International Journal of
Molecular Sciences
Article
Sulforaphane Is Protective against Warm Ischemia/Reperfusion
Injury and Partial Hepatectomy in Rats
Richi Nakatake 1, *,† , Tetsuya Okuyama 1,† , Yuki Hashimoto 1 , Morihiko Ishizaki 1 , Hidesuke Yanagida 1 ,
Hiroaki Kitade 1 , Katsuhiko Yoshizawa 2 , Mikio Nishizawa 3 and Mitsugu Sekimoto 1
Citation: Nakatake, R.; Okuyama, T.;
Hashimoto, Y.; Ishizaki, M.; Yanagida,
H.; Kitade, H.; Yoshizawa, K.;
Nishizawa, M.; Sekimoto, M.
Sulforaphane Is Protective against
Warm Ischemia/Reperfusion Injury
and Partial Hepatectomy in Rats. Int.
J. Mol. Sci. 2024, 25, 579. https://
doi.org/10.3390/ijms25010579
Academic Editors: Joan Roselló-
Catafau, René Adam and Teresa
Carbonell Camós
Received: 30 November 2023
Revised: 27 December 2023
Accepted: 30 December 2023
Published: 1 January 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2024, 25, 579. https://doi.org/10.3390/ijms25010579
1 Department of Surgery, Kansai Medical University, Hirakata 573-1010, Osaka, Japan;
okuyamat@hirakata.kmu.ac.jp (T.O.); hashimoy@hirakata.kmu.ac.jp (Y.H.)
2 Department of Innovative Food Sciences, School of Food Sciences and Nutrition, Mukogawa Women’s
University, 6-46 Ikebiraki-cho, Nishinomiya 663-8558, Hyogo, Japan; yoshizak@mukogawa-u.ac.jp
3 Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi,
Kusatsu 525-8577, Shiga, Japan
* Correspondence: nakatakr@hirakata.kmu.ac.jp; Tel.: +81-72-804-2574
† These authors contributed equally to this work.
Abstract: Sulforaphane (SFN) has various beneficial effects on organ metabolism. However, whether
SFN affects inflammatory mediators induced by warm hepatic ischemia/reperfusion injury (HIRI)
is unclear. To investigate the hepatoprotective effects of SFN using an in vivo model of HIRI and
partial hepatectomy (HIRI + PH), rats were subjected to 15 min of hepatic ischemia with blood inflow
occlusion, followed by 70% hepatectomy and release of the inflow occlusion. SFN (5 mg/kg) or
saline was randomly injected intraperitoneally 1 and 24 h before ischemia. Alternatively, ischemia
was prolonged for 30 min to evaluate the effect on mortality. The influence of SFN on the associated
signaling pathways was analyzed using the interleukin 1β (IL-1β)-treated primary cultured rat
hepatocytes. In the HIRI + PH-treated rats, SFN reduced serum liver enzyme activities and the
frequency of pathological liver injury, such as apoptosis and neutrophil infiltration. SFN suppressed
tumor necrosis factor-alpha (TNF-α) mRNA expression and inhibited nuclear factor-kappa B (NF-κB)
activation by HIRI + PH. Mortality was significantly reduced by SFN. In IL-1β-treated hepatocytes,
SFN suppressed the expression of inflammatory cytokines and NF-κB activation. Taken together, SFN
may have hepatoprotective effects in HIRI + PH in part by inhibiting the induction of inflammatory
mediators, such as TNF-α, via the suppression of NF-κB in hepatocytes.
Keywords: sulforaphane; ischemia/reperfusion injury; partial hepatectomy; primary cultured
hepatocytes; nuclear factor-kappa B
1. Introduction
Hepatic ischemia/reperfusion injury (HIRI) is common in a variety of clinical situa-
tions, including hepatobiliary surgery and liver transplantation. The Pringle maneuver
(PM) is a non-selective inflow occlusion technique that is widely used for blood loss control
during continuous or intermittent portal triad clamping [1,2]. Warm HIRI occurs when hep-
atic inflow is transiently clamped and is a major risk factor for postoperative complications
in the setting of massive hepatic resection [3]. The main molecular mechanisms of HIRI,
including apoptosis, oxidative stress, neutrophil infiltration, and release of inflammatory
cytokines, remain to be elucidated [4,5]. When HIRI is induced, the nuclear factor-kappa B
(NF-κB) pathway is activated, leading to the development of inflammation and exacerba-
tion of liver tissue damage [6,7]. NF-κB is an important transcription factor that regulates
the synthesis of various cytokines and chemokines in immunologic stimuli [8,9]. In the HIRI
process, apoptosis of hepatocytes is the core of liver damage caused by various pathways,
including the NF-κB pathway [10]. Appropriate control of inflammatory responses during
the perioperative period is crucial to prevent organ damage.
https://www.mdpi.com/journal/ijms
 various pathways, including the NF-κB pathway [10]. Appropriate control of inflamma-
