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\begin{document}

\title{Leonurine alleviates ferroptosis in cisplatin-induced acute kidney
injury by activating the Nrf2 signaling pathway}



\author[1]{Jianqiang HU}%
\author[1]{Wenjing  Gu}%
\author[1]{Ning Ma}%
\author[2]{Xiaoye Fan}%
\author[1]{Xinxin Ci}%
\affil[1]{Jilin University First Hospital}%
\affil[2]{Institute of Translational Medicine, The First Hospital, Jilin University}%


\vspace{-1em}



  
  \date{April 27, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Background and purpose: Increasing evidence suggests that ferroptosis
plays a key role in the pathophysiology of acute kidney injury induced
by cisplatin. The Nrf2 signaling pathway regulates oxidative stress and
lipid peroxidation and positively regulates cisplatin-induced AKI
(CI-AKI). However, Nrf2 and its activator leonurine on ferroptosis after
CI-AKI remain unclear. Experimental Approach: The anti-ferroptotic
effects of Nrf2 and its activator leonurine were assessed using a mouse
model of cisplatin-induced AKI. In vitro, the potential effects of
leonurine on erastin- and RSL3-induced HK-2 human PTEC ferroptosis were
examined. Key Results: As expected, Nrf2 deletion induced
ferroptosis-related protein expression and iron accumulation in vivo,
further aggravating CI-AKI. The Nrf2 activator leonurine prevented iron
accumulation and lipid peroxidation and inhibited ferroptosis in vitro,
while these effects were abolished in siNrf2-treated cells. Moreover,
leonurine potently ameliorated cisplatin-induced renal damage, as
indicated by the assessment of SCr, BUN, KIM-1, and NGAL. Importantly,
leonurine activated the Nrf2 antioxidative signaling pathway and
prohibited changes in ferroptosis-related morphological and biochemical
indicators, such as the MDA level, SOD and GSH depletion and GPX4 and
xCT downregulation, in CI-AKI. Moreover, Nrf2 KO mice were more
susceptible to ferroptosis after CI-AKI than control mice, and the
protective effects of leonurine on AKI and ferroptosis were largely
abolished in Nrf2 KO mice. Conclusion and Implications: These data
suggest that the renal protective effects of Nrf2 and its activator
leonurine on CI-AKI are achieved at least partially by inhibiting lipid
peroxide-mediated ferroptosis and highlight the potential of leonurine
as a CI-AKI treatment.%
\end{abstract}%



\sloppy


\textbf{Leonurine alleviates ferroptosis in cisplatin-induced acute
kidney injury by activating the Nrf2 signaling pathway}

Jianqiang Hu \textsuperscript{a\#}, Wenjing Gu \textsuperscript{b\#},
Ning Ma \textsuperscript{a}, Xiaoye Fan \textsuperscript{a}, Xinxin
Ci\textsuperscript{a*}

\textsuperscript{a} Institute of Translational Medicine, The First
Hospital of Jilin University, Changchun, Jilin, 130001, China

\textsuperscript{b} Department of Otolaryngology Head and Neck Surgery,
The First Hospital of Jilin University, Changchun, Jilin 130001, China

\# Jianqiang Hu and Wenjing Gu contributed equally to this work.

*corresponding author.

E-mail address: cixinxin@jlu.edu.cn

Full postal address: Dongminzhu road 519, The First Hospital of Jilin
University, Changchun, Jilin, 130001, China.

\textbf{Data Availability Statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request. Some data may not be made
available becaused of privacy or ethical restrictions.

\textbf{Abstract}

\emph{\textbf{Background and purpose}:} Increasing evidence suggests
that ferroptosis plays a key role in the pathophysiology of acute kidney
injury (AKI) induced by cisplatin. The Nrf2 signaling pathway regulates
oxidative stress and lipid peroxidation and positively regulates
cisplatin-induced AKI (CI-AKI). However, its effect as well as that of
its activator leonurine on ferroptosis after CI-AKI remain unclear.

\emph{Experimental Approach:} The anti-ferroptotic effects of Nrf2 and
its activator leonurine were assessed using a mouse model of
cisplatin-induced AKI. In vitro, the potential effects of leonurine on
erastin- and RSL3-induced HK-2 human PTEC ferroptosis were examined.

\emph{Key Results:} As expected, Nrf2 deletion induced
ferroptosis-related protein expression and iron accumulation in vivo,
further aggravating CI-AKI. The Nrf2 activator leonurine prevented iron
accumulation and lipid peroxidation and inhibited ferroptosis in vitro,
while these effects were abolished in siNrf2-treated cells. Moreover,
leonurine potently ameliorated cisplatin-induced renal damage, as
indicated by the assessment of SCr, BUN, KIM-1, and NGAL. Importantly,
leonurine activated the Nrf2 antioxidative signaling pathway and
prohibited changes in ferroptosis-related morphological and biochemical
indicators, such as the MDA level, SOD and GSH depletion and GPX4 and
xCT downregulation, in CI-AKI. Moreover, Nrf2 KO mice were more
susceptible to ferroptosis after CI-AKI than control mice, and the
protective effects of leonurine on AKI and ferroptosis were largely
abolished in Nrf2 KO mice.

\emph{Conclusion and Implications:} These data suggest that the renal
protective effects of Nrf2 and its activator leonurine on CI-AKI are
achieved at least partially by inhibiting lipid peroxide-mediated
ferroptosis and highlight the potential of leonurine as a CI-AKI
treatment.

\textbf{Key words} : Cisplatin-induced acute kidney injury; Ferroptosis;
Nrf2; Leonurine

\textbf{Abbreviations:} Leo, leonurine; CDDP, cisplatin; AKI, acute
kidney injury; CI-AKI, cisplatin-induced acute kidney injury; HK-2,
Human Kidney-2; SCr, serum creatinine; BUN, Blood Urea Nitrogen; SOD,
Superoxide Dismutase; MDA, malondialdehyde; GSH, glutathione; Nrf2,
nuclear factor erythroid-2; HO-1, Heme Oxygenase-1; NQO1, NAD(P)H
quinone dehydrogenase 1; KIM1, kidney injury molecule-1; NGAL,
Neutrophil gelatinase-associated lipocalin; FTH-1, ferritin heavy chain;
FTL, ferritin light chain; TFR, transferrin receptor 1; GPX4,
glutathione peroxidase 4; XCT, cystine/glutamateantiporter system;
SLC7A11, Solute Carrier Family 7 Member 11; ROS, reactive oxygen
species; LPO, lipid peroxidation; RSL3:(1S,3R)-RSL3; CCK-8, Cell
Counting Kit-8; FBS, fetal bovine serum; DCFH-DA,
2,7-Dichlorodihydrofluorescein diacetate; PBS, phosphate buffered
saline; DMSO, Dimethyl sulfoxide; HE, Hematoxylin and eosin staining;
PCR, Polymerase Chain Reaction; UUO, unilateral ureteral obstruction.

