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\begin{document}

\title{Targeting COVID-19 Based on Pathogenesis}



\author[1]{Zhao Zhong Chong}%
\author[1]{Hongjun Zhang}%
\affil[1]{American Molecular Laboratories, Inc}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
The new coronavirus disease 2019 (COVID-19) caused by severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) are causing health
crisis of the world. To control the pandemic SARS-CoV-2 infection,
world-wide vaccination are under way. However, no specific therapeutic
drugs are available in the treatment of COVID-19 yet. To find the
effectively therapeutic targets for COVID-19, efforts should be focused
on the pathogenesis of COVID-19. SARS-CoV-2 induced COVID-19 involves
the processes of viral entry into the host cells, viral replication in
the host cells, and induction of cytokine storm and cellular damage.
Therefore, the potential targets may include the structural proteins of
the virus that play roles in the pathogenesis, viral replication
associated enzymes, inflammatory cytokines, and signaling proteins that
mediate the induction of cytokine storm. Further exploring this strategy
may benefit us in finding novel therapeutic targets and effective
treatment of COVID-19.%
\end{abstract}%



\sloppy


\textbf{Targeting COVID-19 Based on Pathogenesis}

Zhao-Zhong Chong\textsuperscript{1,2}; Hong-Jun Zhang\textsuperscript{1}

\textsuperscript{1}American Molecular Laboratories, Inc, Vernon Hills,
Illinois

\textsuperscript{2}Institute of Materia Medica, Shandong Academy of
Medical Sciences, Jinan, China

Corresponding to Dr. Zhao-Zhong Chong (zzchong@yahoo.com), American
Molecular Laboratories, Inc., 50 Lakeview Parkway, ste\#127, Vernon
Hills, Illinois 60089. Ph: 847-281-7670.

Number of words in the abstract: 141

Total number of words in the main body of the manuscript: 5931

Total number of references: 150

Number of figures: 3\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Data sharing not applicable -- no new data generated* & Data sharing is
not applicable to this article because no new data were created or
analysed in this review.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Abstract}

The new coronavirus disease 2019 (COVID-19) caused by severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) are causing health
crisis of the world. To control the pandemic SARS-CoV-2 infection,
world-wide vaccination is under way. However, no specific therapeutic
drugs are available in the treatment of COVID-19 yet. To find the
effectively therapeutic targets for COVID-19, efforts should be focused
on the pathogenesis of COVID-19. SARS-CoV-2 induced COVID-19 involves
the processes of viral entry into the host cells, viral replication in
the host cells, and induction of cytokine storm and cellular damage.
Therefore, the potential targets may include the structural proteins of
the virus that play roles in the pathogenesis, viral replication
associated enzymes, inflammatory cytokines, and signaling proteins that
mediate the induction of cytokine storm. Further exploring this strategy
may benefit us in finding novel therapeutic targets and effective
treatment of COVID-19.

\textbf{Key Words:} SARS-CoV-2, Cytokine storm, Toll-like receptors,
Janus kinase, NLRP3 inflammasome

\textbf{INTRODUCTION}

Since December 2019, SARS-CoV-2 infection had hit over 134 million
people worldwide with 2.66 million cases of death until the date of
April 6, 2021. SARS-CoV-2 infection not only causes severe respiratory
impairment, but also leads to complications in other systems of the
body, including cardiac and nervous system, which contribute to the
increased mortality in patients with COVID-19 (Helms et al., 2020; Huang
et al., 2020b; Mao et al., 2020; Thepmankorn et al., 2021). Although the
efforts are underway to control the pandemic with implementation of
vaccination, there is still a lack of specific drugs for the treatment
of COVID-19. However, multiple therapeutic targets have been proposed
and multiple associated therapeutic agents have been tried in the
clinical setting. To explore the pathogenic mechanisms associated with
COVID-19 and thereby find more effectively therapeutic targets are the
urgent task for the control of COVID-19 and reduction of mortality.

The induction of COVID-19 by SARS-CoV-2 infection involve multiple steps
that include viral entry into the host cells, replication of virus, and
induction of pathogenic processes such as cytokine storm. To block
either one of the steps should reduce the severity of infection and
lower the mortality of COVID-19 patients. As a result, targeting these
processes should be an effective strategy to fight against COVID-19. The
review will focus on the following potential target processes and
specific protein targets that are involved in these processes.

\textbf{VIRAL INVASION}

Viral entry into the host cells, such as epithelial cells in respiratory
duct, is the first step for infection. Blockade of the machinery that
mediates cell entry of SARS-CoV-2 can prevent the infection or reduce
the load of the virus and the severity of COVID-19. The following
proteins play important roles in the viral entry into the host cells.

\textbf{2.1. Spike Protein}

The coronavirus structural proteins include the spike glycoprotein
(S-protein), envelope protein (E-protein), membrane protein (M-protein),
and the nucleocapsid protein (N-protein). The S-protein of SARS-CoV-2 is
similar to the coronavirus strain SARS-CoV with over 72\% amino acid
sequence similarity (Chen, Guo, Pan \& Zhao, 2020). SARS-CoV-2 can bind
to angiotensin converting enzyme 2 (ACE2) through the receptor-binding
domain (RBD) of the S-protein (Lan et al., 2020) with a higher affinity
relative to SARS-CoV (Chen, Guo, Pan \& Zhao, 2020). In addition,
SARS-CoV-2 does not use other receptors as other coronavirus, such as
aminopeptidase N and dipeptidyl peptidase-4 (Zhou et al., 2020a) .

The S-protein of SARS-CoV-2 consists of two subunits: a globular S1
domain at the N-terminal region and the membrane-proximal S2 domain. The
RBD within S1 subunit is essential for the virus attachment to host cell
receptor ACE2; while S2 is critical for virus entry by regulating viral
membrane fusion to host cell membrane (Kirchdoerfer et al., 2016; Wrapp
et al., 2020; Yan, Zhang, Li, Xia, Guo \& Zhou, 2020). Both S1-RBD and
S2 domains represent important potential targets for the development of
SARS-CoV-2 vaccine and therapeutic drugs (Huang et al., 2020a). In fact,
many of the currently developed vaccines target S-protein of SARS-CoV-2
(Folegatti et al., 2020; Logunov et al., 2020; Smith et al., 2020; Zhu
et al., 2020). RBD seems to be a good target for SARS-CoV-2 vaccine,
since a recombinant RBD protein of SARS-CoV-2 prepared from insect cells
was reported to induce serum antibodies that could bind the RBD and
neutralize viral infection in nonhuman primates (Walsh et al., 2020;
Yang et al., 2020). The development of monoclonal antibody against or
inhibitors of S-protein might hold great promise to find therapeutic
candidates for COVID-19.

