key: cord-0727802-0p7d3qwt authors: Gümüş, Hüseyin; Erat, Tuğba; Öztürk, İrfan; Demir, Abit; Koyuncu, Ismail title: Oxidative stress and decreased Nrf2 level in pediatric patients with COVID‐19 date: 2022-02-16 journal: J Med Virol DOI: 10.1002/jmv.27640 sha: 3c1938fd8241d35a732dd4e47f5d8bf71a0471b9 doc_id: 727802 cord_uid: 0p7d3qwt The aim of this study was to investigate the change in nuclear factor erythroid 2‐related factor (Nrf2), which plays a critical role in cytoprotection against oxidative stress, in pediatric patients with coronavirus disease 2019 (COVID‐19) infection positivity, and to evaluate the relationship between Nrf2 and oxidative balance. The study included 40 children with confirmed COVID‐19 infection and 35 healthy children. The groups were compared in respect of Nrf2, total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI), in addition to clinical findings of fever, cough, shortness of breath, contact history, and demographic data of age and gender. The mean Nrf2 values and TAS levels were determined to be statistically significantly low (p < 0.001) and the TOS level and OSI were statistically significantly high in the children with COVID‐19 compared to the control group. A significant positive correlation was determined between Nrf2 and TAS (p < 0.01); as the Nrf2 value increased, so the TAS value increased. A significant negative correlation was determined between Nrf2 and TOS and OSI (p < 0.01); as the Nrf2 value increased, there was determined to be a significant decrease in the TOS and OSI values. COVID‐19 infection in pediatric patients causes a decrease in the Nrf2 level. By causing a decrease in the TAS level and an increase in the TOS and OSI levels, the decrease in Nrf2 may explain the tissue damage which can be caused by COVID‐19. Nrf2 is found in the cytoplasm bound to Keap1. In the presence of electrophiles or ROS, the Keap1-Nrf2 complex separates and Nrf2 migrates to the nucleus stimulated by transcription of target genes together with antioxidant response element sequences. Nrf2 controls the expression of genes participating in antioxidant response, redox homeostasis, and mitochondria biogenesis. The activation of these genes protects cells from inflammation. 9 The aim of this study was to determine the relationship between Nrf2 and oxidative balance in pediatric patients with and to determine the roles of these in disease severity. Cases with a negative RT-PCR result despite a contact history or clinical suspicion of COVID-19, cases with a positive RT-PCR result who started treatment but were exposed to cigarette smoke or had a chronic systemic disease such as hypertension, chronic lung disease, diabetes mellitus, congenital heart disease, malignancy, or immune failure, or cases with a recent history of trauma. Nasopharyngeal smear samples were taken by an Ear, Nose, and Throat specialist from cases with suspected COVID-19 infection. The agent was investigated with RT-PCR of the samples collected. Blood sampling. A 2 ml venous blood sample was collected from all the cases in the study on first presentation at hospital before any treatment was started. The blood samples were withdrawn into red top plain blood collection tubes that did not contain any solution. The samples were centrifuged at 1000g for 10 min to separate the plasma. The plasma samples were stored at −80°C until analysis. Nrf2 levels were analyzed according to the commercially available ELISA kit method (BT-LAB). In this method, microplates coated with human Nrf2 antibodies are used. Serum samples are added to these plates and then incubated. After washing the plates, Streptavidinhorseradish peroxidase secondary antibody is added and again incubated. After the next washing, with the addition of substrate, a color is formed. The reaction is stopped by adding a stop solution and absorbance is measured at 450 nm on a microplate reader (Thermo-Go). The total antioxidant status measurements were made using brand commercial kits (Rel Assay Diagnostic Gaziantep) on a microplate reader system (Varioskan Lux; Thermo Scientific). Briefly, free radical reactions were initiated by the Fenton reaction and monitored by absorbance of the dianisidyl radicals. This reaction was measured spectrophotometrically at 660 nm. Using this method, the antioxidative effect was measured as the relative amount of free dianisidyl radicals. 10, 11 The precision of this test has high accuracy (<3% error rate). The data were expressed in mmol Trolox equivalent/L. The total antioxidant status measurements were made using brand commercial kits (Rel Assay Diagnostic Gaziantep) on a microplate reader system (Varioskan Lux; Thermo Scientific). according to the method of Erel. 10, 11 Briefly, oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically (at 530 nm), is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed as μmol H 2 O 2 equivalent/L. (2.5%), and vomiting in 2 (5%). There was no significant difference between the groups in respect of age or gender (p > 0.05). In the COVID-19-positive cases, the TAS and Nrf2 measurements were statistically significantly low (p < 0.001) and the TOS and OSI measurements were statistically significantly high (p < 0.01) ( A statistically significant positive correlation was determined between Nrf2 and TAS (p < 0.01). A statistically significant negative correlation was determined between Nrf2 and TOS and OSI (p < 0.01; Table 3 and Figure 1 ). As oxidative stress plays an important role in the response to infections, it may also be a significant player in the pathogenesis of COVID-19. 12, 13 Studies have shown that oxidative stress regulates the host immune system in viral diseases F I G U R E 1 Correlation graphs between Nrf2 levels and TAS, TOS, and OSI. Nrf2, nuclear factor erythroid 2-related factor; OSI, oxidative stress index; TAS, total antioxidant status; TOS, total oxidant status such as hepatitis B, hepatitis C, herpes simplex virus, and influenza. [14] [15] [16] Oxidative stress has also been found to contribute to the pathogenesis of various pulmonary diseases in children such as pneumonia, asthma, acute bronchiolitis, cystic fibrosis, acute respiratory syndrome, and chronic neonatal pulmonary disease. 17, 18 Studies have reported a potential link between oxidative stress and the pathogenesis, disease severity, and mortality risk of patients infected with SARS-CoV-2. 13, 19 Karsen et al 20 Nfr2 is a primary regulating transcription factor, and induces genes which play a critical role in cytoprotection against oxidative and xenobiotic stresses. 26 In a study by Komaravelli et al. 21 Nrf2 levels were determined to be reduced associated with RSV infection. It was reported that Nfr2 played a significant role in respiratory tract disease caused by RSV, pulmonary damage and the onset of inflammation, and the exacerbation of ROS production, and the reduction in Nfr2 was probably associated with secondary increasing ROS production and oxidative stress. Hybertson et al. 27 showed that Nfr2 activation reduced the rate of viral replication and weakened symptoms by limiting microvascular damage, and could provide the possibility of successful follow-up throughout the cytokine storm, which is a particular problem associated with COVID-19. In the current study, the Nrf2 level was determined to be significantly decreased, together with a significant decrease in TAS, and significantly high TOS and OSI values in pediatric patients with COVID-19. A statistically significant positive correlation was determined between Nrf2 and TAS (p < 0.01), and as Nrf2 increased, so the TAS value also significantly increased. A statistically significant negative correlation was determined between Nrf2 and TOS and OSI (p < 0.01), and as Nrf2 increased, so the TOS and OSI values also significantly decreased. COVID-19 infection in pediatric patients causes a decrease in the level of Nrf2, which creates a defence against oxidative stress and regulates the redox balance. By causing a decrease in TAS level and an increase in TOS and OSI levels, the decrease in Nrf2 in pediatric COVID-19 patients may explain the tissue damage which can be caused by COVID-19. No financial or nonfinancial benefits have been received or will be received from any party related directly or indirectly to the subject of this article. Director-General's opening remarks at the media briefing on COVID-19-11 March. World Health Organization; 2020 COVID-19 coronavirus pandemic: Worldometer, 2021. 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