Nuclear Factor Erythroid 2-Related Factor (NRF2), Heme Oxygenase 1 (HO-1) and Total Oxidant-Antioxidant Status in Patients with COVID-19

Introduction: Studies on nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) levels in COVID-19 patients are limited. This study aimed to investigate the relationship between some biomarkers of oxidant-antioxidant status with COVID-19 disease. Material and methods: The patients older than 18 years of age who


INTRODUCTION
World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a global epidemic on March 11, 2020 and more than 220 countries worldwide are affected by the disease and number of cases reached 615 million while 6,524,568 people died due to the disease worldwide (04/10/2022), making this situation a public health crisis [1,2].Coronaviruses are enveloped Ribonucleic acid (RNA) viruses from the beta-coronavirus genus of the Coronaviridae family.SARS coronavirus-2 (SARS-CoV-2) has been defined as the seventh coronavirus that is pathogenic in humans after other types of coronaviruses, which are seasonal HCoV, SARS-CoV, and MERS-CoV [3].COVID-19 caused by SARS-CoV-2, able to cause different results from an asymptomatic clinical course to acute respiratory distress syndrome (ARDS) or severe pneumonia that may lead to death [4].COVID-19 is characterized by an abnormal host immune response causing excessive inflammatory responses that are revealed by elevated plasma levels of cytokines, chemokines, and C-reactive protein (CRP).This has fatal consequences, as well as causing multi-organ failure and serious damage to the respiratory system [5].Reactive oxygen species (ROS) that occurred after physiological and metabolic activities.This ROSs is removed by enzymatic and non-enzymatic anti-oxidative mechanisms to prevent damages that may occur in the organism [6].Nuclear factor erythroid 2-associated factor 2 (Nrf2) is a transcription factor responsible for the adaptation of cells to oxidative or electrophilic stress.Nrf2 also stimulates expression of heme oxygenase (HO-1) [7].HO-1 has a crucial role in the antioxidant defense system and iron homeostasis.This enzyme can prevent oxidative deoxyribonucleic acid (DNA) damage, which occurs because of cells being exposed to heat shock, changes in ROS or DNA distribution and cell cycle induced by pyrrolidine dithiocarbamate [8].In this study, it was aimed to investigate the relationship of some biomarkers of oxidant-antioxidant status with COVID-19.

STUDY DESIGN AND POPULATION
The patients older than 18 years of age who tested positive for SARS CoV-2 PCR (polymerase chain reaction) between June 2020 -December 2021 with clinical symptoms and signs in the Infectious Diseases Service of Harran Medical Faculty Hospital were included in this study.

INCLUSION CRITERIA
Patients who presented to the emergency department with various symptoms and were diagnosed with COVID-19 because of the reverse transcriptase polymerase chain reaction (RT-PCR) test for SARS COV 2 virus positive were included in the study.Within the scope of this study, patients were grouped according to the diagnostic criteria established in the "COVID-19 (2019-nCoV Disease) Guide", which has been published by the Turkish Ministry of Health and is regularly updated with the latest developments.Accordingly, COVID-19 has been classified into four groups.Group 1 included the presence of mild clinical symptoms; Group 2 included the presence of respiratory symptoms, fever, and radiological pneumonia findings; Group 3 included shortness of breath, oxygen saturation ≤ 93% at rest, respiratory rate (RR) ≥ 30/min, and PaO2/FiO2 ≤ 300 mmHg; and Group 4 included clinical shock status, organ failure or respiratory failure requiring mechanical ventilation [9].

EXCLUSION CRITERIA
Negative results of RT-PCR test for SARS COV 2 virus, diagnosis of COVID-19 who started treatment, history of smoking, hypertension, chronic heart disease, chronic lung disease, malignancy, diabetes mellitus, immunodeficiency or recent trauma were excluded from the study.

SAMPLING AND ANALYSIS
Nasopharyngeal swab was taken from the patients and RT-PCR test was used.

STUDY PROTOCOL
During hospitalization and one week after discharge, 2mL of venous blood was taken from the patients and transferred to serum separator tubes (Becton Dickinson, USA).The tubes were centrifuged at 5000 rpm for 5-10 minutes and the obtained serum sample was stored at -80 degrees.