\textbf{ACE2}

The S-protein of both SARS-CoV and SARS-CoV- has been known to utilize
ACE2 as a receptor for host cell entry (Li et al., 2003; Wang et al.,
2020a). ACE2 is a metallopeptidase that is expressed on major viral
target cells, such as type II pneumocytes and enterocytes (Hamming,
Timens, Bulthuis, Lely, Navis \& van Goor, 2004; Mossel et al., 2008; To
\& Lo, 2004). ACE2 is expressed not only in type II alveolar cells
(AT2), but also in myocardial cells, proximal tubule cells of the
kidney, ileum and esophagus epithelial cells, and bladder urothelial
cells, and as well as in brain cells, establishing the basis for
possible extrapulmonary invasion of SARS-CoV-2.

The catalytic domain of ACE2 binds to S-protein with high affinity (Li
et al., 2003; Wong, Li, Moore, Choe \& Farzan, 2004). Binding of
S-protein to ACE2 triggers conformational rearrangements in S-protein,
which are believed to increase the sensitivity of the S-protein to
proteolytic enzymes at the border between the S1 and S2 subunits for
priming to facilitate the viral entry into the cells.

A clinical-grade soluble recombinant human ACE2 protein (rhACE2) was
shown to inhibit attachment of SARS-CoV-2 to simian Vero-E6 cells and
prevent SARS-CoV-2 infection of engineered human capillary organoids and
kidney organoids (Vero-E 6 cells) (Monteil et al., 2021). However,
whether rhACE2 administration \emph{in vivo} can prevent SARS-CoV-2
infection remains unknown. The roles of ACE2 inhibitors, such as
nicotinamide (Takahashi, Yoshiya, Yoshizawa-Kumagaye \& Sugiyama, 2015),
in SARS-CoV-2 infection should be further investigated.

S-protein can also regulate cellular signing pathways. S-protein has
been shown to activate the mitogen-activated protein kinase (MEK)/
extracellular signal-regulated kinase (ERK) pathway and increase the
downstream chemokine expression through ACE2 (Chen et al., 2010). ANG II
can also reduce the expression of ACE2 mRNA and increase the
extracellular signal-regulated kinase (ERK) 1/ERK2 activity, which was
prevented by the mitogen-activated protein kinase (MAPK) inhibitor
PD98059 in vascular smooth muscle cells (Gallagher, Ferrario \& Tallant,
2008). Further study indicates that MAPK kinase (MEK) inhibitors
(VS-6766, trametinib and selumetinib) reduced ACE2 expression and
attenuated the release of inflammatory cytokines during SARS-Cov-2
infection (Zhou et al., 2020b).

\textbf{2.3. Transmembrane Serine Protease 2} \textbf{(TMPRSS2)}

TMPRSS2, a transmembrane serine protease in airway epithelial cells and
alveolar cells, plays a critical role in viral entry into host cells.
Like ACE2, TMPRSS2 also express in the heart, digestive tract, liver,
kidney, brain, and other organs (Dong et al., 2020).

One key function of TMPRSS2 is to prime the viral S-protein to
facilitate the interaction between S-protein and ACE2, which is
essential for viral infectivity. Both SARS-CoV and SARS-CoV-2 use the
serine protease TMPRSS2 for S protein priming (Hoffmann et al., 2020) .
The binding of RBD within the S1 domain to ACE2 could trigger the
effects of TMPRSS2 on the cleavage of S-protein at the S1 and S2 border
sites and facilitate cell membrane fusion for viral entry (Walls, Park,
Tortorici, Wall, McGuire \& Veesler, 2020).

Inhibition of TMPRSS2 has been shown to block the entry of SARS-CoV-2
(Hoffmann et al., 2020; Sagar et al., 2021). Inhibition of TMPRSS2 by
camostat mesylate in human lung Calu-3 cells significantly reduced
infection of the cells by SARS-CoV-2. Nafamostat mesylate, which has
been demonstrated to inhibit TMPRSS2-dependent host cell entry of
MERS-CoV (Yamamoto et al., 2016), can also prevent SARS-CoV-2 entry into
host cells with roughly 15-fold-higher efficiency than camostat mesylate
(Hoffmann et al., 2020). Another TMPRSS2 inhibitor, \selectlanguage{greek}α-\selectlanguage{english}1antitrypsin,
blocks the SARS-CoV-2 from entering host cells. The clinical efficacy of
these inhibitors is under evaluation for COVID-19.

\textbf{2.4. High-density lipoprotein (HDL) scavenger receptor B type 1
(SR-B1)}

SR-B1 is a cell-surface HDL receptor that mediates the selective uptake
of cholesteryl esters and other lipid components of receptor-bound HDL
particles (Shen, Asthana, Kraemer \& Azhar, 2018). Co-expression of
SR-B1and ACE2 has been found in human pulmonary and extrapulmonary
tissues (Wei et al., 2020).

SR-B1 may be associated with the facilitating the attachment of
SARS-CoV-2 on ACE2-expressing cells. HDL can interact with the S1 domain
of S-protein. Blockade of the cholesterol-binding site on the S1 domain
with either a monoclonal antibody or SR-B1 inhibitors prevents
HDL-enhanced SARS-CoV-2 infection (Wei et al., 2020). Although the
interaction between HDL and S-protein may not the be the major mechanism
of viral entry, blockade of this interaction should be beneficial to
reduce the viral load.

\textbf{3. VIRAL REPLICATION}

\textbf{3.1. RNA dependent RNA polymerase (RdRp)}

SARS-CoV-2 is an RNA virus and its replication is dependent on RdRp.
Besides its 4 structural proteins, SARS-CoV-2 has 16 non-structural
proteins (NSPs). NSP12 is one of the conserved NSPs among coronaviruses
and is a vital enzyme that functions as an RdRp (Perlman \& Netland,
2009; Wu et al., 2020b). In addition, NSP8 functions as a primase that
is associated with RNA synthesis (Perlman \& Netland, 2009). NSP8
interacts with NSP7 for their primer dependent RdRp activity. The
presence of NSP7 and NSP8 is necessary for the binding activity of NSP12
to the template-primer RNA (Yin et al., 2020).

