, and conversely, CVDs risk was intensi ed with CAP. In recent years, CVDs were considered as an outcome of patients admitted to hospital with pneumonia infection (17). After recovery of CAP in addition to the period of the acute infection, there is still the risk of acute cardiovascular events due to systematic in ammation (18).
The initial stage of molecular and cellular stages leading to CVDs is ED (19)(20). OS and in ammation are the two main causes of its creation (21)(22).
Studies indicate the underlying respiratory diseases such as asthma may be effective in the severity of pneumonia injuries. Asthma, whose main feature is chronic in ammation in the airway wall (8), is the most common chronic respiratory disease in children, especially in developing countries.
In this study, TNF-α was signi cantly higher in children with pneumonia and asthma than pneumonia and healthy children. Studies indicate the in ammatory process associated with ED exacerbates the severity of the consequences of CAP (23). Also, recent evidence suggests a critical role for pneumonia infection in the pathogenesis of atherosclerosis by exacerbating OS, in ammation, and ED. Increasing the proin ammatory cytokine TNF-α as a consequence of pneumonia induce ED by various mechanisms, such as increasing the endothelial permeability and reducing the endothelium-dependent relaxation. It increased vascular endothelial growth factor (VEGF) as the endothelial permeability mediator and diminishing the half-life of mRNA encoding for endothelial nitric oxide synthase and decreasing nitric oxide production (24,25).
In this study, VCAM-1 and PAI-I as two biomarkers of ED were signi cantly higher in children with pneumonia and asthma than the children with pneumonia only. Also, they were signi cantly more in children with pneumonia than healthy children. OS and in ammation are closely linked with each other. In ammatory mediators lead to OS, and reciprocally, OS increases the production of in ammatory mediators with the activation of NF-kB and AP-1 (26). NF-κB and AP-1 are involved in the activation of pro-in ammatory molecules, such as VCAM-1 and PAI-I (27).

Conclusions
Signi cant changes in OS, in ammation, and ED biomarkers occur in asthma children with pneumonia compared with pneumonia children without asthma and healthy children. Our ndings amplify the growing evidence supporting the concept that endothelial activation, in ammation, and OS play an important mechanistic role of effects asthma in the pathogenesis of pneumonia. Treatment with antioxidants and anti-VCAM-1 pharmacological agents may help reduce outcomes in these children.