Reply to Letter to Editor by Elumalai et al. re : "Ginger (Zingiber officinale roscoe) extract could upregulate the renal expression of NRF2 and TNFα and prevents ethanol-induced toxicity in rat kidney"

Thanks for your valuable comment
on the article entitled: “Ginger
(Zingiber officinale roscoe) extract
could upregulate the renal expression
of NRF2 and TNFα and prevents
ethanol-induced toxicity in rat kidney”.
We reviewed your comment. In the
beginning, I think it is necessary to
first give an overview of NRF2 and its
expression.
Nuclear factor erythroid 2–related
factor 2 (Nrf2) is a transcription factor
that regulates important antioxidant
and phase II detoxification genes
against oxidative stress (Tonelli et al.,
2017). Nrf2 is involved in the
regulation of (1) production,
utilization, and regeneration of
glutathione (GSH) and thioredoxin (TXN),
(2) nicotinamide adenine dinucleotide
phosphate (NADPH) regeneration, (3) heme
and iron metabolism, (4) reactive oxygen
species (ROS) and xenobiotic detoxification
(Tonelli et al., 2017). Nrf2 activity is
subjected to regulation at various levels
including protein stability, transcription, and
post-transcription (Tonelli et al., 2017; Li et
al., 2019). Regulation of Nrf2 protein
stability level occurs mainly by keap-1-
dependent and keap-1-independent
pathways. In addition to the modulation of
Nrf2 protein stability, regulation of Nrf2
signaling occurs at the transcriptional level.
The transcriptional factors involved include
the aryl hydrocarbon receptor (AhR), NFκB, and Nrf2 itself (Li et al., 2019). The
transcription of the Nrf2 gene is found to be
activated by AhR (Li et al., 2019), and the
expression of AhR may be decreased by
ethanol (Zhang et al., 2012). Nrf2 gene
promoter also contains a binding site for NFκB, and NF-κB subunits p50 and p65 induce
transactivation of the Nrf2 gene (Rushworth
et al., 2012). This explains the activation of
Nrf2 by NF-κB-activating inflammatory
cytokines. Although NF-κB activates Nrf2,
Nrf2 activation attenuates NF-κB signaling,
suggesting a cross-talk between Nrf2 and
NF-κB (Cuadrado et al., 2014). Inhibition of
NF-κB signaling by Nrf2 may contribute, at
least partly, to the anti-inflammatory
function of Nrf2 activators, such as
sulforaphane (Sun et al., 2015). How Nrf2
suppresses NF-κB signaling remains
Akbari
 AJP, Vol. 13, No. 1, Jan-Feb 2023 4
unknown. It is suggested that Nrf2
activation may shift the cellular redox
status to a more reducing state due to
increased expression of antioxidants
(Li et al., 2019), and we know that
ethanol or its metabolic products may
disrupt these conditions by producing
different species of free radicals
(Comporti et al., 2010). Nrf2
autoregulation is another mechanism
that regulates the transcription of this
gene. Due to the presence of ARE-like
sequences in the promoter region of
the Nrf2 gene, Nrf2 may activate its
own gene expression, leading to
increased production of Nrf2 protein
(Kwak et al., 2002). This represents a
positive feedback mechanism. On the
other hand, Nrf2 may stimulate Keap1
gene expression for its own
degradation (Lee et al., 2007). This
negative feedback is a mechanism to
control the undue expression of Nrf2
and uncontrolled Nrf2 signaling (Lee
et al., 2007). In fact, these descriptions
show that the expression of this gene
can be controlled by different and
interconnected cellular mechanisms
that are involved in various
pathophysiological events (Tebay et
al., 2015; Wu et al., 2012). In vivo and
in vitro studies showed that
interventions such as ethanol
consumption (Wu et al., 2012, Gong
and Cederbaum, 2006a, Dong et al.,
2008), smoking (Knörr-Wittmann et
al., 2005), or exposure to heavy metals
(He et al., 2007; Korashy and El-Kadi,
2006) have led to different changes in
the expression of this gene. The
examination of each of these studies
shows contradictory results regarding
the expression of this gene, similar to
our study. In addition, we were aware,
based on studies by other researchers
and previous studies on the expression
of this gene that the results presented
in our article may have occurred
contrary to what has been expected so
far. Nevertheless, we reported what we
achieved. In addition, it should be noted that
despite the results of all in vivo and in vivo
studies, we face a complex and dynamic
biological system in the face of harmful
interventions that can individually produce
adaptations and compensatory responses,
and conflicting results that justify many
unexpected data. Therefore, reporting a
result in a study that is different from other
studies is not far from the mind and is
usually seen in many studies in different
fields.
To answer to the question “why the
expression of this gene is increased in the
presence of ethanol? Despite the hypotheses
that exist in this field”, we have not yet
encountered a study that has been conducted
specifically for this purpose. However, Gong
et al. (2006) reported that the induction of
CYP2E1 by ethanol is one pathway through
which, ethanol generates oxidative stress.
They also suggested that the levels of protein
and mRNA Nrf2 are increased when
CYP2E1 is elevated, and Nrf2 plays a key
role in the adaptive response against
increased oxidative stress caused by
CYP2E1 (Gong and Cederbaum, 2006a).
Dong et al. (2008) also reported that
maternal ethanol treatment increased both
Nrf2 protein levels and Nrf2-ARE binding in
mouse embryos. It has also resulted in a
moderate increase in the mRNA expression
of Nrf2 downstream target genes (Dong et
al., 2008). Because exposure to ethanol
results in the generation of ROS which are
known to activate Nrf2 (Kensler et al.,
2007), the observed Nrf2 activation was
expected. This response is not unique to
ethanol-exposed embryos. Similar effects
have been observed in cells treated with a
number of other toxic chemicals, including
heavy metals (He et al., 2007; Korashy and
El-Kadi, 2006), cigarette smoke (KnörrWittmann et al., 2005), and arachidonic acid
(Gong and Cederbaum, 2006b). Of particular
interest to this study is that an increase in
Nrf2 protein has also been observed in livers
and hepatocytes of alcohol-fed mice and rats
(Gong and Cederbaum, 2006a).
Ginger, ethanol and renal NRF2 expression
AJP, Vol. 13, No. 1, Jan-Feb 2023 5
However, another contradiction that
can be seen in the results of this study
is the decrease in the activity of
antioxidant enzymes despite the high
level of expression of the Nrf2 gene.
These results can be easily described
and interpreted. A decrease in the
activity of antioxidant enzymes is
actually due to their use to scavenge free
radicals produced due to incomplete ethanol
metabolism and the increase in the
expression of the Nrf2s gene actually
indicates a compensatory response to
improve the response capacity of the
antioxidant system and increase the activity
of these enzymes