\textbf{Introduction}

Acute kidney injury (AKI), a common and severe syndrome characterized by
tubular damage and sudden loss of renal function within hours, is a
global health problem with high morbidity and mortality(Hoste et al.,
2018). As an antitumor drug, cisplatin is widely used in the treatment
of solid tumors and is one of the main causes of AKI in patients with
cancer(Yang et al., 2018). Because the exact molecular mechanisms of
CI-AKI are not fully understood, strategies for the prevention and
treatment of AKI are limited. Tubular cell injury and death play vital
roles in the initiation and progression of AKI(Deng et al., 2021;
Linkermann et al., 2014). Previous studies focused on the induction of
renal tubular cell apoptosis and necrosis by cisplatin, but specific
inhibitors of both apoptosis and necrosis failed to completely prevent
or stop CI-AKI(Herzog et al., 2012; Tristao et al., 2012). Ferroptosis,
a new type of ``regulated cell death'', was recently reported to be
involved in the pathological processes of cisplatin-induced AKI (Hu et
al., 2020). Thus, elucidating the mechanisms by which cisplatin induces
ferroptosis will have important clinical value for alleviating AKI.

In contrast to other regulatory cell death mechanisms, such as
apoptosis, necrosis and pyrogen death, ferroptosis is a newly discovered
regulatory type of cell death caused by iron-dependent lipid
peroxidation (Stockwell et al., 2017) (Deng et al., 2021). In the case
of ferroptosis, low FTH-1 expression and TFR overexpression lead to
excessive iron accumulation, which in turn promotes ROS production via
Fenton reactions (Dixon and Stockwell, 2014). At the same time, the
function of system XC is disrupted during ferroptosis, thereby
decreasing the production of glutathione (GSH) and inhibiting the
antioxidant capacity of GPX4 (Wang et al., 2020) (Maiorino et al.,
2018). Thus, ROS have been demonstrated to a central role in the entire
ferroptosis process (Park and Chung, 2019). In cisplatin-induced AKI,
the generation of excess ROS can cause oxidative stress and lead to
tubular cell damage. Nrf2, the most important nuclear transcription
factor for antioxidative stress, regulates not only the expression of a
series of signaling proteins and enzymes to maintain cellular redox
homeostasis but also many important genes related to iron storage and
transport (Dodson et al., 2019). Previous studies have highlighted the
role of Nrf2 in alleviating lipid peroxidation and ferroptosis and
further elucidated the role of the Nrf2-lipid peroxidation-ferroptosis
axis in neurodegenerative and cardiovascular diseases(Dodson et al.,
2019). Many Nrf2 activators have been reported to alleviate
cisplatin-induced nephrotoxicity by inhibiting ROS generation (Fan et
al., 2020). In addition, ferrostatin-1, the most potent inhibitor of
ferroptosis, was shown to significantly reduce cisplatin-induced HK-2
cell death and attenuate CI-AKI in mice(Deng et al., 2019). Considering
the close relationship among ROS, Nrf2 and ferroptosis, we proposed that
controlling ferroptosis via Nrf2 activation might improve CI-AKI.

Leonurine, a major active alkaloid compound found only in motherwort,
has been successfully extracted and depurated\textsuperscript{(Li et
al., 2020)}. Leonurine was recently shown to regulate various
pathological processes, including oxidative stress, inflammation,
fibrosis, and apoptosis, as well as a variety of metabolic disorders.
Leonurine protects against LPS-induced acute renal injury and fibrosis
induced by unilateral ureteral obstruction (UUO) in mice(Cheng et al.,
2015). However, an alleviating effect of leonurine on CI-AKI has not
been reported. At the same time, some studies have shown that leonurine
can alleviate aging and ischemic stroke in mice by activating the Nrf2
pathway(Chen et al., 2019; Xie et al., 2019), but its effect on
ferroptosis has not been investigated. Herein, we first investigated
whether the levels of Nrf2 are directly related to ferroptosis
sensitivity in mice with CI-AKI. We then investigated the potential of
leonurine to protect against ferroptosis in vitro and in vivo. Finally,
using Nrf2 siRNA and Nrf2 knockout (KO) mice, we further assessed the
underlying mechanisms by which leonurine protects against ferroptosis
and AKI in vitro and in vivo.

\textbf{2. Methods}

\textbf{2.1 Materials}

Leonurine (98.24\%) was purchased from PuFeiDe Biotech Co. (Chengdu,
China). Erastin and RSL3 were purchased from Selleckchem (Shanghai,
China). Cisplatin was purchased from Med Chem Express (New Jersey, USA).
Primary antibodies against \selectlanguage{greek}β-\selectlanguage{english}actin, KIM1, NGAL, GPX4, XCT, FTL, FTH-1,
TFR, Nrf2, HO-1, and NQO1 were purchased from Abcam (Cambridge MA, USA),
Cell Signaling Technology (Danvers, Massachusetts, USA), R\&D Systems
(Minnesota, USA) and Proteintech (Wuhan, China). The BCA protein assay
kit was purchased from Thermo Fisher Scientific (MA, USA). The iron
assay kit was purchased from BioAssay Systems (Northern California Bay
Area, USA). Calcein-acetoxymethyl (AM) was purchased from Beyotime
Biotechnology (Jiangsu, China). DMEM/F-12, penicillin, streptomycin and
trypsin EDTA, used for cell culturing, were purchased from hyclone
(Logan, Utah, USA). Dimethyl sulfoxide (DMSO) and DCFH-DA were purchased
from Sigma (St. Louis, Missouri, USA). Cell Counting Kit-8 (CCK-8) was
purchased from Bimake (Houston, USA). Nrf2 siRNA was obtained from
Hanbio Biotechnology Co., Ltd. (Shanghai, China). SYBR Green I was
purchased from Roche (Basel, Switzerland).

\textbf{2.2 Cell treatment}

HK-2 cells were cultured in DMEM/F12 supplemented with 10\% fetal bovine
serum (FBS) and 100 U/ml penicillin/streptomycin at 37  and 5\%
CO\textsubscript{2}. After being cultured in the absence or presence of
different concentrations of leonurine for 1 h, the cells were treated
with different concentrations of RSL3 or erastin for 24 h and then
collected or stained for subsequent experiments.

\textbf{2.3 Cell viability}

Cell viability was determined using the CCK-8 assay. HK-2 cells were
inoculated into 96-well culture plates (1\selectlanguage{ngerman}×10\textsuperscript{4}/well)
and treated with leonurine and cisplatin for 24 h. Thereafter, 10 \selectlanguage{greek}μ\selectlanguage{english}L of
CCK-8 reagent was added to each well, and the plate was cultured at 37 
for 1 h. The OD value at 450 nm was then measured with an
enzyme-labeling instrument to determine the cell survival rate.

\textbf{2.4 Analysis of intracellular ROS levels}

After planking and drug treatment for 24 h, the cells were mixed with
DCFH-DA (1 \selectlanguage{greek}μ\selectlanguage{english}M) and incubated in the dark at room temperature for 30
minutes. After washing the cells with PBS three times, they were
observed by a fluorescence microscope. The OD value was determined by an
enzyme-labeling instrument, and the ROS content was determined.

\textbf{2.5 Analysis of lipid peroxidation (LPO)}

A Liperfluo assay kit was used to detect the lipid peroxide content in
HK-2 cells. After washing three times, the cells were mixed with 1 \selectlanguage{greek}μ\selectlanguage{english}M
Liperfluo, incubated in the dark at room temperature for 30 minutes, and
then washed with PBS three times. Lipid peroxide was finally measured
and evaluated by confocal microscopy.