Since there are 96.35\%, 98.8\% and 97.5\% similarities in NSP12, NSP7
and NSP8 between SARS-CoV and SARS-CoV-2, respectively (Ruan, Yang,
Wang, Jiang \& Song, 2020), inhibitors of the NSP12--NSP7--NSP8 complex
for SARS-CoV may also inhibit the complex of SARS-CoV-2 (Yin et al.,
2020). Therefore, the NSP12--NSP7--NSP8 complex and RdRp activity
inhibitor remdesivir has also been investigated in SARS-CoV-2 (Yin et
al., 2020).

Remdesivir was originally developed for the treatment for of Ebola virus
infection. Remdesivir is covalently incorporated into the primer strand
of the virus at the first replicated base pair of RdRp to prevent the
chain elongation (Yin et al., 2020). It functions through its active
form, metabolite remdesivir triphosphate to compete with the
incorporation of nucleotide counterparts and inhibits transcription of
viral RNA. Although remdesivir has not shown to reduce the mortality in
COVID-19 patients, it appears to accelerate the recovery when its
administration was initiated early (Beigel et al., 2020). Remdesivir has
been authorized in the USA for patients with severe COVID-19 since a NIH
clinical trials demonstrated that it can accelerate the recovery and
shorten the hospital stay of severe COVID-19 patients (Beigel et al.,
2020). A recent report indicates that remdesivir given within 9 days
from symptom-onset was associated with decrease in mortality in
moderate-to-severe COVID-19 patients (Mehta, Bansal, Bysani \& Kalpakam,
2021).

However, the effectiveness of remdesivir is far from convincing
(Brouqui, Giraud-Gatineau \& Raoult, 2020). An earlier clinical trial in
China did not show significant benefit of remdesivir in severe COVID-19
patients (Wang et al., 2020b). The discrepancy of results between China
and the USA has been ascribed to the difference in genetic backgrounds
of patients with COVID-19 (Wang, Cui, Ouyang, Zhan, Guo \& Yin, 2020).
More effective inhibitors of SARS-CoV-1 RdRp are required for better
management of COVID-19.

\textbf{3.2. Viral Proteases}

The 3-chymotrypsin-like protease (3CLPro) and a papain-like protease
(PLPro) function to process NSPs including RdRp, and therefore,
inhibition of these proteases should interfere the activity of RdRp
(Rathnayake et al., 2020). The antiviral and cell protection efficacy of
3CLPro inhibition has been illustrated in simian Vero cells infected by
SARS-CoV-2 (Jin et al., 2020).

Several candidates have been found to have inhibitory effects on 3CLPro.
Anti-hepatitis C virus (HCV) drug ravidasvir has the ability to bind and
inhibit the 3CLPro of SARS-CoV-2 (Bera, 2021). Similarly, HCV protease
inhibitors paritaprevir and simeprevir were also identified as potential
inhibitors of SARS-CoV-2 3CLPro (Alamri et al., 2020). Using
computational molecular modeling to screen FDA approved drugs and
subsequent studying for their inhibitory effects on SARS-CoV-2 3CLpro
enzyme \emph{in vitro} , boceprevir, ombitasvir, paritaprevir,
tipranavir, ivermectin, and micafungin were found to exhibit inhibitory
effect towards 3CLpro enzymatic activity (Mody et al., 2021).

The efficacy of 3CLPro inhibitors in COVID-19 need to be proved.
Lopinavir is a highly potent inhibitor of the human immunodeficiency
virus (HIV) protease essential for intracellular HIV assembly.
Concomitant oral administration of lopinavir and ritonavir, which blocks
the metabolism of lopinavir, increases the anti-viral potency of
lopinavir. However, COVID-19 patients receiving the combined treatment
lopinavir and ritonavir yielded no significant benefit compared to
patients treated with standard-care (Cao et al., 2020a). More specific
inhibitors of proteases for SARS-CoV-2 should be evaluated in COVID-19,
since a study indicated that lopinavir and ritonavir did not significant
inhibit 3ClPro in an \emph{in vitro} enzymatic assays (Mahdi, Motyan,
Szojka, Golda, Miczi \& Tozser, 2020). The aforementioned 3CLPro
inhibitors may benefit COVID-19 patients, which is needed to be proved
in future clinical trials.

In regard of PLPro, activity profiling and crystal structures of
inhibitor study indicated that there is a very high level of sequence
and structural similarity between SARS-CoV and SARS-CoV-2 PLPro in the
substrate binding pocket, suggesting that SARS-CoV PLPro inhibitors can
possibly inhibit SARS-CoV-2 PLPro (Rut et al., 2020). Non-covalent small
molecule SARS-CoV PLPro inhibitors has been shown to inhibit SARS-CoV-2
PLPro and display antiviral activity in a SARS-CoV-2 infection model
(Klemm et al., 2020). Biochemical, structural, and functional
characterization investigation revealed that SARS-CoV and SARS-CoV-2
PLPro share 83\% sequence identity but exhibit different host substrate
preferences. SARS-CoV-2 PLPro preferentially bind to the ubiquitin-like
interferon-stimulated gene 15 protein (ISG15), cleaving ISG15 from
interferon responsive factor 3 (IRF3) and attenuating type I interferon
responses. Whereas SARS-CoV PLPro predominantly targets ubiquitin
chains. Inhibition of SARS-CoV-2 PLPro impairs the virus-induced
cytopathogenic effect, maintains the antiviral interferon pathway and
reduces viral replication in infected cells (Shin et al., 2020).

\textbf{N-protein}

To prevent the replication of SARS-CoV-2 RNA, the N-protein of the virus
may also be the potential target. N-proteins comprise three domains,
including an N-terminal RNA-binding domain (NTD), a Ser/Arg rich central
linker region and a C-terminal dimerization domain (CTD). The N-proteins
plays a major role in packing the viral RNA into viral
ribonucleoproteins (McBride, van Zyl \& Fielding, 2014). It also helps
in viral RNA transcription and replication (Cong et al., 2020). The NTD
functions for RNA-binding and the CTD domain plays a role in
oligomerization, while the central linker is necessary for primary
phosphorylation (Kang et al., 2020). However, targeting N-protein for
vaccine should be careful, since the increased titer of IgG antibody
against N-protein has been associated with poor prognosis, increasing
ICU admission and longer hospital stay (Batra et al., 2021). But the
higher N-protein antibody titer may indicate the higher viral load and
more severe disease, which cause the poor outcomes, not the N-protein
antibody itself leads to the poor prognosis. Further studies could be
performed to clarify the underlying association.