MEASUREMENT OF TOTAL ANTIOXIDANT STATUS (TAS)
Commercial kits (Rel Assay Diagnostic Gaziantep, Turkey) were used in a microplate reader system (Varioskan Lux, Thermo Scientific, USA) to measure total antioxidant status.The data were expressed as mmol Trolox equivalent/L.

MEASUREMENT OF TOTAL OXIDANT STATUS (TOS)
Commercial kits (Rel Assay Diagnostic Gaziantep, Turkey) were used in a microplate reader system (Varioskan Lux, Thermo Scientific, USA) to measure total oxidant status.The data were expressed as μmol H2O2 equivalent/L.

CALCULATION OF OXIDATIVE STRESS INDEX (OSI)
Firstly, TAS units results were converted to mmol/L, and then following formula was used: OSI (arbitrary unit) = TOS (mmol H2O2 equivalent/L)/TAS (mmol Trolox equivalent/L).
2.6.5.STATISTICAL ANALYSIS SPSS version 22.0 (SPSS Inc., Chicago, IL) program was used for statistical analysis.Power analysis was performed to determine sample size using G*Power v3.1.9.7.Effect size was selected as 0.75 (to detect the medium-sized differences),  as 0.05 and power of the test as 0.80.Descriptive statistics were summarized as number, median (minimum-maximum), mean, percentage, and standard deviation.Shapiro-Wilk (Since n=16 ≤30) test was used for investigating the conformity of variables to normal distribution.Continuous variables were analyzed with either Paired Samples T-Test or Wilcoxon test depending on homogeneity and distribution of the data.Pearson correlation was used to examine the relationship between parameters.Correlation coefficient (r) was found because of Pearson correlation analysis.p-value that is less than 0.05 was considered as statistically significant.

ETHICAL CONSIDERATIONS Approval of the Harran University Faculty of Medicine
Ethics Committee was obtained for the study (21/06/2021 HRU/21.12.10).