\textbf{2.6 Animal experiments}

Female C57BL/6 mice (8-10 weeks, 20-22g) and Nrf2 KO mice were purchased
from Vital River (Beijing, China) and Jackson Laboratory (Bar, Harbor,
USA), respectively. After one week of acclimatization, CI-AKI was
established in female C57BL/6 mice. The experimental animals were
divided into five groups as follows (n=10/group): control (0.1\% DMSO in
normal saline), cisplatin (20 mg/kg), cisplatin (20 mg/kg)+leonurine (50
mg/kg and 100 mg/kg) and leonurine only (100 mg/kg dissolved in 0.1\%
DMSO). After fasting, the mice in the cisplatin (20 mg/kg) + leonurine
group were treated with 50 mg/kg and 100 mg/kg leonurine by gavage and
intraperitoneally injected with cisplatin (20 mg/kg) one hour later to
induce acute renal injury. Mice in the control, cisplatin and leonurine
groups were treated with 0.1\% DMSO in normal saline, cisplatin (20
mg/kg) and leonurine (100 mg/kg dissolved in 0.1\% DMSO), respectively.
On the second and third days, the mice in the cisplatin (20
mg/kg)+leonurine and leonurine only groups were administered leonurine
by gavage once more. On the fourth day, all the mice were sacrificed,
and kidney and blood samples were collected for further experiments. The
mice were housed in a controlled living environment and provided
adequate amounts of food and water.

\textbf{2.7 Hematoxylin and eosin staining (HE)}

Renal tissue portions were fixed overnight with 4\% paraformaldehyde,
embedded in paraffin, sectioned, stained with HE, and then observed
under an optical microscope (Olympus, Japan). The degree of renal injury
was evaluated in three random fields per sample by grading the renal
tissue injury as follows: 0-10\%, 0; 11-25\%, 1; 26-50\%, 2; 51-75\%, 3;
and 76-100\%, 4. At least five renal regions on each slide were randomly
selected.

\textbf{2.8 Immunohistochemistry and enhanced Prussian blue staining}

For immunohistochemistry analysis, the slides were sequentially
incubated with 5\% bovine serum albumin for 1 h, with a primary antibody
targeting F4/80 overnight, and then with the appropriate secondary
antibody. After incubation with the secondary antibody, the cells were
observed under an optical microscope. Enhanced Prussian blue staining
was used to detect the iron deposition in renal tissue. Mouse kidney
tissues were fixed with 3\% glutaraldehyde overnight, washed three
times, and sectioned at a thickness of 4 \selectlanguage{greek}μ\selectlanguage{english}m for prussian blue staining.
After washing with PBS, nuclei were stained with hematoxylin, and the
iron deposition in the tissues was finally observed under an optical
microscope.

\textbf{2.9 Transmission electron microscopy}

Morphological changes in renal tissue mitochondria were observed by
electron microscopy. In brief, the tissues were fixed with pentanediol,
sectioned with an ultrathin microtome, dehydrated, and stained many
times. Images were then obtained with a transmission electron
microscope, and more than 5 different visual fields from each slice were
observed.

\textbf{2.10 RNA isolation and real-time PCR}

Total RNA was extracted from fresh renal tissue by using the TRIzol
method and then reverse transcribed into cDNA. The cDNA content was
detected by real-time quantitative PCR to determine the gene
transcription levels in the tissue. Samples from five independent
experiments were utilized, and each sample was subjected to real-time
PCR in triplicate. The primers used in this study are provided in Table
S1.

\textbf{2.11 Assays of renal functional parameters as well as GSH, MDA
and SOD}

The levels of BUN and SCr, which can indicate the level of renal
function, were detected in serum samples, whereas the levels of GSH, MDA
and SOD were detected in renal tissue and cell suspensions. The above
mentioned indices were detected by BUN, SCr, GSH, MDA and SOD assay kits
according to the manufacturers' instructions.

\textbf{2.12 Iron content analysis}

The intracellular iron level was measured by a calcein-AM kit. After
cell plating and drug treatment for 24 h, HK-2 cells were treated with
AM (2 \selectlanguage{greek}μ\selectlanguage{english}M) in the dark at 37  for 30 minutes. Next, the cells were
washed and analyzed with a fluorescence microscope. To perform
fluorescence imaging of Fe\textsuperscript{2+} in living cells, HK-2
cells were inoculated into confocal dishes and cultured overnight at 37
 in a 5\% CO\textsubscript{2} incubator. FerroOrange working solution
(1 \selectlanguage{greek}μ\selectlanguage{english}M), purchased from Dojindo (Kumamoto, Kyushu, Japan), was added to
the cells, which were cultured in a 5\% CO\textsubscript{2} incubator at
37  and then observed by a fluorescence confocal microscope. In vivo,
an iron assay kit was used to detect the iron ion content in mouse
serum, which was determined by detecting the absorbance at 405 nm.

\textbf{2.13 Western blot assay}

Fresh cells or kidney tissues were collected and lysed on ice for 30
minutes. Equal amounts of protein (20 \selectlanguage{greek}µ\selectlanguage{english}g) were separated by 10-15\%
SDS-PAGE, and the separated proteins were transferred onto PVDF
membranes and blocked with 5\% nonfat powder milk in PBS buffer for 1 h
at room temperature. The cells were incubated with primary antibodies
against \selectlanguage{greek}β-\selectlanguage{english}actin, KIM1, NGAL, GPx4, xCT, FTL, FTH-1, TFR, Nrf2, HO-1 and
NQO1 overnight at 4\selectlanguage{ngerman}°C and then with an HRP-conjugated secondary antibody
(1:5000) at room temperature for 1 h. The relative expression levels
were determined with Image J software.

\textbf{2.14 RNA interference}

HK-2 cells were transfected with Nrf2 siRNAs for 72 h, after which
Western blotting was performed with an anti-Nrf2 antibody (1:1000) to
assess their transfection efficiencies. The siRNA with the highest
transfection efficiency was selected for the cell treatments described
above and for subsequent cell experiments.

\textbf{2.15 Statistical analysis}

The data were statistically analyzed by SPSS 19.0 (IBM) and are
expressed as the means ± SEMs. The experimental data were compared by
one-way analysis of variance (ANOVA). A P value \textless{} 0.05 was
considered statistically significant. The sample size needed for
statistical analysis was determined to be at least 5 animals in each
group (N=5, where n refers to the number of independent data points).

\textbf{3. Results}

\textbf{3.1 Nrf2 KO aggravates cisplatin-induced kidney injury and iron
accumulation}

Nrf2 and its regulatory genes are reported to be involved in preventing
the progression of ferroptosis, but their regulatory effect on
ferroptosis in CI-AKI has not been clarified. As shown in Fig. 1A-D,
Nrf2 KO aggravated cisplatin-induced kidney injury, which was
characterized by elevated levels of Bun and SCr as well as more
morphological damage, including necrosis, brush edge loss, cast
formation, tubular degeneration, and vacuolization. Nrf2 targets play
key roles in mediating iron metabolism, which is important for the
initiation of ferroptosis. Therefore, we investigated the changes in the
iron levels in Nrf2 KO mice and found that cisplatin injection increased
the iron levels in Nrf2 KO mice compared with wild-type mice (Fig. 1E).
In addition, lower expression levels of the antioxidant response genes
Nrf2, HO-1 and NQO1 were observed in Nrf2 KO mice compared with
wild-type mice. Interestingly, loss of Nrf2 not only increased TFR
expression but also led to a compensatory increase in FTH-1 and FTL
(Fig. 1F-H).