Recently, the sequences that interact with B and T cells in the NTD
domain have been found by single immuno-informatics and structure-based
drug discovery techniques (Kwarteng, Asiedu, Sylverken, Larbi, Sakyi \&
Asiedu, 2021), suggesting that N-protein might be associated with
overactive immune responses and the development of the NTD inhibitors
may hold some promises. The role of N-protein inhibition in the
treatment of anti-SARS-CoV-2 should be further evaluated.

\textbf{4. CYTOKINE STORM}

The cytokine storm during SARS-CoV-2 infection has been associated with
the severity and mortality in COVID-19 patients. The impaired acquired
immune responses and uncontrolled inflammatory innate responses to
SARS-CoV-2 may cause cytokine storm (Hu, Huang \& Yin, 2021; Thepmankorn
et al., 2021). During SARS-CoV-2 infection, the interleukin-6 (IL-6) and
tumor necrosis factor-\selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}) are the most frequently reported
cytokines that are significantly increased (Qin et al., 2020;
Thepmankorn et al., 2021). Other cytokines that are increased but not
exclusively include granulocyte stimulating factor (G-CSF), interferon
gamma-induced protein 10 (IP-10), monocyte chemoattractant protein
(MCP)-1, MCP-3, macrophage inflammatory protein 1\selectlanguage{greek}α \selectlanguage{english}(MIP-1A), cutaneous
T-cell attracting chemokine (CTACK), IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α, \selectlanguage{english}and
IL-1\textasciitilde{}10 and 18 (Hadjadj et al., 2020). To prevent
cytokine storm should be an important strategic measure for the
treatment of COVID-19 (Lariccia, Magi, Serfilippi, Toujani, Gratteri \&
Amoroso, 2020).

Corticosteroids have been used in COVID-19 pneumonia to reduce systemic
inflammation; however, they may increase the risk of delayed viral
elimination and secondary infection. Since cytokines are the effectors
in the cytokine storm, the specific cytokine antagonists should reduce
cytokine induced damage when they are used for the treatment of
COVID-19. Either monoclonal antibodies against cytokines or
pharmacological inhibitors of cytokines will fall into this category.

\textbf{4.1. IL-6}

IL-6 plays multiple biological roles, which include enhancing the
synthesis of inflammatory response proteins, such as C-reactive protein
(CRP), regulating both T and B immune cells to increase immune
responses, and promoting the production of vascular endothelium growth
factor (VEGF), which aggravate the inflammatory damage by inducing
angiogenesis and impairing the vascular integrity. In addition to its
immune and inflammatory regulatory activities, IL-6 also upregulates the
activation of the coagulation pathway, increasing the thrombotic events
(D'Alessandro et al., 2020).

The proinflammatory function of IL-6 is mediated through a series of
cell signaling pathways. IL-6 binds to its receptor (IL-6R) to initiate
intra cellular signaling\emph{.} Two forms of IL-6R have been found:
membrane IL-6 receptor (mIL-6R) and soluble IL-6 receptor (sIL-6R). The
binding of IL-6 to both receptor results in the dimerization and
activation of the glycosylated type I membrane protein of 130--150 kDa
(gp130). IL-6/mIL-6R medicated activation of gp130 induces IL-6 classic
signaling pathway; while IL-6/sIL-6R induced the activation of gp130
leads to activation of IL-6 trans-signaling pathway (Ebihara, Matsuda,
Nakamura, Matsuda \& Murakami, 2011). Classic IL-6 signaling is
generally regarded as anti-inflammation and protective pathway, while
IL-6 trans-signaling is mainly pro-inflammatory pathway (Garbers,
Aparicio-Siegmund \& Rose-John, 2015). Blockade of IL-6 trans-signaling,
not the full blockade of IL-6 signaling, prevents inflammation
(Barkhausen et al., 2011).

The dimerization of signaling receptor gp130 mediates the activation of
Janus kinases (JAKs) and subsequent activation of phosphatase Src
homology domains containing tyrosine phosphatase-2 (SHP-2), the
ras/raf/MAPK pathway, signal transducer and activator of transcription
factor-3 STAT-3) (Wang S, Zhang W 2016), and PI3K/Akt (Zegeye et al.,
2018), which are translocated into the nucleus to activate target genes
(Figure 1).

IL-6 has been closely associated with SARS-CoV-2 induced cytokine storm.
IL-6 is one of the key cytokines that is frequently reported to be
increased in COVID-19 patients with hypercytokinemia (Kaman, Azmy,
Chichra, Britto-Leon \& Price, 2021). The level of IL-6 is valuable as a
prognostic parameter for the disease severity in COVID-19 patients (Liu
et al., 2020). Increasing IL-6 concentrations are associated with the
requirement of ventilatory support, the progression of ARDS, and the
risk of death (Herold et al., 2020; Wu et al., 2020a). Although a
positive correlation between IL-6 and CRP exists in COVID-19 patients,
IL-6 appears to be a better predictor for the disease progression, since
IL-6, not CRP, levels were significantly lower in survivors than in
non-survivors of all age groups of COVID-19 patients, suggesting that
IL-6 is predictive of in-hospital mortality after SARS-CoV-2 infection
(Santa Cruz et al., 2021; Zhang et al., 2020).

Since IL-6 functions through its receptor and the monoclonal antibody
against IL-6R has been developed to prevent the activation of IL-6R by
IL-6. Tocilizumab is a monoclonal antibody against IL-6R that has been
approved by the US FDA for the treatment of cytokine release syndrome.
It is also rational to expect tocilizumab to be capable of attenuating
the cytokine storm induced by SARS-CoV-2 (Pelaia, Calabrese, Garofalo,
Bruni, Vatrella \& Pelaia, 2021).

Primary data using tocilizumab demonstrated some benefits for critical
COVID-19 patients. Tocilizumab can rapidly resolve the symptoms. In
severe COVID-19 patients, fever returned to normal on the first day
after tocilizumab administration; other symptoms were improved
remarkably within a few days: 75.0\% of patients reduced their
requirement of oxygen intake within 5 d; and the percentage of
lymphocytes in peripheral blood returned to normal in 52.6\% of patients
on the fifth day after treatment (Xu et al., 2020).