tory responses during the perioperative period is crucial to prevent organ damage.
Int. J. Mol. Sci. 2024, 25, 579 In recent years, interest in the nutritional and medicinal value of sulforaphane (SFN; 2 of 15
Figure 1A) has grown. SFN, which has been identified in broccoli [11], is one of the most
fascinating phytochemicals known because it protects cells under aerobic conditions from
toxicIncarcinogens,
recent years, electrophiles,
interest in theand oxidants that
nutritional and damage
medicinal DNAvaluethrough the induction
of sulforaphane (SFN; of
the network of phase 2 detoxifying and antioxidant enzymes and
Figure 1A) has grown. SFN, which has been identified in broccoli [11], is one of the most the suppression of in-
flammatoryphytochemicals
fascinating responses [12,13]. knownThebecause
principal cytoprotective
it protects properties
cells under aerobicofconditions
SFN are medi- from
ated by
toxic upregulation
carcinogens, of the transcription
electrophiles, factorthat
and oxidants NF-E2-related
damage DNA factor 2 (Nrf2)
through the and by anti-
induction
inflammatory
of the networkmechanisms, such as the and
of phase 2 detoxifying inhibition of the NF-κB
antioxidant enzymes pathway
and the [14,15]. However,
suppression of
there are few reports
inflammatory responses on the efficacy
[12,13]. Theofprincipal
SFN in improving hepatic
cytoprotective abnormalities
properties of SFN in humans
are me-
to date.by
diated Many animal studies
upregulation of the have been performed
transcription to demonstrate
factor NF-E2-related the hepatoprotective
factor 2 (Nrf2) and by
effects of SFN against hepatotoxic chemicals, such as cisplatin
anti-inflammatory mechanisms, such as the inhibition of the NF-κB pathway [14,15]. [16], microcystin [17], car-
How-
bon tetrachloride
ever, there are few[18], acetaminophen
reports on the efficacy[19],ofand
SFND-galactosamine
in improving hepatic and lipopolysaccharide
abnormalities in
(LPS) combination
humans to date. Many [20].animal
Furthermore, SFN been
studies have has been suggested
performed to be potentthe
to demonstrate in preventing
hepatopro-
lifestyleeffects
tective diseases caused
of SFN by excess
against alcoholchemicals,
hepatotoxic consumption such[21] and high-energy
as cisplatin diets [22,23].
[16], microcystin [17],
These hepatoprotective
carbon tetrachloride [18], effects have been
acetaminophen suggested
[19], to occur because
and D-galactosamine of the superior in-
and lipopolysaccharide
(LPS)
ductioncombination
capacity of[20].SFNFurthermore,
for phase II SFN has been suggested
cytoprotective to be as
proteins, such potent in preventing
detoxification and
lifestyle
antioxidantdiseases caused
enzymes, bythe
via excess alcoholofconsumption
activation [21] and
the transcription high-energy
factor—Nrf2. diets [22,23].
Regarding the
These hepatoprotective
anti-inflammatory effectsofhave
properties SFN,been
SFN suggested
inhibits thetoexpression
occur because of the superior
of inflammatory factorsin-
duction capacity factor-alpha,
(tumor necrosis of SFN for phase II cytoprotective
TNF-α; interleukin 1β,proteins,
IL-1β; and such as detoxification
IL-6) and inflammatory and
antioxidant enzymes,
mediator enzymes via the activation
(inducible nitric oxideofsynthase
the transcription factor—Nrf2.
and cyclooxygenase Regarding
2) and the activa-the
anti-inflammatory properties ofmouse
tion of NF-κB in LPS-treated SFN, SFN
RAWinhibits the expression[24]
264.7 macrophages of inflammatory
and goat mammary factors
(tumor
epithelialnecrosis factor-alpha,
cells [25]. The inhibitionTNF-α; interleukin
of TNF-α 1β, IL-1β; and
and inflammatory IL-6) and
mediators inflammatory
(nitric oxide and
mediator
prostaglandin E2) production has been proposed as a useful approach for theand
enzymes (inducible nitric oxide synthase and cyclooxygenase 2) the acti-
treatment of
vation of NF-κB in LPS-treated mouse RAW 264.7 macrophages
various inflammatory diseases and a potential chemoprevention strategy [25,26]. [24] and goat mammary
epithelial cells
In this [25]. the
study, Thehepatoprotective
inhibition of TNF-α effectsandofinflammatory mediators
SFN were examined (nitric
using twooxide and
models:
prostaglandin
an in vivo warm E2 )HIRI
production has been
and a partial proposed model
hepatectomy as a useful
(HIRIapproach
+ PH) and forantheintreatment
vitro model of
various inflammatory diseases and a potential
of liver injury using primary rat hepatocyte cultures. chemoprevention strategy [25,26].