\textbf{3.2 Nrf2 KO mice are susceptible to cisplatin-induced lipid
peroxidation and ferroptosis}

Nrf2 is a key reliever of lipid peroxidation and ferroptosis, and
abnormal Nrf2 signaling is thought to aggravate the disease by
regulating lipid peroxides and ferroptosis. We investigated lipid
peroxidation and ferroptosis in Nrf2 KO mice and found that their
malondialdehyde (MDA) levels were increased to a greater extent than
those in the WT mice, while the GSH levels were significantly decreased
in Nrf2 KO mice after cisplatin injection compared to those in the WT
mice (Fig. 2A-B). We further examined the protein expression of GPX4 and
xCT, biomarkers of ferroptosis, to clarify the role of Nrf2 in
ferroptosis. As shown in Fig. 2C-F, both the mRNA and protein levels of
GPX4 and xCT were significantly decreased in Nrf2 KO mice compared with
WT mice.

\textbf{3.3 Leonurine inhibits iron accumulation and ferroptosis in HK-2
cells}

HK-2 cells were treated with different doses of erastin or RSL3,
classical inducers of ferroptosis, to determine the exact concentrations
that inhibited cell growth. As shown in Fig. 3A and 3B, erastin (30 \selectlanguage{greek}μ\selectlanguage{english}M)
and RSL3 (0.4 \selectlanguage{greek}μ\selectlanguage{english}M) inhibited cell viability in a dose-dependent manner.
Importantly, leonurine treatment significantly ameliorated the effects
of erastin and RSL3 on cell death (Fig. 3C-D). Then, we used a
fluorescent probe calcein-AM, to detect changes in cellular labile iron
in cells based on its ability to be rapidly quenched when bound to
cellular iron. As shown in Fig. E, RSL3 treatment markedly decreased the
fluorescence signals, indicating that cellular iron levels were
increased by RSL3 treatment but reversed by leonurine treatment. We next
detected Fe\textsuperscript{2+} in HK-2 cells using FerroOrange. As
shown in Fig. 3F, the Fe\textsuperscript{2+} content was increased after
RSL3 treatment but decreased after leonurine treatment. We further
evaluated the protein expression of key factors regulating cellular iron
homeostasis. As shown in Figure 3G and 3H, RSL3 significantly increased
the protein levels of TFR, FTH-1 and FTL, and these trends were reversed
by leonurine treatment.

\textbf{3.4 Leonurine inhibited ROS generation and lipid peroxidation
and restored the expression of ferroptosis-related genes}

Free iron can interact with ROS to form lipid radicals that rapidly
react with oxygen to induce lipid peroxidation. We first investigated
intracellular ROS using the sensitive fluorescent probe DCFH-DA,
revealing that ROS generation was induced by RSL3 but reversed by
leonurine (Fig. 4A). In addition, we used the Liperfluo probe to detect
lipid peroxides and found that lipid peroxidation was increased by RSL3
treatment but decreased by leonurine (Fig. 4B). To further confirm that
the leonurine-regulated ferroptosis blockade was associated with lipid
peroxidation, we examined the cellular MDA and GSH levels. RSL3
treatment significantly downregulated GSH and upregulated MDA, and these
trends were reversed by leonurine treatment (Fig. 4C to 4D). Next, we
further evaluated the regulatory effect of leonurine on the expression
of antioxidant and ferroptosis-related target molecules. RSL3 treatment
significantly decreased the GPX4 and xCT expression compared to that in
the control group, but leonurine restored these changes in a
dose-dependent manner (Fig. 4E-F). Moreover, leonurine dose-dependently
upregulated the protein expression of Nrf2, NQO1 and HO-1 (Fig. 4G).

\textbf{3.5 Knockdown Nrf2 abolishes the effect of leonurine on ROS
generation, lipid peroxidation and ferroptosis}

Considering the role of Nrf2 in preventing lipid peroxidation and
ferroptosis, we attempted to investigate the potential for Nrf2 to
mediate the protective effect of leonurine on ferroptosis. Control or
Nrf2 siRNA was transiently transfected into cells, and Nrf2 protein
expression was shown to be significantly inhibited in the Nrf2 siRNA
group (Fig. 5A-B). As shown in Fig. 5C, the protective effect of
leonurine on RSL3-induced cell death was alleviated in Nrf2 knockdown
cells. Furthermore, the protective effects of leonurine against ROS
generation, MDA production and GSH depletion were nearly abolished in
Nrf2 knockdown cells (Fig. 5D-F). The inhibitory effect of leonurine on
cellular iron levels was almost eliminated in Nrf2 knockdown cells as
determined by calcein-AM detection (Fig. 5G). Next, we examined the
protein expression of key factors regulating cellular iron homeostasis
and ferroptosis. As shown in Fig. 5H-J, the regulatory effects of
leonurine on TFR, FTH1, GPX4 and XCT are nearly eliminated in Nrf2
knockdown cells.

\textbf{3.6 Leonurine protects against CI-AKI}

Based on the effect of leonurine on ferroptosis in vitro, we further
evaluated its potential effects on CI-AKI in vivo. Compared to those in
the control group, the SCr and BUN levels in mice receiving the
cisplatin injection were significantly increased; however, the levels
were reversed by leonurine treatment (Fig. 6A-B). HE staining of renal
tissue further indicated the protective effect of leonurine on kidney
damage. The severe tubular dilatation, marked tubular epithelial cell
edema, and obvious cast formation induced by cisplatin were inhibited by
leonurine treatment (Fig. 6C-D). In addition, the levels of two other
known tubule damage biomarkers, KIM-1 and NGAL were elevated by
cisplatin injection and reduced by leonurine treatment (Fig. 6E-F).
Macrophages have been shown to be the primary cause of inflammatory
infiltration in AKI. F4/80 staining was used to further evaluate the
macrophage infiltration in cisplatin-challenged kidneys. As shown in
Fig. 6G, the number of F4/80-positive cells was dramatically increased
in the cisplatin treatment group but significantly decreased in the
leonurine treatment group.

\textbf{3.7 Leonurine alleviates iron accumulation in CI-AKI}

Based on the critical ability of iron to initiate ferroptosis, we next
assessed the changes in renal iron deposition. Compared to the control,
cisplatin induced iron deposition, and both low-dose and high-dose
leonurine significantly alleviated the increase in the iron deposition
(Fig. 7A). We next found that the renal iron levels were increased by
cisplatin treatment but reversed by leonurine treatment (Fig. 7B). The
expression level of TFR, a membrane protein affecting iron transport,
was significantly increased in the model group and inhibited by
leonurine treatment. Moreover, the expression levels of FTH-1 and FTL
were upregulated in cisplatin-treated mice but downregulated by
leonurine treatment (Fig. 7C-D). Similar to the protein trends, the mRNA
levels of TFR and FTH-1 were increased after cisplatin administration
and decreased after leonurine treatment (Fig. 7E-F).

\textbf{3.8 Leonurine suppresses lipid peroxidation and ferroptosis in
CI-AKI}

Next, we assessed the effects of leonurine on cisplatin-induced lipid
peroxidation and ferroptosis in vivo. Compared to the control, cisplatin
significantly increased the MDA content and decreased the GSH and SOD
contents in the kidney, while leonurine obviously reversed the above
changes (Fig. 8A-C). In addition, the protein levels of GPX4 and xCT
were significantly downregulated by cisplatin treatment but
dose-dependently restored by leonurine treatment. Moreover, the protein
levels of~Nrf2 and its regulated~downstream antioxidases NQO1 and HO-1
were activated by leonurine treatment (Fig. 8D-F). Similar to the
protein levels, the mRNA levels of GPX4 and xCT were decreased in the
renal tissues of the model group. Interestingly, leonurine treatment
significantly increased the transcript levels of GPX4 and xCT (Fig.
8G-H). Moreover, electron microscopic observations revealed
mitochondrial damage in renal tissue cells of the model group,
manifested as mitochondrial rupture and disappearance of mitochondrial
cristae, and leonurine treatment significantly alleviated this damage
(Fig. 8I).