DISCUSSION
In this study, we examined the relationship between COVID-19 and oxidative stress.TOS and OSI levels decreased after the treatment while TAS, Nrf2, and HO increased.These results were statistically significant.The decrease in oxidative stress indicators throughout the process in COVID-19 and the increase in TAS, which is an indicator of anti-oxidative state, as well as in Nrf2 and HO-1, which are indicators of anti-oxidative defense response, suggest that the body tries to prevent ROS-related oxidative stress via Nrf2 and HO-1.Therefore, it could be suggested that oxidative stress may have a key role in COVID-19.Oxidative stress is defined as "the situation in which oxidation occurs due to the loss of balance between the body's antioxidant systems" [10].ROS and reactive nitrogen species (RNS) are by products of several cellular processes.Under normal physiological conditions, the balance between ROS and antioxidant levels is maintained at the cellular level.However, these powerful oxidants (free radicals) may pose harmful effects when the redox balance is lost; they are often associated with the onset of various lifestyle diseases.Such harmful effects are caused by free radicals that attack various cells and contain uneven oxygen and nitrogen damaging DNA, proteins, and lipids [11].
Although various measurement methods have been developed for TOS and TAS, there is no generally accepted reference method yet.In this study, we used a novel automatic measurement method developed by Erel to determine total antioxidant status and total oxidant status of the plasma.This method is advantageous since it is simple, inexpensive, reliable, and very sensitive.Considering the indices, the literature provides some but the most widely used one is the OSI, which was proposed in 2003.This index has changed little over time, but all conducted studies conclude and agree that it is a ratio between TOS and TAS expressed as arbitrary units [12][13][14][15][16]. Karkhanei et al. [17] performed a case-control study aiming to investigate the association between some oxidative stress biomarkers and COVID-19, and the patients were divided into four groups as non-intensive care, intensive care and intubated, intensive care but non-intubated, and healthy without COVID-19 diagnosis.This study found TOS to be significantly higher in those three groups compared to the control group.On the other hand, TAS level was not different in non-intensive care patients compared to the control group but was significantly higher in patients followed up in intensive care unit compared to the control group.Mehri et al. [18] found that the mean serum TOS concentration was significantly higher in COVID-19 patients when compared to the control group and they stated that this elevation might have an important role in the mechanism of disease development.Aykaç et al. [19] determined that serum TOS levels were significantly higher in patients diagnosed with COVID-19.
In a case-control study conducted by Gümüş et al. [20] to investigate the relationship between COVID-19 and oxidative stress in pediatric patients, Nrf2 and TAS levels were found to be lower and TOS and OSI levels higher in the patient group, and it was stated that this might be related to tissue damage in COVID-19.In our study, we determined that TOS value decreased after the treatment compared to pre-treatment.The pre-treatment TAS value increased significantly in the post-treatment period.The pre-treatment OSI value decreased significantly.Accordingly, the decrease in TOS, which is among oxidative stress parameters, and the increase in TAS, which reflects the antioxidative state, suggest that anti-oxidative mechanisms step in during the disease and endeavor to reduce oxidative damage.
Various studies have been conducted on the relationship between HO-1 enzyme level and viral infections.Su et al. [21] carried out a study comparing the relationship between desaturation and heme oxygenase in patients with COVID-19 diagnosis.The authors observed that heme and HO-1 enzyme levels increased significantly in the low SpO2 group, and they believed that the increased HO-1 in the low oxygenation group may reduce inflammation and provide benefit in terms of survival in this group of patients.L.-L.Ma et al. [22] emphasized the antiviral effect of HO-1 against influenza viruses.The authors of the relevant study demonstrated that cobalt protoporphyrin (CoPP), a potent inducer of HO-1 like hemin, interacts with IRF3 and HO-1 and then inhibits the replication of influenza A virus by expression of IFNa/β.In another study, the same mechanism was observed in the reduction of viral replication and lung inflammation following HO-1 induction and IFNa/β expression in the infected lung in human respiratory syncytial virus infection [23].HO-1-mediated type I IFN response is assumed to be able to control many other viral infections such as hepatitis B/C virus, Ebola virus, and human immunodeficiency virus by inhibiting virus replication [24].In our study, we detected that the pretreatment HO-1 increased significantly in the post-treatment period.This result suggests that HO-1 enzyme may have antiviral effects in the pathogenesis of COVID-19 and may contribute to the reduction of oxidative stress.As a transcription factor, Nrf2 regulates certain genes directly defending against oxidative stress, including catalases, numerous peroxidases, superoxide dismutase, and enzymes synthesizing glutathione [25].Attending to the human coronavirus HCoV-229E associated with cold and lung disease, deficient expression of Nrf2 target gene glucose-6-phosphate dehydrogenase (G6PDH) increases ROS production, viral gene expression, and particle production.It is also observed that lung biopsies of COVID-19 patients showed that the Nrf2 pathway is suppressed and the pharmacological inducers of Nrf2 inhibit the replication of SARS-CoV2 and the inflammatory response [26][27][28][29].The current study demonstrated significantly increased Nrf2 after treatment of COVID-19.This increase in Nrf2 level points out that the Nrf2 pathway may be an important factor in reducing oxidative stress in COVID-19 infection.It is also known that Nrf2 has a role in stimulating the expression of HO-1 [7].
There are many studies investigating the inflammatory parameters in COVID-19 prognosis [30][31][32].However, this study is one of the rare studies investigating oxidative stress in COVID-19.Similarly, Binici et al. [31] investigated superoxide dismutase, glutathione peroxidase, glutathione, total thiol, natural thiol, disulfide, oxidative DNA damage, and malondialdehyde levels in 35 COVID-19 cases and 35 control volunteers.In this study, serum superoxide dismutase, glutathione peroxidase, malondialdehyde, 8hydroxy-2-deoxyguanosine/10⁶, disulfide levels were higher, and glutathione, total thiol, and natural thiol levels were lower in COVID-19 group compared to the healthy control group.In addition, there was a negative correlation between 8-hydroxy-2-deoxyguanosine/10⁶ deoxyguanosine and glutathione, natural thiol, and total thiol, and a positive correlation with disulfide [30].Our results showed that the statistically significant increase in both Nrf2 and HO-1 levels supports our anticipation.
The important limitations of this study were the small patient population, the absence of severe patients, and the inability to compare with healthy volunteers.

CONCLUSIONS
COVID-19 is still a global problem due to the resulting high morbidity and mortality.Considering the pre-treatment and post-treatment results relating to TAS, TOS, OSI, Nrf2, and HO-1 oxidative stress biomarkers in our study, we think that oxidative stress may play an important role in the pathophysiology of COVID-19.However, there were no clinically severe patients in our study.Large-scale studies including patients with severe COVID-19 are needed.