THE INFLUENCE OF FLAVONOID COMPOSITIONS ON NRF2 TRANSCRIPTION FACTOR EXPRESSION IN CASE OF INFECTIONS TRIGGERED BY INFLUENZA A VIRUS AND TRANSMISSIBLE GASTROENTERITIS CORONAVIRUS

Background. Viral infections trigger transcription factors, including Nrf2, which regulate the expression of genes related to cytokines, chemokines


Introduction
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor initially discovered as a master regulator of redox homeostasis in cells [1].Interplaying with NF-B pathway, Nrf2 coordinates cellular response to oxidative stress and inflammation [2].Under oxidative stress, Nrf2 dissociates from its suppressor Kelch-like-ECH-associated protein 1 (KEAP1) in cytoplasm and relocates to the nucleus.
There, it binds to antioxidant response elements (AREs) in the genes encoding proteins that regulate oxidative stress, diminish inflammation, and confer other cytoprotective effects at the transcriptional level [3].Keap1-Nrf2-ARE pathway interacts with NF-B pathway regulating the response to stress and inflammation in such a way that the decreased expression of Nrf2 upregulates NF-κB expression by various mechanisms that is important for the balance of pro-and anti-inflammatory factors [2].
Nrf2, like other transcription factors, plays an important role in regulating the body's protective systems and maintaining cell homeostasis due to its versatile effects.It now became clear that the effects of Nrf2 are broader than its involvement in the antioxidant response.In particular, Nrf2 is essential for the regulation of innate immunity [4].The experiments with Nrf2-deficient mice demonstrated Nrf2 involvement in regulation of neutrophil gene expression as well as genes related to phagocytosis, Fc receptor function, complement and immunoglobulin regulation during acute bacterial infections [5].
Recently, the new roles have been attributed to Nrf2 including regulation of antiviral responses [6].Nrf2 activation has been demonstrated in many viral infections, and different mechanisms of its activation have been proved [7][8][9].The mechanisms of pro-and antiviral activities of Nrf2 seem to be diverse.Nrf2's role in antiviral immunity seems to be partially associated with the regulation of the interferon response by inhibiting certain signaling components of interferon-inducing pathways [10,11].However, the prevailing belief is that Nrf2 primarily contributes to viral reproduction mostly by interferon-independent mechanisms [6].
Nrf2 seems to play a dual role in viral infections depending both on the type of the virus and the clinical or experimental setting.Many studies suggest a protective role of Nrf2 in viral infections.The sequels of the experimental viral infection were more pronounced in Nrf2-deficient mice [12].The use of Nrf2 activators in vitro provides the cytoprotective effect and inhibits viral replication [13].The in vitro antiviral effects associated with Nrf2 activation have been demonstrated for a broad range of viruses of different taxonomic groups such as influenza virus, Zika virus, herpes simplex virus, SARS-CoV2, etc. [6,9,10].Nevertheless, the protective role of Nrf2 in viral infections cannot be considered universally consistent taking into account several reports associating Nrf2 activation with the progression of viral infection [9,14].
Nrf2 transcription is induced by various substances of different classes.Various phenolic compounds including flavonoids are among the natural Nrf2 activators [13,15].Luteolin, apigenin, quercetin, myricetin, rutin, naringenin, epicatechin, and genistein have been experimentally proved as Nrf2 inducers [16].On the other hand, these substances have proved to possess antiviral activity against the broad spectrum of viruses [17].It is of interest to assess the Nrf2-inducing capacity in the experi-mental systems of virus infection where antiviral activity of these substances was demonstrated.
The study of the dynamics of Nrf2 activation in viral infections associated with pneumonia is of particular importance taking into account the role of Nrf2 not only in viral infection but also in the accompanying pathological processes in the organs and tissues.Recently, we confirmed the antiviral effects of Proteflazidum ® and its constituents in vivo in case of influenza virus-induced pneumonia in mice and in in vitro infection with transmissible gastroenteritis coronavirus (TGEV) that belongs to alpha-coronaviruses (unpublished data).
The aim of the present study was to research Nrf2 activation in influenza virus infection in vitro and TGEV infection in vivo and the effects of flavonoid compositions Proteflazidum ® and Protoil on the Nrf2 expression in the setting of influenza virus infection in vivo and transmissible gastroenteritis coronavirus (TGEV) infection in vitro.