\textbf{3.9 The renal protective effect of leonurine on CI-AKI is
dependent on Nrf2 in mice}

We further evaluated whether the protective effect of leonurine on
CI-AKI was dependent on Nrf2 using Nrf2 KO mice. As shown in Fig. 9A and
B, leonurine significantly decreased the cisplatin-induced upregulation
of SCr and BUN in the sera of wild-type mice, but these effects were
nearly abolished in Nrf2 KO mice. Renal HE staining and protein
expression of KIM1 and NGAL further demonstrated that the protective
effect of leonurine on kidney damage was almost abrogated in Nrf2 KO
mice (Fig. 9C-E, G). Moreover, the expression levels of Nrf2 and the
downstream regulatory factors HO-1 and NQO1 were obviously increased in
wild-type mice but not in Nrf2 KO mice (Fig. 9E-F).

\textbf{3.10 Nrf2 deficiency prohibits the regulatory effect of
leonurine on ferroptosis}

We further investigated whether the ability of leonurine to inhibit
ferroptosis, as well as its cytoprotective potential, was dependent on
Nrf2. In Nrf2 KO mice, cisplatin significantly increased the Fe and MDA
levels and decreased the GSH levels compared to those in the wild-type
mice (Fig. 10A-C). Moreover, treatment with leonurine markedly reversed
the above changes in wild-type mice, while this effect was almost
abolished in the Nrf2 KO mice. Moreover, the alleviating effects of
leonurine on FTH-1 and TFR were eliminated (Fig. 10D-E). The protective
effects of leonurine on the cisplatin-induced expression of the
ferroptosis-related proteins GPX4 and xCT were also abolished in Nrf2 KO
mice (Fig. 10D-F). DAB staining indicated that the reduced iron
deposition in the leonurine treatment group was restored by the KO of
Nrf2 (Fig. 10G). Furthermore, electron microscopy analysis revealed that
the mitochondrial damage mitigated by leonurine was reversed in Nrf2 KO
mice (Fig. 10H).

\textbf{Discussion and conclusions}

Ferroptosis and its related components are now better understood, and
Nrf2 has been shown to play a key role in mediating this process. In
particular, the antioxidant, iron, and intermediate metabolic statuses
of cells are mediated by Nrf2 target genes. While the mechanisms of Nrf2
and ferroptosis in cisplatin-induced AKI have been elucidated, the
relationship between them remains unclear(Hu et al., 2020; Ikeda et al.,
2021; Shelton et al., 2013). In this study, we aimed to determine
whether the nuclear transcription factor Nrf2, a critical regulator of
the cellular antioxidant response, could inhibit cisplatin-induced
ferroptosis in subjects with kidney injury. Moreover, we evaluated
whether leonurine, an Nrf2 activator, could alleviate cisplatin-induced
kidney injury by inhibiting Nrf2-mediated lipid peroxidation and
ferroptosis.

Iron has been reported to play a key role in cisplatin-induced
nephrotoxicity both in vitro and in vivo (Baliga et al., 1998). The
abnormal accumulation of iron produces large amounts of free radicals
that damage DNA, proteins, and other biomolecules (Stoyanovsky et al.,
2019). Nrf2 is a key factor regulating the cellular antioxidant
response, as it controls the expression of HO-1, NQO1 and other genes
related to antioxidant stress. In addition to its important role in
maintaining cellular redox balance, Nrf2 helps to mediate lipid
peroxidation and ferroptosis (Abdalkader et al., 2018). The injection of
cisplatin significantly increased lipid peroxidation and ROS production,
which were reversed by the activation of Nrf2 or its downstream target
genes. Notably, increased lipid oxidation and downstream Nrf2 target
inactivation significantly enhance the overall protein lipid oxidation
and ferroptosis in disease environments with low Nrf2, further promoting
disease progression (La Rosa et al., 2021). However, whether Nrf2 levels
are directly related to ferroptosis sensitivity in cisplatin-induced AKI
has not been clarified. To elucidate the effect of Nrf2 on ferroptosis
sensitivity in CI-AKI, we analyzed the iron accumulation, lipid
peroxidation and expression of ferroptosis-related proteins in
cisplatin-treated wild-type and Nrf2 KO mice. Both the wild-type and
Nrf2 KO mice exhibited altered iron accumulation, upregulated MDA
levels, and downregulated protein levels of GPX4 and xCT, which are
biomarkers of ferroptosis. However, the abovementioned biomarkers were
altered more significantly in Nrf2 KO mice than in the wild-type mice
(Figs. 1-2). Therefore, we propose for the first time that the
inhibition of Nrf2 aggravates ferroptosis and further enhances the
progression of CI-AKI.

Considering the mitigated effect of Nrf2 on ferroptosis, targeting the
upstream regulators of the ferroptotic cascade, including dysregulated
iron levels and ROS production, by pharmacologically modulating the Nrf2
signaling pathway remains one of the best strategies for treating
ferroptosis-related pathologies. We found that leonurine, an Nrf2
activator, significantly increased the cell viability and decreased the
iron accumulation, ROS and cellular lipid ROS induced by erastin and
RSL3 (Fig. 3). Iron is an essential element that is regulated by a
variety of proteins. In general, iron is loaded onto transferrin, which
binds to transferrin receptor 1 (TFR) on the cytoplasmic membrane and
delivers iron to numerous tissues via endocytosis (Torti and Torti,
2013). Excess iron is stored in the protein ferritin, which is composed
of 24 subunits of FTH-1 and FTL. Here, leonurine significantly reduced
the protein expression levels of TFR, FTH-1 and FTL induced by RSL3
(Fig. 3). Next, we measured the levels of several other well-established
biomarkers of ferroptosis, including GSH and lipid peroxidase-derived
MDA, as well as those of GPX4 and xCT. As GSH depletion can trigger
ferroptosis and MDA is the end-product of ferroptosis, we herein showed
that leonurine inhibited the GSH depletion induced by RSL3 and the
upregulation of MDA. Importantly, xCT and its key component Slc7a11 are
responsible for the generation of intracellular GSH in response to
oxidative stress. Inhibition of xCT reduces the level of GSH and the
activity of glutathione peroxidase 4 (GPX4), thereby increasing lipid
peroxidation. GPX4 deficiency is considered to be a biomarker of
ferroptosis, and the depletion of GPX4 induces the ferroptosis of
numerous renal tubular epithelial cells (Friedmann Angeli et al., 2014)
(Seibt et al., 2019). In the present study, the downregulation of xCT
and GPX4 induced by RSL3 was reversed by leonurine treatment in a
dose-dependent manner (Fig. 4). Based on the ability of Nrf2 to prevent
lipid peroxidation and ferroptosis, we hypothesized that the inhibitory
effect of leonurine on ferroptosis is mediated by Nrf2. We further
showed that the siRNA-mediated knockdown of Nrf2 ameliorated the effects
of leonurine on the ROS production, lipid peroxidation, cellular iron
level and expression of ferroptosis-related proteins induced by RSL3
(Fig. 5). In summary, our data showed that leonurine significantly
activated Nrf2 and inhibited the ferroptosis of HK-2 cells induced by
RSL3. The above effects were not observed after Nrf2 knockdown, which
indicates that the protective effect of leonurine on ferroptosis in
vitro may be mediated via the Nrf2 pathway.