Figure 1.
Figure 1. Study
Studydesign.
design.(A)(A)
Molecular
Molecularstructure of of
structure sulforaphane (SFN).
sulforaphane In In
(SFN). the the
hepatic ische-
hepatic is-
mia/reperfusion injury and partial hepatectomy (HIRI + PH) group, the rats underwent
chemia/reperfusion injury and partial hepatectomy (HIRI + PH) group, the rats underwent 70% 70%
hepatectomy following HIRI after vehicle administration preoperatively. In the HIRI + PH and SFN
groups, the rats were administered SFN (5 mg/kg) 1 and 24 h before HIRI + PH treatment. (B) Blood
and liver samples were collected at the indicated time after HIRI + PH treatment. (C) Survival was
monitored for 168 h after HIRI + PH treatment.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 15
Int. J. Mol. Sci. 2024, 25, 579 3 of 15

hepatectomy following HIRI after vehicle administration preoperatively. In the HIRI + PH and SFN
groups,In the
thisrats werethe
study, administered SFN (5 mg/kg)
hepatoprotective 1 and
effects 24 h were
of SFN beforeexamined
HIRI + PH treatment.
using two(B) Blood
models:
and liver samples were collected at the indicated time after HIRI + PH treatment.
an in vivo warm HIRI and a partial hepatectomy model (HIRI + PH) and an in vitro model (C) Survival was
monitored for 168 h after HIRI + PH treatment.
of liver injury using primary rat hepatocyte cultures.

2.
2. Results
Results
2.1.
2.1. SFN
SFN Decreases
Decreases Pathological
Pathological Changes
Changes in
in Liver
Liver Injury
Injury
The livers
The livers of of HIRI
HIRI ++ PH-treated
PH-treated rats
rats were
were examined
examinedhistologically
histologicallyafter
after33hh andand 66 hh
(Figure 1B).
(Figure 1B). Figure
Figure 2A–E2A–E present
present representative
representative images images ofof the
the hematoxylin
hematoxylin and and eosin
eosin
(H&E)-stained sections.
(H&E)-stained sections. Liver
Liverinjury
injury was
was characterized
characterized by by hemorrhagic
hemorrhagic manifestations,
manifestations,
focal necrosis,
focal necrosis, and and inflammatory
inflammatory cellcell infiltration
infiltration (Figure
(Figure 2B,D)
2B,D) compared
compared to to the
the control
control
(Figure 2A). Ballooning degeneration of the hepatocytes was also observed.
(Figure 2A). Ballooning degeneration of the hepatocytes was also observed. SFN reduced SFN reduced
the frequency
the frequency and and extent
extent of
of these
these pathological
pathological changes
changes (Figure
(Figure 2C,E).
2C,E). Damage
Damage was was graded
graded
using the
using the Suzuki
Suzuki scorescore in
in Figure
Figure 2F;
2F; SFN
SFN significantly
significantly reduced
reduced the
the grade
grade of
of liver
liver injury
injury by
by
0.25-fold at
0.25-fold at 33 hh (p
(p == 0.044)
0.044) and
and by
by 0.38-fold
0.38-fold atat 66 hh (p
(p <<0.001),
0.001),respectively,
respectively, compared
compared with with
HIRI ++PH-only
HIRI PH-onlytreatment.
treatment.