Flavonoid compositions
Proteflazidum ® composition, developed and produced by Ecopharm Research-and-Production Company, Ukraine, contains ethanol extract from wild gramineous plants Deschampsia caespitosa L. (tussock grass) and Calamagrostis epigeios L. (reed grass).The content of flavonoids is not less than 0.32 mg/ml calculated as rutin equivalent.The composition comprises O-and C-glycosides of tricin, lutheolin, apigenin, quercetin, rhamnasine as well as their aglycons) [18].
Protoil composition consists of the dense extract prepared via vacuum concentration of Proteflazidum, but ethanol here is substituted with the sunflower oil and polyethylene glycol 400.The content of flavonoids is not less than 3.5 mg/mL.The samples of Protoil were provided by Ecopharm Research-and-Production Company, Ukraine.
The TGEV strain D 52-5 (BRE 79 ) was provided generously by Dr. Hubert Laude (Molecular Virology Laboratory of Biotechnology Center, INRA, France).The attenuated TGEV variant was obtained following 100 passages in continuous porcine cell lines [22].Prior to the study, the attenuated TGEV was adapted to Jurkat cells.All viral materials were stored at 70 C prior to experimental studies.

Experimental animals
The outbred white mice, body weight -14-18 g, sourced from the experimental animals' vivarium of the L.V. Gromashevsky Institute of Epidemiology and Infectious Diseases, were used in the study.The animals were kept under controlled conditions of temperature (22 to 24 C), relative humidity (40% to 70%), lighting (12-hour lightdark cycle) and fed a standard pellet diet.

Design of in vivo experiment
The mice were infected intranasally with 10 LD 50 of the mouse-adapted influenza virus A/FM/1/47 (H1N1).One hour after infection, the mice of experimental groups were exposed to flavonoid compositions.Proteflazidum ® was administered intraperitoneally at a 1:100 dilution in a volume of 0.2 ml.Protoil was fed by gavage at a 1:100 dilution in a volume of 0.2 ml.Flavonoids were also administered to non-infected mice according to the same scheme.The mice of experimental groups as well as virus-infected and intact mice were sacrificed on Day 1 and Day 4. The experimental setting comprised 6 groups of animals with 6 mice in each group.Thus, at each experimental point (Day 1 and Day 4), studies were carried out on 3 mice.
RNA was isolated from lung homogenates of the untreated or flavonoid-treated virus-infected mice on Days 1 and 4 postinfection and Nrf2 expression was assessed by RT-PCR.RNA isolated from the lungs of the intact mice served as the negative control.The relative content of Nrf2-specific RNA was analyzed by the end-point dilution method.The log of the threshold dilution that did not reveal specific transcript was taken as a measure of its content in the initial sample.

Bioethical norms
The study was performed according to the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986) and was approved by the Institutional Animal Care and Use Committee.The experimental procedures with the animals were carried out in accordance with the provisions of the Law of Ukraine "On the Protection of Animals from Cruelty" ( 2006

Cell lines
Human Jurkat cell line of T-lymphoblastic leukemia was obtained from the Depository of Cell Lines and Tumor Strains of the R.E.Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National Academy of Sciences of Ukraine.The cells were grown in RPMI-1640 medium with 10% fetal calf serum (FCS) (Sigma, USA) in a 5% CO 2 -humidified atmosphere and subcultured twice a week.For experimental studies, the cells were seeded in 24-well microplates and each experimental point was repeated in four replicates.

RT-PCR analysis
The nucleotide sequences of the target genes (mouse and human Nrf2) were searched from GenBank database (https://www.ncbi.nlm.nih.gov/nucleotide/).The selected sequences were aligned and analyzed, and specific primers were construed based on Vector NTI v.10.0.1 software (Invitrogen, USA) and Basic Local Alignment Search Tool (BLAST) (National Center for Biotechnology Information (NCBI), USA), except for the sequences for reverse and forward primers for mouse Nrf2 that were taken from [23].All the primers were synthesized by Generi Biotech (Czech Republic, http://www.generi-biotech.com/).All primers used in the study are listed in Table 1.
RNA from cells was isolated with "GeneJet RNA Purification Kit" (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

RNA concentration was measured on a Nano
Drop 1000C spectrophotometer (Thermo Fisher Scientific, USA).
The relative content of the specific transcript in RNA samples was determined by end-point dilution method.For each sample, (real-time) RT-PCR was performed in tenfold serial dilutions, in four replicates each.The log of the threshold dilution of the initial RNA sample that did not produce the specific signal (Ct exceeding 38 in our experimental setting) was taken as the relative measure of the content of the specific transcript in the initial sample.