As reported, leonurine is a therapeutic candidate for LPS-induced AKI
and renal fibrosis (Cheng et al., 2015; Xu et al., 2014). However, the
potential effects of leonurine on cisplatin-induced AKI have not yet
been elucidated. Our results showed that leonurine significantly
decreased the serum levels of Bun and SCr induced by cisplatin as well
as the levels of KIM1 and NGAL and the histological extent of kidney
injury (Fig. 6). Considering the abovementioned inhibitory effect of
leonurine on ferroptosis induced by RSL3 in vitro, we further examined
its effects on related ferroptosis biomarkers in the kidney. Consistent
with the in vitro results, leonurine significantly inhibited the
cisplatin-induced increases in iron accumulation and the TFR, FTL and
FTH-1 levels in the kidney (Fig. 7). Furthermore, leonurine inhibited
morphological and biochemical changes in factors related to ferroptosis,
such as MDA, SOD and GSH depletion and the downregulation of GPX4 and
xCT in cisplatin-induced AKI. In addition, leonurine markedly
upregulated the expression of Nrf2, HO-1 and NQO1 (Fig. 8). Given that
Nrf2 is a principal regulator of antioxidant responses that suppresses
ferroptosis, we further investigated the potential for Nrf2 deficiency
to ameliorate the protective effects of leonurine against
cisplatin-induced ferroptosis. We treated Nrf2 KO mice with leonurine
and then observed the histological and molecular parameters of
cisplatin-induced AKI. The treatment of Nrf2 KO mice with leonurine did
not rescue cisplatin-induced renal damage, as determined by the serum
levels of Bun and Cre, the levels of KIM1 and NGAL, and the histological
extent of kidney injury (Fig. 9). Furthermore, the treatment of Nrf2 KO
mice with leonurine did not inhibit ferroptosis-related morphological or
biochemical changes in cisplatin-induced AKI, as reflected by the MDA
levels, SOD and GSH depletion, and downregulation of GPX4 and xCT (Fig.
10). Taken together, our results support that leonurine provides renal
protection predominantly by activating the Nrf2-mediated inhibition of
ferroptosis.

In conclusion, our study revealed the substantial significance of
upregulating Nrf2 to prevent ferroptosis, thereby alleviating CI-AKI.
This conclusion is derived from three key findings. First, Nrf2 KO mice
were more susceptible to cisplatin-induced renal injury and ferroptosis.
Second, the protective effect of leonurine against cisplatin-induced AKI
was achieved by activating the antioxidant signaling molecule Nrf2 and
inhibiting ferroptosis-related morphological and biochemical changes.
Finally, the treatment of Nrf2 KO mice and Nrf2 siRNA HK-2 cells with
leonurine nearly failed to rescue cisplatin- and RSL3-induced
ferroptosis and renal injury in vivo and in vitro. The present study
furthers our understanding of the mechanism by which Nrf2 inhibits
ferroptosis and provides a strategy for investigating the
antiferroptotic activity of Nrf2 activators in the context of
cisplatin-induced AKI.

\textbf{Acknowledgments}

The study was supported by grants from the National Natural Science
Foundation of China (Grant No. 81970576) and the Natural Science
Foundation of Jilin (No. 20200201378JC and No. JLSCZD2019-065).

\textbf{Author contributions}

Xinxin Ci designed the study, Jianqiang Hu, Wenjing Gu, Ning Ma and
Xiaoye Fan carried out experiments and analyzed the data; Jianqiang Hu
and Wenjing Gu drafted and revised the paper. The final version of the
paper has been approved by all authors.

\textbf{Conflicts of interests}

All authors declare no competing interests.

\textbf{References:}

Abdalkader M., Lampinen R., Kanninen K. M., Malm T. M. and Liddell J. R.
(2018). Targeting Nrf2 to Suppress Ferroptosis and Mitochondrial
Dysfunction in Neurodegeneration. \emph{Front Neurosci,} 12,466. doi:
10.3389/fnins.2018.00466.

Baliga R., Zhang Z., Baliga M., Ueda N. and Shah S. V. (1998). In vitro
and in vivo evidence suggesting a role for iron in cisplatin-induced
nephrotoxicity. \emph{Kidney Int,} 53,394-401. doi:
10.1046/j.1523-1755.1998.00767.x.

Chen P., Chen F. and Zhou B. H. (2019). Leonurine ameliorates
D-galactose-induced aging in mice through activation of the Nrf2
signalling pathway. \emph{Aging (Albany NY),} 11,7339-7356. doi:
10.18632/aging.101733.

Cheng H., Bo Y., Shen W., Tan J., Jia Z., Xu C. and Li F. (2015).
Leonurine ameliorates kidney fibrosis via suppressing TGF-beta and
NF-kappaB signaling pathway in UUO mice. \emph{Int
Immunopharmacol,}25,406-415. doi: 10.1016/j.intimp.2015.02.023.

Deng F., Sharma I., Dai Y., Yang M. and Kanwar Y. S. (2019).
Myo-inositol oxygenase expression profile modulates pathogenic
ferroptosis in the renal proximal tubule. \emph{J Clin
Invest,}129,5033-5049. doi: 10.1172/JCI129903.

Deng F., Zheng X., Sharma I., Dai Y., Wang Y. and Kanwar Y. S. (2021).
Regulated cell death in cisplatin-induced AKI: relevance of myo-inositol
metabolism. \emph{Am J Physiol Renal Physiol,} 320,F578-F595. doi:
10.1152/ajprenal.00016.2021.

Dixon S. J. and Stockwell B. R. (2014). The role of iron and reactive
oxygen species in cell death. \emph{Nat Chem Biol,} 10,9-17. doi:
10.1038/nchembio.1416.

Dodson M., Castro-Portuguez R. and Zhang D. D. (2019). NRF2 plays a
critical role in mitigating lipid peroxidation and
ferroptosis.\emph{Redox Biol,} 23,101107. doi:
10.1016/j.redox.2019.101107.

Fan X., Wei W., Huang J., Peng L. and Ci X. (2020). Daphnetin Attenuated
Cisplatin-Induced Acute Nephrotoxicity With Enhancing Antitumor Activity
of Cisplatin by Upregulating SIRT1/SIRT6-Nrf2 Pathway. \emph{Front
Pharmacol,} 11,579178. doi: 10.3389/fphar.2020.579178.

Friedmann Angeli J. P., Schneider M., Proneth B., Tyurina Y. Y., Tyurin
V. A., Hammond V. J., Herbach N., Aichler M., Walch A., Eggenhofer E.,
Basavarajappa D., Radmark O., Kobayashi S., Seibt T., Beck H., Neff F.,
Esposito I., Wanke R., Forster H., Yefremova O., Heinrichmeyer M.,
Bornkamm G. W., Geissler E. K., Thomas S. B., Stockwell B. R., O'Donnell
V. B., Kagan V. E., Schick J. A. and Conrad M. (2014). Inactivation of
the ferroptosis regulator Gpx4 triggers acute renal failure in
mice.\emph{Nat Cell Biol,} 16,1180-1191. doi: 10.1038/ncb3064.