In severe to critical COVID-19 disease with hypercytokinemia in ICU,
tocilizumab significantly reduced inflammatory response illustrated by
the decrease in CRP, decreased the requirement of respiratory support,
and lowered the mortality (Nasa et al., 2020). The cases with
unfavorable outcomes after tocilizumab treatment were linked to the
failure in reducing the level of CRP (Luo, Liu, Qiu, Liu, Liu \& Li,
2020). In addition, since IL-6 is the main inducer of CRP production in
the liver during the acute phase response (Sproston, El Mohtadi, Slevin,
Gilmore \& Ashworth, 2018), tocilizumab seemed to be more effective in
patients with markedly elevated CRP (\textgreater{} 200 mg/L) . Systemic
review of the online data suggests that addition of tocilizumab to the
standard of care might reduce mortality in severe COVID-19; however, the
inconsistency in the effectiveness exists among patients with reduced
severity (Boregowda, Perisetti, Nanjappa, Gajendran, Kutti Sridharan \&
Goyal, 2020; Gorgolas Hernandez-Mora et al., 2021).

The severity of COVID-19 is an important parameter that affects the
effectiveness of tocilizumab, since tocilizumab given to severe, not
critical (requirement of intubation or ICU admission), SARS-CoV-2
pneumonia patients increased the requirement of intubation and elevated
the mortality. In contrast, in patients with COVID-19 pneumonia and
life-threatening acute respiratory distress syndrome (ARDS) requiring
ventilatory support, intravenous administration of tocilizumab
significantly improve the patients' respiratory conditions and
downregulate the inflammatory markers (Conrozier et al., 2020; Toniati
et al., 2020). However, two phase III clinical trials include an earlier
one by Roche (Roche, 2020) and a recent report (Rosas et al., 2021), the
use of tocilizumab in hospitalized patients with severe COVID-19
pneumonia did not result in significantly better clinical status or
lower mortality.

In conclusion, the effectiveness of tocilizumab might be subjected to
various factors, such as the severity of the disease and the
inflammatory status. The value of tocilizumab in the treatment of
COVID-19 should be further evaluated. One side effect of tocilizumab is
that its administration in COVID-19 patients significantly increased
IL-6 levels (Antwi-Amoabeng, Kanji, Ford, Beutler, Riddle \& Siddiqui,
2020). Tocilizumab only blocks IL-6R without effects on the production
of IL-6. Blockade of IL-6R might reduce the IL-6 consumption that leads
to an increase in IL-6 (Nishimoto, Terao, Mima, Nakahara, Takagi \&
Kakehi, 2008). The concern is whether higher IL-6 levels after
tocilizumab treatment in COVID-19 patients would have adverse effects or
exacerbate the CNS inflammation due to impermeability of BBB to
tocilizumab (Zhang et al., 2020).

Since it is the trans IL-6 pathway that has been associated with
inflammation, the specific antagonists for this pathway might increase
the efficacy and reduce the side effects. The development of regulators
of trans-IL-6 signaling pathway may hold better promise for the
treatment of COVID-19.

\textbf{4.2. IL-1}

IL-1 that include IL-1\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}is also an important inflammatory
cytokine. IL-1\selectlanguage{greek}α \selectlanguage{english}is released from dying epithelial and endothelial cells,
while IL-1\selectlanguage{greek}β \selectlanguage{english}comes from activated macrophages, monocytes, and
neutrophils. IL-1 is primarily associated with acute and chronic
inflammation. In the inflammatory cascade, IL-1 is an upstream cytokine
that promotes the activation of other cytokines including IL-6 and TNF
-\selectlanguage{greek}α \selectlanguage{english}(Nieto-Torres et al., 2014).

IL-1 receptor (IL-1R) antagonists have also been used in trial treatment
in COVID-19 patients. Anakinra is a protein inhibitor of IL-1R. In
COVID-19 patients with respiratory insufficiency and hyperinflammation,
pneumonia, or ARDS, application of anakinra significantly reduced the
mortality (Cavalli \& Dagna, 2021; Franzetti et al., 2021; Pontali et
al., 2021). Intravenous administration of anakinra (5 mg/kg twice a day)
can significantly decrease serum CRP and improve respiratory function in
COVID-19 patients with ARDS (Cauchois et al., 2020; Cavalli et al.,
2020). Subcutaneous application of anakinra (100 mg twice a day for 72
h, then 100 mg daily for 7 days) also decreased the mortality in
COVID-19 patients with respiratory improvement (Huet et al., 2020).

Similarly, the human IL-1R monoclonal antibody, canakinumab can
attenuate the systemic inflammation and improve respiratory function in
patients with bilateral pneumonia and hyperinflammation after SARS-CoV-2
infection (Ucciferri et al., 2020).

The development of cytokine antagonists is good strategy, and this type
of drugs might benefit patients in reducing the specific cytokine
induced inflammatory damage. However, cytokine storm induced by
SARS-CoV-2 involves multiple cytokines, it is impossible for one
cytokine antagonist to block all the inflammatory responses. Anyway,
cytokine antagonists have shown some benefit in reducing symptoms and
are deserved to be evaluated in the treatment of COVID-19.

\textbf{5. CYTOKINE ASSOCIATED SIGNALING PATHWAYS}

\textbf{5.1. Toll-like Receptors (TLRs)}

TLRs are the major pattern recognition receptors (PRRs) that can
identify pathogen-associated molecular patterns (PAMPs) and endogenous
damage-associated molecular patterns (DAMPs). The function of TLRs is
closely related to process of inflammatory cytokine release. Expressed
on monocytes, macrophages, dendritic cells, neutrophils, B cells, T
cells, fibroblasts, endothelial cells, epithelial cells, and even on
neurons, TLRs are type I transmembrane proteins that contain three
structural domains: a leucine-rich repeats (LRRs) motif as an
extracellular domain, a transmembrane domain, and a cytoplasmic
Toll/IL-1R (TIR) domain. The LRRs motif is responsible for identifying
PAMPs, while the TIR domain interacts with signal transduction adaptors
to initiate downstream signaling pathways (Kawasaki \& Kawai, 2014). .

TLR mediated cell signaling pathway plays a significant role in cytokine
storm-associated diseases including severe COVID-19. Upon the
stimulation by microbial-associated molecular patterns (MAMPs) of the
pathogens or DAMPs of the host cells, TLRs are activated. Bacteria,
fungi, protozoa, and viruses all express MAMPs (Goulopoulou, McCarthy \&
Webb, 2016). After activation, TLRs recruit cytoplasmic TIR
domain-containing adaptor proteins, such as myeloid differentiation
primary-response 88 (MyD88) and TIR-containing adapter-inducing
interferon-\selectlanguage{greek}β \selectlanguage{english}(TRIF), leading to the activation of nuclear factor
(NF)-\selectlanguage{greek}κ\selectlanguage{english}B, MAPKs, or interferon-regulatory factor (IRF). Subsequently, the
transcription of genes that are responsible for synthesis and the
release of proinflammatory cytokines are activated, promoting the
release of cytokines, such as including TNF-\selectlanguage{greek}α, \selectlanguage{english}and IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, IL-18,
chemokines, and type I IFNs (Costa et al., 2018; Vogelpoel et al., 2015)
(Figure 2).