Nrf2 activation quantity assay
"Nrf2 Transcription Factor Assay Kit (Colorimetric)" (Abcam, USA) was used to quantify Nrf2 activation in nuclear extracts.This assay is a high throughput assay to quantify Nrf2 activation in nuclear extracts.It combines a quick ELISA (enzymelinked immunosorbent assay) format with a sensitive and specific non-radioactive assay for transcription factor activation.The nuclear extracts were prepared from cell homogenate or cell culture with "Nuclear Extraction Kit" (Abcam, USA) according to the manufacturer's instructions.Protein concentration in the nuclear extract was measured by Bradford method [24] using Bradford Assay Kit (Sigma, USA).The absorbance was read on Bio-Tek Elx 800 ELISA reader at 450 nm vs. 655 nm [25].

Statistical processing
The data of ELISA analysis were expressed as mean  standard error of the mean (M  SEM).The groups compared were small and statistical power may not be sufficient to detect significant differences between groups.The non-parametric Mann -Whitney U test was used despite the normal distribution of the data.The significance of the differences was analyzed by Microsoft Excel and Microcal Origin software.The difference was considered as significant if р  0.05.
The data for determining the Nrf2-specific RNA content (in the form of the logarithm of the threshold dilution) were expressed as mean  standard error of the mean (M  SEM) [26].

Effects of flavonoid compositions and influenza virus infection on Nrf2 expression in lung of mice
The threshold points shown in Table 2 are indicative of the ballpark estimation of Nrf2 RNA expression changes due to virus infection and/or flavonoid treatment.On Day 1 after infection, level of Nrf2 in virus-infected mice's lungs tends to increase within a 100-fold range.Application of Protoil but not Proteflazidum ® resulted in the increase of Nrf2 expression in the intact mice (the trend within 10-fold range).Notably, in virus-infected mice treated with Proteflazidum ® or Protoil, the Nrf2 expression in lungs tended to decrease.On Day 4 after infection, the patterns were similar except for Proteflazidum ® .

Effects of flavonoid compositions and TGEV infection on Nrf2 expression in Jurkat cells
The expression of Nrf2 was analyzed in vitro in Jurkat cells infected with TGEV and/or treated with Proteflazidum ® or Protoil.Cells were infected  Treatment arm -log of end-point (threshold) dilution Non-infected cells (negative control) 3.00 ± 0.00 Virus-infected cells 5.25 ± 0.71 Non-infected cells + Proteflazidum ® 3.00 ± 0.00 Non-infected cells + Protoil 3.25 ± 0.71 Virus-infected cells + Proteflazidum ® 3.00 ± 0.00 Virus-infected cells + Protoil 3.25 ± 0.71 Notes.The relative content of the specific transcript in RNA samples is expressed as the log of end -point dilution of RNA in RT-PCR incubation mixture that did not detect the presence of the target gene sequences.All ten-fold dilutions of the initial RNA were assayed in four replicates.Following virus absorption, the medium containing Proteflazidum ® at a 1:100 dilution or Protoil at a 1:1000 dilution was added to ensure equal concentrations of the active substances.Control cells were incubated in a standard RPMI-1640 medium.Following one-day culture, cells were collected and RNA was isolated.Ten-fold dilutions of the initial RNA were analyzed with RT-PCR with primers specific for human Nrf2.The threshold points are presented in Table 3. Proteflazidum ® and Protoil had no impact on Nrf2 expression in intact Jurkat cells.TGEV infection led to the increase of Nrf2 expression within 100-fold range.Nevertheless, in TGEV-infected cells treated with either Proteflazidum ® or Protoil the level of Nrf2 expression returned to the baseline level in the intact cells.