Figure
Figure 2.2. Effects
Effectsofofsulforaphane
sulforaphane(SFN)(SFN) onon
hepatic
hepatic pathological
pathologicalchanges
changesin the livers
in the of hepatic
livers of hepaticis-
chemia/reperfusion injury and partial hepatectomy (HIRI + PH)−treated rats. Representative liver
ischemia/reperfusion injury and partial hepatectomy (HIRI + PH)−treated rats. Representative
histology of HIRI + PH treatment. Liver sections of (A) naïve rats and rats treated with (B,D) HIRI
liver histology of HIRI + PH treatment. Liver sections of (A) naïve rats and rats treated with
+ PH and (C,E) HIRI + PH and SFN were stained with hematoxylin and eosin (H&E) (magnification
(B,D) HIRI
×200; bar + PH
= 100 andNote
µm). (C,E)the
HIRI + PH
areas ofand SFN
focal were stained
necrosis with
(arrows), hematoxylin cell
inflammatory and infiltration
eosin (H&E)(white(mag-
nification × 200; bar = 100 µm). Note the areas of focal necrosis (arrows), inflammatory
asterisk), massive hemorrhage (white arrowheads), and ballooning degeneration of the hepatocytes cell infiltration
(whitearrowheads)
(black asterisk), massive
in HIRIhemorrhage
+ PH rats. (white arrowheads),
(F) Suzuki and
histological ballooning
grading in thedegeneration of thein
livers. The values hepa-
the
tocytes
bar (black
graphs arrowheads)
represent in HIRI
the mean + PH rats.
± standard (F) Suzuki
deviation (SD;histological
n = 3–4 ratsgrading in the livers.
per time-point The values
per group). *p
<in0.05
the and ** p < 0.01
bar graphs versusthe
represent HIRI
mean+ PH−only rats.deviation (SD; n = 3–4 rats per time-point per group).
± standard
* p < 0.05 and ** p < 0.01 versus HIRI + PH−only rats.
In Figure 3A–E, representative images of liver sections stained by terminal deoxynu-
In Figure
cleotidyl 3A–E, representative
transferase-mediated images ofnick-end
deoxyuridine liver sections stained
labeling by terminal
(TUNEL) deoxynu-
are shown. HIRI
+cleotidyl transferase-mediated
PH increased apoptosis in deoxyuridine nick-end
the hepatocytes at 6 hlabeling
(Figure(TUNEL) are shown.
3D), whereas SFN HIRI + PH
markedly
 Int. J.J. Mol.
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decreasedapoptosis
increased apoptosisinbythe
0.39-fold compared
hepatocytes at 6 hto HIRI +3D),
(Figure PH-only treatment
whereas (p = 0.002;
SFN markedly Figure
decreased
3E,F).
apoptosis by 0.39-fold compared to HIRI + PH-only treatment (p = 0.002; Figure 3E,F).

Figure 3.
Figure 3. Effects
Effects ofof sulforaphane
sulforaphane (SFN) on apoptosis
(SFN) on apoptosis in in the
the livers
livers of
of hepatic
hepatic ischemia/reperfusion
ischemia/reperfusion
injury and partial hepatectomy (HIRI + PH)−treated rats. Representative liver histology of HIRI +
injury and partial hepatectomy (HIRI + PH)−treated rats. Representative liver histology of
PH treatment. Liver sections of (A) naïve rats and rats treated with (B,D) HIRI + PH and (C,E) HIRI
HIRI + PH treatment. Liver sections of (A) naïve rats and rats treated with (B,D) HIRI + PH and
+ PH and SFN were stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine
(C,E) HIRI
nick-end + PH and
labeling SFN were
(TUNEL) stained with
(magnification ×200;terminal
bar = 100deoxynucleotidyl
µm). Note the brown transferase-mediated de-
nuclei in the positive
oxyuridine
cells (blacknick-end labeling
arrowheads). (E)(TUNEL) (magnification
The numbers ×200; bar = cells
of TUNEL−positive 100 µm). Note themillimeter
per square brown nuclei
werein
the positive
counted. Thecells (black
values arrowheads).
in the bar graphs(F) The numbers
represent of TUNEL
the mean −positive
± standard cells per
deviation (SD;square millimeter
n = 3–4 rats per
time-point
were perThe
counted. group).
values** pin<the
0.01bar
versus HIRI
graphs + PH−only
represent rats. ± standard deviation (SD; n = 3–4 rats
the mean
per time-point per group). ** p < 0.01 versus HIRI + PH−only rats.
Next, the effects of HIRI + PH treatment on neutrophil infiltration in the liver were
Next, by
evaluated theimmunostaining
effects of HIRI +forPHmyeloperoxidase
treatment on neutrophil infiltration
(MPO) (Figure in the
4). HIRI liver
+ PH were
mark-
evaluated by immunostaining
edly increased for myeloperoxidase
neutrophil infiltration (MPO)
in the liver at (Figure 4A,B,D),
6 h (Figure 4). HIRI +whereas
PH markedly
SFN
increased
markedly neutrophil infiltrationinfiltration
decreased neutrophil in the liver(by
at 60.40-fold,
h (Figurep4A,B,D), whereas
= 0.002; Figure SFN
4F). markedly
A summary
decreased neutrophil infiltration (by 0.40-fold,
of the results is provided in Supplementary Table S2. p = 0.002; Figure 4F). A summary of the
results is provided in Supplementary Table S2.