In SARS-CoV-2 induced cytokine storm, TLR4 seems to play a role
(Aboudounya \& Heads, 2021). Recombinant proteins of SARS-CoV-2 has been
demonstrated to promote the expression of pro-inflammatory
cytokines/chemokines and NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling activation in human primary
peripheral blood mononuclear cells and monocyte-derived macrophages
(Sohn et al., 2020). Activation of TLR4 in lung macrophages resulted in
a concentration- and time-dependent increase in the production of
chemokines and cytokines (Grassin-Delyle, Abrial, Salvator, Brollo,
Naline \& Devillier, 2020). In patients with COVID-19, the expression of
TLR 4 and NF-\selectlanguage{greek}κ\selectlanguage{english}B target genes was upregulated (Sohn et al., 2020). Among
TLRs, TLR4 is most likely to be involved in recognizing MAMP from
SARS-CoV-2 to induce inflammatory responses (Grassin-Delyle, Abrial,
Salvator, Brollo, Naline \& Devillier, 2020). In addition, TLR4 may bind
to and interact with S-protein to promote the expression of ACE2,
facilitating the viral entry into the host cells (Choudhury \&
Mukherjee, 2020). Antagonists of TLR4 may attenuate the cytokine storm
and reduce the viral entry in severe COVID-19 patients (Choudhury, Das,
Patra \& Mukherjee, 2021).

\textbf{5.2. Angiotensin 2 (Ang II)}

The renin-angiotensin system (RAS) plays an important role in regulating
vascular and kidney functions. In this system, angiotensin I (Ang I) is
generated by cleavage of angiotensinogen by renin, and then ACE converts
Ang I to Ang II. ACE2 functions to converse Ang II to Ang 1-7 (Figure
2). Ang II activates Ang II receptor type 1 (AT1R) and Ang II receptor
type 2 (AT2R). Activation of AT1R induces detrimental effects, such as
inflammation with release of cytokines, fibrosis, and impaired redox
balance, and vasoconstriction (Eguchi, Kawai, Scalia \& Rizzo, 2018),
while activation of AT2R leads to anti-inflammatory, anti-fibrotic, and
vasodilation (D'Ardes et al., 2020). Ang II can also increase the
recruitment of immune cells to the injury sites and enhances the release
of inflammatory cytokines (Nataraj et al., 1999). Ang II may regulate
TLR4 mediated pathway by upregulating the expression of TLR4 and the
downstream pathways (Wu et al., 2009).

Ang II induced cell signaling pathway is a potential mechanism
underlying the cytokine storm caused by SARS-CoV-2. The binding of
S-protein of SARS-CoV-2 to ACE2 on the cell surface caused
downregulation of ACE2 expression. The reduced expression and activity
of ACE2 resulted in an increase in Ang II, leading to elevated
inflammatory responses (Figure 2). The Ang II regulators to balance the
Ang II double edged function might benefit COVID-19.

\textbf{5.3. Janus-associated kinase (JAK)}

The JAK family is one of ten recognized families of non-receptor
tyrosine kinases. Mammalian members of Jaks family are Jak1, Jak2, Jak3
and Tyrosine kinase 2 (Tyk2). JAK-STAT-pathway mediates the downstream
signals of multiple cytokines and inhibitors of JAK/STAT have been used
in the treatment of inflammatory diseases (El Jammal, Gerfaud-Valentin,
Seve \& Jamilloux, 2020; Jamilloux, El Jammal, Vuitton,
Gerfaud-Valentin, Kerever \& Seve, 2019). JAK associates with cytokine
receptors to mediate cytokines induced signaling pathways. JAK1 and JAK2
regulate cell signaling pathways of cytokines, such as IL-2, IL-4, IL-7,
IL-9, and IL-15, that share common \selectlanguage{greek}γ \selectlanguage{english}chain (\selectlanguage{greek}γ\selectlanguage{english}c) as receptor subunit
(Moradian et al., 2020; Pesu, Laurence, Kishore, Zwillich, Chan \&
O'Shea, 2008). JAK1 also regulate cell signaling downstream of IL-6,
which activate their receptor to initiate the dimerization of receptor
subunit gp130 (Barkhausen et al., 2011). JAK2 activation is essential
for the signaling transduction of erythropoietin and other growth
factors (Maiese, Chong, Shang \& Wang, 2012).

Since the multiple cytokines are involved in SARS-CoV-2 induced cytokine
storm, JAK inhibitors have been therapeutically used in COVID-19
patients (Seif et al., 2020). Ruxolitinib is a potent and selective
inhibitor of JAK1/2 with some levels of inhibitory effects on TYK2 and
JAK3. COVID-19 patients received ruxolitinib plus standard-of-care
treatment showed a faster clinical improvement on CT images with
decreased levels of cytokines including IL-6, nerve growth factor \selectlanguage{greek}β,
\selectlanguage{english}IL-12, migration inhibitory factor, MIP-1\selectlanguage{greek}α, \selectlanguage{english}MIP-1\selectlanguage{greek}β, \selectlanguage{english}and VEGF, although
it was not associated with significantly accelerated clinical
improvement in severe cases (Cao et al., 2020b). Treatment with
ruxolitinib in COVID-19 patients with severe systemic hyperinflammation,
who suffered from progression to ARDS and multiorgan failure,
significantly reduced the inflammatory score with sustained clinical
improvement (La Rosee \& La Rosee, 2020). For an example, ruxolitinib,
given to a 65-year-old Asian woman with COVID-19-induced ARDS, not only
potently reduced ARDS-associated inflammatory blood cytokine levels such
as IL-6 and the acute phase protein ferritin, but also associated with a
rapid respiratory and cardiac improvement and clinical stabilization
(Neubauer et al., 2020).