Effects of Proteflazidum ® on Nrf2 activation in TGEV-infected or intact Jurkat cells
To confirm the data on the Nrf2 expression at the level of mRNA, the level of Nrf2 protein expression in TGEV-infected or intact Jurkat cells treated with Proteflazidum ® was assessed by ELISA technique.Cells were infected with 100 TCD 50 TGEV as described above and after 1-h virus absorption, RPMI-1640 medium with 2% FCS was added with or without Proteflazidum ® at a 1:200 or 1:400 dilution.One day later, the cells were washed, the nuclear fraction was extracted and Nrf2 protein content was measured by ELISA technique.The results are presented in the Figure .TGEV infection resulted in the substantial activation of Nrf2 protein.Proteflazidum ® itself did * ** ** not activate Nrf2 in our experimental setting.Nevertheless, similarly to the Nrf2 gene expression data, Proteflazidum ® decreased significantly Nrf2 protein expression in TGEV-infected cells with a trend of the dose dependence.

Discussion
The Nrf2 pathway is an important cell signaling mechanism in maintaining redox homeostasis.Bacterial and viral infections are associated with oxidative stress contributing to their pathogenesis.Therefore, studying Nrf2 activation in the course of different viral infections could be important for outlining the dual role of this pathway in antiviral protection as well as in pathogenesis of viral infections at the level of tissues and organs of the host since Nrf2 activation influences the expression of various Nrf2-regulated genes, alterations in which are observed during viral infections.
While the role of Keap1-Nrf2-ARE pathway in the antioxidant protection of cells is well established, the recent studies explore its involvement in viral infection and immunity [6].Sometimes this role may be dualistic depending on virus, cell type, or stage of infection.Infection by most viruses including influenza viruses induces Nrf2 expression [27].Nrf2 not only exerts direct antiviral effect in influenza virus-infected cells but also protects the lungs from virus-induced lung injury [28].Several viruses, however, promote Nrf2 degradation that facilitates viral replication [29].Recent research has explored the role of Nrf2 activation in suppressing SARS-CoV2 replication, including dampening the response to pro-inflammatory cytokines [30].In several viral infections, the dualistic role of Nrf2 activation is traced since it affects both virus replication and pathogenesis of viral infection.In general, the effect of virus infection on Nrf2 activation depends on the specific mechanisms of virus-cell interaction, in particular on the contribution of apoptosis as a factor of antiviral protection and the role of the inflammatory response that could be advantageous for viral propagation.Therefore, an analysis of the mechanisms of Nrf2 activation and the Keap1-Nrf2-ARE pathway modulation in different viral infections is important.
We have demonstrated Nrf2 activation in two different models: influenza virus infection in vivo and alphacoronavirus (TGEV) infection in vitro.In our study, as in [27], in vivo infection with accompanying pneumonia came amid with Nrf2 mRNA up-regulation in lung cells detected both on Day 1 and Day 4 of infection.It should be noted that in our experimental model, all infected mice died on Day 5 post-infection.The Nrf2 activation in our experimental model did not depend on the stage of influenza virus infection contrary to that for herpes virus infection stated in [31].Our data correspond to the work [27], where it was shown that the influenza A virus increases the production of ROS and the expression of Nrf2, as well as its translocation to the nucleus.Note that experiments [27] were carried out in vitro in alveolar type II cells isolated from human lungs, and the Nrf2 expression assessment was carried out after 48 hours.
TGEV infection in vitro also was accompanied with Nrf2 mRNA up-regulation detected on Day 1 post-infection.The activation of Nrf2 in our study was confirmed also at the level of protein expression.The induction of Nrf2 in TGEV-infected cells in vitro was shown by Wang et al. [32].It should be mentioned that in case of SARS-CoV-2, strong inhibition of Nrf2 in the lungs was demonstrated at the late stages of infection [33,34].
Polyphenols including various flavonoids are among known Nrf2 inducers.On the other hand, many polyphenols possess antiviral activity when it comes to various viral infections [17].Several different mechanisms of the antiviral effects of flavonoids in addition to their antioxidant activities have been studied.In particular, flavonoids can directly inhibit virus infection by impeding virus attachment, entry, replication, and release [35], although the mechanisms related to Nrf2 activation/inhibition are less researched.The possible mechanisms of Nrf2 activation include the disruption of interaction between Keap1 and Nrf2 [36] and different forms of Nrf2 protein modifications [16].
Anti-influenza activity of natural polyphenols including flavonoids has been reviewed elsewhere [37].Such flavonoid substances as epigallocatechin, quercetin and its glycosides effectively inhibit replication of influenza virus in vitro [17].Natural flavonoids effectively inhibit replication of coronaviruses [38].The contribution of Nrf2 pathway to antiviral effects of flavonoids and various herbal phenolic compounds remains to be elucidated in detail.Recent findings support the role of various Nrf2 activators in inhibiting influenza virus replication [39].Catechin flavonoids have been shown to inhibit TGEV-induced ROS and cytopathic effect [40].The association of the anti-TGEV effects of the natural phenolic compound eugenol with the decreased Keap-1 expression and significant increase of Nrf2 expression has been recently demonstrated [32].Note that the data [32], in contrast to the design of our study, relate to in vivo conditions.That is why the comparison of our data and the data of [32] is extremely difficult: in terms of both the dose dependence and the complex effect of the viral infection and herbal medicine on the animal's body.Obviously, further in vivo studies of the anti-TGEV effects of the drugs under study should include different treatment conditions in order to determine the appropriate dose range and frequency of use.The effects of flavonoids on Keap1-Nrf2-ARE pathway depend on the concentration of these substances since high concentrations may cause the reverse effect, therefore inhibiting Nrf2 production [41].Form of flavonoid substances and their compositions are also of a great significance.In our study, Protoil composition, not Proteflazidum ® , increased Nrf2 expression in the lungs of mice.Nevertheless, in influenza virus-infected mice, on Day 1 postinfection, both Protoil and Proteflazidum ® , decreased Nrf2 expression in the lungs as compared to virus-infected mice, which were not treated with these flavonoid compositions.The effects of both flavonoid compositions on Nrf2 expression in the intact Jurkat cells on Day 1 have not been detected, while both compositions down-regulated this expression in TGEV-infected cells.These trends have been confirmed when Nrf2 expression in this experimental system was analyzed at the protein level.