2.2. SFN Attenuates Cell Damage in the Livers of HIRI + PH-Treated Rats
HIRI + PH resulted in a rapid increase in aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) serum levels after reperfusion. In contrast, AST (6 h) and ALT (3 h)
serum levels were reduced by 0.47-fold (p = 0.018) and 0.24-fold (p = 0.007), respectively, by
treatment with SFN compared to HIRI + PH-only treatment (Figure 5).
 Int.J.J.Mol.
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Figure 4. Effects of sulforaphane (SFN) on neutrophil infiltration in the livers of hepatic ische-
mia/reperfusion injury and partial hepatectomy (HIRI + PH)−treated rats. Representative liver his-
tology of HIRI + PH treatment. The liver sections of (A) naïve rats and rats treated with (B,D) HIRI
+ PH and (C,E) HIRI + PH and SFN were stained with anti-myeloperoxidase (MPO) antibody (mag-
nification ×200; bar = 100 µm). Note the brown staining in the positive cells (black arrowheads). (F)
The numbers of MPO−positive cells per square millimeter were counted. The values in the bar graph
Figure 4.
represent
Figure 4. Effects
the ofofsulforaphane
mean
Effects ± standard (SFN)
deviation
sulforaphane on(SD;
(SFN) neutrophil
on infiltration
n = 3–4 rats
neutrophil in the
in livers
per time-point
infiltration of hepatic
perlivers
the group). p <ische-
of *hepatic0.05is-
versus
mia/reperfusion
HIRI injury and partial hepatectomy (HIRI + PH)−treated rats. Representative
+ PH−only rats.injury and partial hepatectomy (HIRI + PH)−treated rats. Representative liver his-
chemia/reperfusion
tology of HIRI + PH treatment. The liver sections of (A) naïve rats and rats treated with (B,D) HIRI
liver histology of HIRI + PH treatment. The liver sections of (A) naïve rats and rats treated with (B,D)
+ PH and (C,E) HIRI + PH and SFN were stained with anti-myeloperoxidase (MPO) antibody (mag-
2.2.
HIRI SFN
nification Attenuates
+ PH×200;
and bar
(C,E) Cell
HIRI
= 100 +Damage
µm). PH andthe
Note inbrown
SFNthewere
Livers of HIRI
stained
staining the+anti-myeloperoxidase
with
in PH-Treated
positive Rats arrowheads).
cells (black (MPO) antibody(F)
(magnification
HIRI +ofPH
The numbers ×MPO−positive
200; bar = 100incells
resulted µm). Note
a rapid
per the brown
increase
square staining
in
millimeter wereincounted.
aspartate the aminotransferase
positive cells (black
The values in thearrowheads).
(AST)
bar graphand ala-
(F) The
representnumbers
the meanof MPO
± −
nine aminotransferase (ALT) serum levels after reperfusion. In contrast, AST (6bar
positive
standard cells
deviation per
(SD; square
n = 3–4 millimeter
rats per were
time-point counted.
per The
group). *values
p < in
0.05 the
versus h) and
HIRI +represent
graph PH−only the
rats.mean ± standard deviation (SD; n = 3–4 rats per time-point per group). * p < 0.05
ALT (3 h) serum levels were reduced by 0.47-fold (p = 0.018) and 0.24-fold (p = 0.007),
versus HIRI + PH−only rats.
respectively, by treatment
2.2. SFN Attenuates with
Cell Damage SFN
in the compared
Livers of HIRI to HIRI + PH-only
+ PH-Treated Rats treatment (Figure 5).
HIRI + PH resulted in a rapid increase in aspartate aminotransferase (AST) and ala-
A 3000
AST
nine aminotransferase
B ALT
(ALT) serum levels after reperfusion. In contrast, AST (6 h) and
†† 600 ††
ALT (3 h) serum levels were reduced by 0.47-fold (p = 0.018) and 0.24-fold (p = 0.007),
2000 by treatment
respectively, †† with SFN compared to HIRI††+ PH-only treatment (Figure 5).
400
U/L