Baricitinib is an oral JAK inhibitor. Clinical studies have demonstrated
the ability of baricitinib to reduce the viral titers and decrease IL-6
level with resolution of fever and cough symptoms in COVID-19 patients
(Richardson, Ottaviani, Prelle, Stebbing, Casalini \& Corbellino, 2020;
Stebbing et al., 2021). In COVID-19 pneumonia, baricitinib treatment
reduced the serum levels of IL-6, IL-1\selectlanguage{greek}β, \selectlanguage{english}and TNF-\selectlanguage{greek}α, \selectlanguage{english}promoted the
recovery of circulating T and B cell frequencies, increased antibody
production against the SARS-CoV-2 S-protein, and reduced patients' need
for oxygen therapy (Bronte et al., 2020). In moderate to severe
SARS-CoV-2 pneumonia, baricitinib has add-on effect when combined with
corticosteroids in improving pulmonary function (Rodriguez-Garcia,
Sanchez-Nievas, Arevalo-Serrano, Garcia-Gomez, Jimenez-Vizuete \&
Martinez-Alfaro, 2021). Baricitinib exerts both anti-inflammatory and
anti-viral activities effects and one additional advantage is its
ability to cross BBB into the CNS (Richardson, Ottaviani, Prelle,
Stebbing, Casalini \& Corbellino, 2020).

\textbf{5.4. NOD-like Receptor Pyrin Domain Containing 3}

As a well characterized member of the NOD-like receptor family of the
innate immune system, NOD-like receptor pyrin domain containing 3
(NLRP3) has been implicated in chronic inflammation associated with
obesity, diabetes, cancer, etc. NLRP3 contains a pyrin domain, a
nucleotide-binding site (NBS) domain, and a leucine-rich repeat (LRR)
motif. The NLRP3 inflammasome is a multi-protein complex that recruits
pro-caspase-1 \emph{via} the adaptor, apoptosis-associated speck-like
protein containing caspase activation and recruitment domains (ASC), and
then cleave the cytokine precursor pro-IL-1\selectlanguage{greek}β \selectlanguage{english}into mature IL-1\selectlanguage{greek}β \selectlanguage{english}and then
of pro-IL-18 into IL-18 (He, Hara \& Nunez, 2016). The following release
of other inflammatory cytokines, such as IL-6, TNF- \selectlanguage{greek}α, \selectlanguage{english}prostaglandins
and leukotrienes (Abderrazak et al., 2015) contributes to the induction
of cytokine storm. Inhibition of NLRP3 has been shown to attenuate the
severity of inflammatory diseases (Tomani et al., 2020).

SARS-CoV-2, similar to SARS-CoV, encodes viroporins that have been shown
to activate NLRP3 (Chen, Moriyama, Chang \& Ichinohe, 2019). Ang II,
which can be reduced by ACE2, has been shown to induce the activation of
NLRP3 activation in renal tubular epithelial cells (Wen et al., 2016).
The binding of S-protein to ACE2 to reduce the availability of ACE2 may
increase Ang II, which is a possible mechanism that SARS-CoV-2 induces
the activation of NLRP3. Downregulation of ACE2 after SARS-CoV-2
infection has been observed (Zhang, Penninger, Li, Zhong \& Slutsky,
2020). The different scenarios of NLRP3 activation in COVID-19 may
reflect different level of immune responses (van den Berg \& Te Velde,
2020). However, the role of NLRP3 in inflammatory response and
effectiveness of NLRP3 inhibitors in COVID-19 patients with
hyperinflammation remains to be elucidated.

Colchicine, a currently used anti-gout drug, has anti-inflammatory
effect by inhibiting neutrophil chemotaxis and inflammasome. The
anti-inflammasome function of colchicine is mediated through its
inhibiting activity of NLRP3 inflammasome (Liang, Zhou, Tong, Chen, Ren
\& Zhao, 2019). Colchicine has been shown to not only reduce the
inflammatory cytokines, but also decrease the expression of NLRP3
inflammasome (Fujisue et al., 2017).

Treatment with colchicine reduced cytokines and lowered the rate of
clinical deterioration in COVID-19 patients (Deftereos et al., 2020;
Gandolfini et al., 2020). In hospitalized COVID-19 patients with
pneumonia, treatment with colchicine was associated with reduced
mortality and accelerated recovery (Manenti et al., 2021). A recent
randomized trial indicated that colchicine 1 mg for 1-3 days followed by
0.5 mg/day for 14 days is effective as a proactive anti-inflammatory
therapy in hospitalized COVID-19 patients and viral pneumonia (Mareev et
al., 2021). The systemic review of total of eight studies with 5778
COVID-19 patients demonstrated that the administration of colchicine was
associated with improved outcomes of COVID-19 (Hariyanto, Halim,
Jodhinata, Yanto \& Kurniawan, 2021).

\textbf{5.5. The nuclear Factor Erythroid 2-Related Factor 2 (NRF2)}

NRF2, a transcription factor, upregulates the genes that are associated
with antioxidative stress and mitochondrial biogenesis. Cellular stress
activates NRF2 leading to its translocation to the nucleus, where it
binds to the antioxidant response element (ARE) to initiate the
transcription of antioxidant genes to protect cells against inflammatory
responses (Ahmed, Luo, Namani, Wang \& Tang, 2017). NFR2 also has a gene
repressing activity that inhibits the transcription of cytokine genes,
resulting in a decrease in the expression of the inflammatory cytokines
IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α \selectlanguage{english}in human macrophages (Kobayashi et al., 2016). In
contrast, NRF2 knockout mice showed increased levels of proinflammatory
cytokines in response to lipopolysaccharide stimulation (Thimmulappa et
al., 2006a). In addition, NRF2 induces the expression of heme oxgenase-1
(HO-1) and increases the activity of HO-1 (Reichard, Motz \& Puga,
2007). HO-1 functions to catalyze the degradation of heme into carbon
monoxide (CO), free iron, and biliverdin, which then is converted to
bilirubin by biliverdin reductase. Free heme is pro-inflammatory, while
CO, bilirubin, and HO-1 itself have significant anti-inflammatory
effects (Vijayan, Wagener \& Immenschuh, 2018). CO can inhibit the
production of proinflammatory cytokines, such as TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β,
\selectlanguage{english}through mediating p38MPAK pathway (Otterbein et al., 2000). Increased
NRF2-dependent HO-1 expression has been associated with
anti-inflammatory activity (Kuhn et al., 2011). Moreover, NRF2 has been
shown to induce the quinone oxidoreductase (NQO1) expression and thereby
inhibit NLRP3 inflammasome activation (Liu et al., 2017). NRF2 also
inhibits NF-\selectlanguage{polish}ĸ\selectlanguage{english}B transcriptional activity, since NRF2 knockdown
significantly increases NF-\selectlanguage{polish}ĸ\selectlanguage{english}B-dependent gene transcription (Thimmulappa
et al., 2006b). The downstream target of NRF2, HO-1 can also inhibit
NF-\selectlanguage{polish}ĸ\selectlanguage{english}B activity (Bellezza et al., 2012). In response to oxidative stress,
activated I\selectlanguage{polish}ĸ\selectlanguage{english}B kinase (IKK) promotes the phosphorylation and degradation
of I\selectlanguage{polish}ĸ\selectlanguage{english}B. In normal condition, NF-\selectlanguage{polish}ĸ\selectlanguage{english}B is trapped in the cytoplasm by I\selectlanguage{polish}ĸ\selectlanguage{english}B
binding. The loss of I\selectlanguage{polish}ĸ\selectlanguage{english}B frees NF-\selectlanguage{polish}ĸ\selectlanguage{english}B, which is translocated to nucleus
to promote the gene transcription of pro-inflammatory cytokines, such as
IL-6, TNF-\selectlanguage{greek}α, \selectlanguage{english}an IL-1 (Lawrence \& Fong, 2010)(Figure 3).