Herzog C., Yang C., Holmes A. and Kaushal G. P. (2012). zVAD-fmk
prevents cisplatin-induced cleavage of autophagy proteins but impairs
autophagic flux and worsens renal function. \emph{Am J Physiol Renal
Physiol,} 303,F1239-1250. doi: 10.1152/ajprenal.00659.2011.

Hoste E. A. J., Kellum J. A., Selby N. M., Zarbock A., Palevsky P. M.,
Bagshaw S. M., Goldstein S. L., Cerda J. and Chawla L. S. (2018). Global
epidemiology and outcomes of acute kidney injury. \emph{Nat Rev
Nephrol,} 14,607-625. doi: 10.1038/s41581-018-0052-0.

Hu Z., Zhang H., Yi B., Yang S., Liu J., Hu J., Wang J., Cao K. and
Zhang W. (2020). VDR activation attenuate cisplatin induced AKI by
inhibiting ferroptosis. \emph{Cell Death Dis,} 11,73. doi:
10.1038/s41419-020-2256-z.

Ikeda Y., Hamano H., Horinouchi Y., Miyamoto L., Hirayama T., Nagasawa
H., Tamaki T. and Tsuchiya K. (2021). Role of ferroptosis in
cisplatin-induced acute nephrotoxicity in mice. \emph{J Trace Elem Med
Biol,} 67,126798. doi: 10.1016/j.jtemb.2021.126798.

La Rosa P., Petrillo S., Turchi R., Berardinelli F., Schirinzi T., Vasco
G., Lettieri-Barbato D., Fiorenza M. T., Bertini E. S., Aquilano K. and
Piemonte F. (2021). The Nrf2 induction prevents ferroptosis in
Friedreich's Ataxia. \emph{Redox Biol,} 38,101791. doi:
10.1016/j.redox.2020.101791.

Li Y. Y., Lin Y. K., Liu X. H., Wang L., Yu M., Li D. J., Zhu Y. Z. and
Du M. R. (2020). Leonurine: From Gynecologic Medicine to Pleiotropic
Agent. \emph{Chin J Integr Med,} 26,152-160. doi:
10.1007/s11655-019-3453-0.

Linkermann A., Chen G., Dong G., Kunzendorf U., Krautwald S. and Dong Z.
(2014). Regulated cell death in AKI. \emph{J Am Soc
Nephrol,}25,2689-2701. doi: 10.1681/ASN.2014030262.

Maiorino M., Conrad M. and Ursini F. (2018). GPx4, Lipid Peroxidation,
and Cell Death: Discoveries, Rediscoveries, and Open
Issues.\emph{Antioxid Redox Signal,} 29,61-74. doi:
10.1089/ars.2017.7115.

Park E. and Chung S. W. (2019). ROS-mediated autophagy increases
intracellular iron levels and ferroptosis by ferritin and transferrin
receptor regulation. \emph{Cell Death Dis,} 10,822. doi:
10.1038/s41419-019-2064-5.

Seibt T. M., Proneth B. and Conrad M. (2019). Role of GPX4 in
ferroptosis and its pharmacological implication. \emph{Free Radic Biol
Med,} 133,144-152. doi: 10.1016/j.freeradbiomed.2018.09.014.

Shelton L. M., Park B. K. and Copple I. M. (2013). Role of Nrf2 in
protection against acute kidney injury. \emph{Kidney Int,} 84,1090-1095.
doi: 10.1038/ki.2013.248.

Stockwell B. R., Friedmann Angeli J. P., Bayir H., Bush A. I., Conrad
M., Dixon S. J., Fulda S., Gascon S., Hatzios S. K., Kagan V. E., Noel
K., Jiang X., Linkermann A., Murphy M. E., Overholtzer M., Oyagi A.,
Pagnussat G. C., Park J., Ran Q., Rosenfeld C. S., Salnikow K., Tang D.,
Torti F. M., Torti S. V., Toyokuni S., Woerpel K. A. and Zhang D. D.
(2017). Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism,
Redox Biology, and Disease. \emph{Cell,} 171,273-285. doi:
10.1016/j.cell.2017.09.021.

Stoyanovsky D. A., Tyurina Y. Y., Shrivastava I., Bahar I., Tyurin V.
A., Protchenko O., Jadhav S., Bolevich S. B., Kozlov A. V., Vladimirov
Y. A., Shvedova A. A., Philpott C. C., Bayir H. and Kagan V. E. (2019).
Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic
or random free radical reaction? \emph{Free Radic Biol Med,}133,153-161.
doi: 10.1016/j.freeradbiomed.2018.09.008.

Torti S. V. and Torti F. M. (2013). Iron and cancer: more ore to be
mined. \emph{Nat Rev Cancer,} 13,342-355. doi: 10.1038/nrc3495.

Tristao V. R., Goncalves P. F., Dalboni M. A., Batista M. C., Durao Mde
S., Jr. and Monte J. C. (2012). Nec-1 protects against nonapoptotic cell
death in cisplatin-induced kidney injury. \emph{Ren Fail,} 34,373-377.
doi: 10.3109/0886022X.2011.647343.

Wang L., Liu Y., Du T., Yang H., Lei L., Guo M., Ding H. F., Zhang J.,
Wang H., Chen X. and Yan C. (2020). ATF3 promotes erastin-induced
ferroptosis by suppressing system Xc(.). \emph{Cell Death
Differ,}27,662-675. doi: 10.1038/s41418-019-0380-z.

Xie Y. Z., Zhang X. J., Zhang C., Yang Y., He J. N. and Chen Y. X.
(2019). Protective effects of leonurine against ischemic stroke in mice
by activating nuclear factor erythroid 2-related factor 2
pathway.\emph{CNS Neurosci Ther,} 25,1006-1017. doi: 10.1111/cns.13146.

Xu D., Chen M., Ren X., Ren X. and Wu Y. (2014). Leonurine ameliorates
LPS-induced acute kidney injury via suppressing ROS-mediated NF-kappaB
signaling pathway. \emph{Fitoterapia,} 97,148-155. doi:
10.1016/j.fitote.2014.06.005.

Yang Y., Adebali O., Wu G., Selby C. P., Chiou Y. Y., Rashid N., Hu J.,
Hogenesch J. B. and Sancar A. (2018). Cisplatin-DNA adduct repair of
transcribed genes is controlled by two circadian programs in mouse
tissues. \emph{Proc Natl Acad Sci U S A,} 115,E4777-E4785. doi:
10.1073/pnas.1804493115.

\textbf{Figure legends:}

\textbf{Fig. 1 Nrf2 KO mice exhibited more severe renal injury and iron
metabolism disruption after cisplatin stimulation than wild-type
mice.}C57BL/6 mice with or without Nrf2 gene KO were treated by an
intraperitoneal injection of cisplatin (20 mg/kg) or vehicle. Three days
later, the mice were sacrificed, and their serum and renal tissues were
collected. The serum BUN (A) and SCr (B) levels were then detected. (C)
and (D) HE staining indicated renal tissue damage. (E) The serum
contents of iron ions were measured to assess the iron metabolism in the
mice. (F to H) The protein levels of Nrf2, NQO1, HO-1, FTH-1, FTL and
TFR in renal tissues were analyzed by Western blot. All experiments were
repeated 5 times, P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**}[?]
0.01 versus the control group, P\textsuperscript{\#} [?] 0.05 and
P\textsuperscript{\#\#} [?] 0.01 versus the WT CDDP group. WT, wild-type
C57BL/6 mice; KO, Nrf2 KO C57BL/6 mice.