Conclusions
To sum up, we have studied the effects of two forms of flavonoid compositions with identical constituents and different solvents in two experimental models of viral infection (influenza virus infection in vivo and TGEV infection in vitro) on the Nrf2 expression.Both general and specific effects for these two experimental systems have been revealed.Both viruses up-regulate Nrf2 expression significantly.Flavonoid compositions have only slight effect on Nrf2 expression in the intact cells while in virus infection both in vivo and in vitro they counteract the extensive up-regulation of Nrf2 expression due to viral infection.

Interests disclosure
Alexander Galkin is the Editor-in-Chief and Svitlana Rybalko is the member of the editorial board of the journal Innovative Biosystems and Bioengineering, they did not participate in the editorial evaluation and decision-making on the publication of the article.Viktor Atamaniuk reports being employee of Ecopharm Research-and-Production Company; the author declares that he has no competing interests.The other authors have no conflicts of interest to disclose.

Funding
The study was conducted within the budget funding of the National Academy of Medical Sciences of Ukraine.
) and the provisions of the Directive 2010/63/EU of the European Parliament and the Council of the European Union "On the Protection of Animals Used for Scientific Purposes" (2010).All animal-related procedures were performed in accordance with the ethical standards of the Animal Ethics Committee, L.V. Gromashevsky Institute of Epidemiology and Infectious Diseases of the National Academy of Medical Sciences of Ukraine (No. AEC/25/2023).

Figure :
Figure:Expression of Nrf2 protein in nuclear extracts of Jurkat cells infected with TGEV and ciltured with/without Proteflazidum ® for 1 day; * -the difference is significant compared to intact cells, p  0.05; ** -the difference is significant compared to virusinfected cells, p  0.05

Table 1 :
List of primers used in the study

Table 2 :
Expression of Nrf2 gene in the lungs of mice following influenza virus infection and/or treatment with flavonoid compositions (Proteflazidum ® or Protoil)

Table 3 :
Expression of Nrf2 gene in Jurkat cells infected with TGEV and treated with flavonoid compositions (Proteflazidum ® or Protoil)