As a result, proposed use of NRF2 inducers to prevent development of an
excessive inflammatory response in COVID-19 patients is rationale
(Zinovkin \& Grebenchikov, 2020). Recently, NRF2 agonist
4-octyl-itaconate and dimethyl fumarate has been demonstrated to
attenuate inflammatory responses to SARS-CoV2 infection (Olagnier et
al., 2020). NRF2 agonists may be potentially valuable candidates in the
treatment of SARS-CoV-2 infection.

\textbf{6. CONCLUSION}

Multiple pathogenic processes are involved in COVID-19 after SARS-CoV-2
infection. The associated proteins or signals ruling in the viral entry
into the host cells, viral replication in host cells, and induction of
cytokine storm might function as therapeutic targets for COVID-19.
Further elucidation of these processes and underlying mechanisms may
find novel therapeutic targets, which is a critical strategy for
developing treatments of COVID-19.

\textbf{CONFLICT OF INTEREST}

The authors declare no conflicts of interest.

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COVID-19 Patients. Biochemistry (Mosc) 85\textbf{:} 833-837.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} IL-6 mediates a series of cell signaling pathways.
IL-6 binds to the membrane IL-6 receptor (mIL-6R) and soluble IL-6
receptor (sIL-6R). The binding of IL-6 to both receptors results in the
dimerization and activation of the glycosylated type I membrane protein
of 130--150 kDa (gp130). The dimerization of signaling receptor gp130
mediates the activation of Janus kinases (JAKs) and subsequent
activation of phosphatase Src homology domains containing tyrosine
phosphatase-2 (SHP-2), the ras/raf/mitogen-activated protein kinase
(MAPK) pathway, signal transducer and activator of transcription
factor-3 (STAT-3), and PI3K/Akt, which are translocated into the nucleus
to activate target genes. IL-6/mIL-6R medicated activation of gp130
induces IL-6 classic signaling pathway, leading to anti-inflammatory
biological activities; while IL-6/sIL-6R induced the activation of gp130
leads to activation of IL-6 trans-signaling pathway that results in
pro-inflammatory responses.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
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\textbf{Figure 2.} Angiotensin II (Ang II) is involved in the diction of
cytokine storm during SARS-CoV-2 infection. In the renin-angiotensin
system, renin cleaves angiotensinogen into angiotensin I (Ang I), which
is converted into Ang II by the angiotensin-converting enzyme (ACE).
While ACE2 functions to converse Ang II to Angiotensin 1-7 (Ang 1-7).
The binding of the S-protein of SARS-CoV-2 to ACE2 on the cell surface
causes the downregulation of ACE2 expression, resulting in an increase
in Ang II expression. Ang II binds to and activates Ang II receptor type
1 (AT1R) and Ang II receptor type 2 (AT2R). Activation of AT1R induces
the release of cytokines and induction of inflammation, promotes
fibrosis, and impairs redox balance, and induces vasoconstriction. In
contrast, activation of AT2R leads to anti-inflammatory, anti-fibrotic,
redox balance, and vasodilation. In addition, Ang II upregulates the
expression of TLR4 and the downstream pathways. After activation, TLRs
recruit cytoplasmic TIR domain-containing adaptor proteins such as
myeloid differentiation primary-response 88 (MyD88) and TIR-containing
adapter-inducing interferon-\selectlanguage{greek}β \selectlanguage{english}(TRIF), leading to the activation of
nuclear factor (NF)-\selectlanguage{greek}κ\selectlanguage{english}B, mitogen-activated protein kinases (MAPKs), or
interferon-regulatory factor (IRF). Subsequently, the transcription of
genes that are responsible for the synthesis and the release of
proinflammatory cytokines are activated, promoting the release of
cytokines and type I interferons (IFNs).

\textbf{Figure 3.} The role of nuclear factor erythroid 2-related factor
2 (NRF2) induced cell signaling. In response to oxidative stress,
activated NRF2 represses the genes that are associated with the
transcription of cytokine genes, resulting in a decrease in the
expression of the inflammatory cytokines IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α. \selectlanguage{english}In
addition, NRF2 induces the expression of heme oxygenase-1 (HO-1) and
increases the activity of HO-1. HO-1 functions to catalyze the
degradation of heme into carbon monoxide (CO), free iron, and
biliverdin, which then is converted to bilirubin by biliverdin
reductase. Free heme is pro-inflammatory, while CO, bilirubin, and HO-1
itself have significant anti-inflammatory effects. CO can inhibit the
production of proinflammatory cytokines, such as TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β,
\selectlanguage{english}through mediating p38MPAK pathway. Moreover, NRF2 can induce the
expression of quinone oxidoreductase (NQO1) that inhibits NLRP3
inflammasome activation. In response to oxidative stress, activated I\selectlanguage{polish}ĸ\selectlanguage{english}B
kinase (IKK) promotes the phosphorylation of I\selectlanguage{polish}ĸ\selectlanguage{english}B, resulting in the
release and nuclear translocation of NF-\selectlanguage{polish}ĸ\selectlanguage{english}B, which then promote the gene
transcription of pro-inflammatory cytokines, such as IL-6, TNF-\selectlanguage{greek}α, \selectlanguage{english}and
IL-1. NRF2 can inhibit NF-\selectlanguage{polish}ĸ\selectlanguage{english}B transcriptional activity directly or
through activating HO-1.

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