\textbf{Fig. 2 Cisplatin-stimulated Nrf2 KO mice exhibited more severe
lipid peroxidation than wild-type mice.} Renal tissues were collected
for the measurement of (A) MDA and (B) GSH. (C) and (D) The protein
expression levels of GPX4 and xCT in renal tissues were analyzed by
Western blot. (E) and (F) The mRNA levels of GPX4 and SLC7A11 in renal
tissues were detected by real-time quantitative PCR. All experiments
were repeated 5 times, P\textsuperscript{*} [?] 0.05 and
P\textsuperscript{**} [?] 0.01 versus the control group,
P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#} [?] 0.01 versus
the WT CDDP group.

\textbf{Fig. 3 Leonurine protects HK-2 cells from ferroptosis.} HK-2
cells were treated with gradient concentrations of erastin (A) or RSL3
(B) for 24 h, and the cell survival rate was detected by the CCK-8
assay. (C) and (D) After 24 h of treatment with or without leonurine
(100 \selectlanguage{greek}µ\selectlanguage{english}M), HK-2 cells were stimulated with erastin (30 \selectlanguage{greek}µ\selectlanguage{english}M) or RSL3 (0.4
\selectlanguage{greek}µ\selectlanguage{english}M), and the cell survival rate was detected by the CCK-8 assay at 24 h.
(E) The cell iron content was then measured by calcein-AM staining and
measurement of the OD value. (F) The above mentioned cells were stained
with FerroOrange and detected by confocal microscopy. (G and H) The
protein levels of TFR, FTL and FTH-1 in HK-2 cells were analyzed by
Western blot. All experiments were repeated 5 times,
P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**} [?] 0.01 versus the
control group, P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#}[?]
0.01 versus the RSL3-treated group.

\textbf{Fig. 4 Leonurine alleviates lipid peroxidation induced by RSL3
in HK-2 cells.} (A) The ROS content in HK-2 cells was measured by
DCFH-DA. (B) Liperfluo staining, as observed by confocal microscopy, was
used to detect lipid peroxidation in HK-2 cells. HK-2 cells were
harvested, and the levels of (C) GSH and (D) MDA were detected. (E to G)
The protein levels of xCT, GPX4, Nrf2, NQO1 and HO-1 in HK-2 cells were
analyzed by Western blot. All experiments were repeated 5 times,
P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**} [?] 0.01 versus the
control group, P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#}[?]
0.01 versus the RSL3-treated group.

\textbf{Fig. 5 The inhibitory effect of leonurine on HK-2 cell
ferroptosis is dependent on Nrf2 gene expression.} (A and B) The
efficiency of Nrf2 knockdown in HK-2 cells was analyzed by Western blot.
(C) The CCK-8 assay was performed to determine the survival rate of
cells. The contents of (D) GSH and (E) MDA in HK-2 cells were detected
by appropriate kits. (F) The ROS level in HK-2 cells was measured by
DCFH-DA. (G) The iron content in HK-2 cells was detected by calcein-AM
staining. (H to J) The protein levels of FTH-1, TFR, xCT and GPX4 in
HK-2 cells were analyzed by Western blot. All experiments were repeated
5 times, P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**} [?] 0.01
versus the control group, P\textsuperscript{\#} [?] 0.05 and
P\textsuperscript{\#\#} [?] 0.01 versus the RSL3-treated group. NS, no
specificity.

\textbf{Fig. 6 Leonurine alleviates cisplatin-induced acute renal injury
in vivo.} The animals were grouped based on the treatment administered.
On the fourth day, the mice were sacrificed, and their blood and kidneys
were harvested. The levels of (A) BUN and (B) SCR in renal tissues were
detected. (C-D) HE staining was performed to determine the degree of
renal injury, and the renal injury indices were calculated according to
the standard method. (E-F) The protein levels of KIM1 and NGAL in renal
tissues were analyzed by Western blot. (G) F4/80 staining was performed
to determine the degree of renal tissue inflammation. All experiments
were repeated 5 times, P\textsuperscript{*} [?] 0.05 and
P\textsuperscript{**} [?] 0.01 versus the control group,
P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#} [?] 0.01 versus
the WT CDDP group.

\textbf{Fig. 7 Leonurine reverses the cisplatin-induced iron metabolism
disruption in the mouse kidney.} (A) DAB staining was performed to
detect the iron deposition in renal tissues. (B) The iron content in
renal tissues was detected by an iron assay kit. (C and D) The protein
levels of TFR, FTL and FTH-1 in renal tissues were analyzed by Western
blot. (E and F) The mRNA levels of FTH-1 and TFR in renal tissues were
measured by real-time quantitative PCR. All experiments were repeated 5
times, P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**} [?] 0.01
versus the control group, P\textsuperscript{\#} [?] 0.05 and
P\textsuperscript{\#\#} [?] 0.01 versus the WT CDDP group.

\textbf{Fig. 8 Leonurine alleviates cisplatin-induced lipid peroxidation
via the Nrf2 pathway.} The levels of (A) MDA, (B) GSH and (C) SOD in
renal tissues were detected by the appropriate assay kits. (D-F) The
protein levels of GPX4, xCT, Nrf2, NQO1 and HO-1 in renal tissues were
analyzed by Western blot. The mRNA levels of (G) GPX4 and (H) SLC7A11 in
renal tissues were measured by real-time quantitative PCR. (I) The
mitochondrial structure of renal cells was observed under an electron
microscope. All experiments were repeated 5 times, P\textsuperscript{*}[?]
0.05 and P\textsuperscript{**} [?] 0.01 versus the control group,
P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#} [?] 0.01 versus
the WT CDDP group.

\textbf{Fig. 9 Leonurine alleviates cisplatin-induced renal injury in
vivo, and this phenomenon is dependent on Nrf2 gene expression.} C57BL/6
mice with or without Nrf2 gene KO were subjected to the same treatments
as described for the abovementioned animal experiments. (A and B) The
levels of BUN and SCr in mouse kidneys were detected. (C and D) HE
staining was performed to detected changes in renal tissue damage based
on the evaluation of renal injury criteria. (E to G) The protein levels
of Nrf2, NQO1, HO-1, KIM1 and NGAL in renal tissues were analyzed by
Western blot. All experiments were repeated 5 times,
P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**} [?] 0.01 versus the
control group, P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#}[?]
0.01 versus the WT CDDP group. NS, no specificity.

\textbf{Fig. 10 The effect of leonurine on alleviating cisplatin-induced
ferroptosis in vivo depends on Nrf2 gene expression.} The levels of (A)
Fe\textsuperscript{2+}, (B) GSH and (C) MDA in renal tissues were
measured by the appropriate assay kits. (D-F) The protein levels of
FTH-1, TFR, GPX4 and xCT in renal tissues were analyzed by Western blot.
(G) The iron deposition in renal tissues was detected by DAB staining.
(H) Morphological changes in renal cell mitochondria were observed by
electron microscopy. All experiments were repeated 5 times,
P\textsuperscript{*} [?] 0.05 and P\textsuperscript{**} [?] 0.01 versus the
control group, P\textsuperscript{\#} [?] 0.05 and P\textsuperscript{\#\#}[?]
0.01 versus the WT CDDP group, NS, no specificity.

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Fig. 10

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