U/L

A 3000 AST * B ALT

1000 600200 ††
†† **
††
20000 †† 400 0
HIRI + PH - + + + * + HIRI + PH - + + + +
U/L

U/L

1000
SFN - - + - + 200 SFN - **
- + - +
Time0(h) 0 3 3 6 6 Time
0
(h) 0 3 3 6 6
HIRI + PH - + + + +
Figure 5.5.Effects of sulforaphane
Effects of sulforaphane
HIRI + PH - + + + +
(SFN)(SFN)
on liver
ondamage and functions
liver damage in hepaticinischemia/reperfu-
and functions hepatic is-
sion SFN
injury and - -
partial+ - +
hepatectomy (HIRISFN+ - -
PH)−treated+ - +
chemia/reperfusion injury and partial hepatectomy (HIRI + PH)−treated rats. The serum (A)
rats. The serum levels of aspartate
levels of
(A) Time
aspartate(h)aminotransferase
aminotransferase 0 3 3 6 (AST)
(AST) and 6
(B) Time
alanine
and (h) 0 aminotransferase
3 3 6 6 (ALT) were measured at the
aminotransferase
(B) alanine (ALT) were measured at the indicated
time aftertime
indicated HIRI
after+ HIRI
PH +treatment. TheThe
PH treatment. values
valuesininthe
the bar graphsrepresent
bar graphs represent the mean
the mean ± standard
± standard
Figure 5. Effects of sulforaphane (SFN) on liver damage and functions in hepatic ischemia/reperfu-
deviation andnpartial
sion injury(SD; = 3–4 rats per time-point
hepatectomy (HIRI per p < 0.01
group). †† rats.
+ PH)−treated Theversus
serumnaïve
levelsrats. * paspartate
of (A) < 0.05 and
** p < 0.01 versus HIRI + PH − only rats.
aminotransferase (AST) and (B) alanine aminotransferase (ALT) were measured at the indicated
time after HIRI + PH treatment. The values in the bar graphs represent the mean ± standard
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 15

Int. J. Mol. Sci. 2024, 25, 579 deviation (SD; n = 3–4 rats per time-point per group). †† p < 0.01 versus naïve rats. * p < 0.05 and **
6 of 15
p < 0.01 versus HIRI + PH−only rats.

2.3.
2.3. SFN
SFN Affects
Affects Inflammation-,
Inflammation-, Apoptosis-,
Apoptosis-, and
and Liver
Liver Generation-Related
Generation-Related Gene Gene Expression
Expression in
in
HIRI ++ PH-Treated
PH-Treated Rats
The HIRI ++ PHPH and
and SFN
SFN groups
groups had
had significantly lower mRNA expression of TNF-α
(3 h, 0.52-fold, p = = 0.012,
0.012, Figure
Figure 6A)
6A) and
and C-X-C
C-X-C motif
motif chemokine
chemokine ligandligand 11 (CXCL1;
(CXCL1; 66 h,
h,
0.27-fold,
0.27-fold, pp==0.007,
0.007,Figure
Figure6B) genes
6B) genes than thethe
than expression
expressionin the HIRI
in the + PH+group.
HIRI The HIRI
PH group. The
+HIRI
PH + and
PHSFN
andgroup had significantly
SFN group higherhigher
had significantly mRNA expression
mRNA in theinIL-10
expression gene gene
the IL-10 than
the
thanexpression in the
the expression in HIRI + PH
the HIRI group
+ PH group(6 h, 2.21-fold,
(6 h, 2.21-fold,p =p =
0.013,
0.013,Figure
Figure6C).
6C).Moreover,
Moreover,
mRNA expression of myeloid cell leukemia leukemia 1 (MCL1),
(MCL1), a member
member of the Bcl-2 Bcl-2 family, was
significantly enhanced by SFN (3 h, 1.84-fold, p = 0.032; 0.032; 66 h, 1.87-fold, pp ==0.005,
h, 1.87-fold, 0.005,Figure
Figure6D).
6D).
significantly increased
SFN significantly increased hepatocyte
hepatocyte growth
growth factor
factor (HGF)
(HGF) mRNAmRNA expression
expression atat 6 h
(2.67-fold, p < 0.001, Figure 6E).

A TNF-α B CXCL1 C IL-10 *

1.5 †† 3 †† 2

expression
expression

expression

Relative
Relative

Relative

1 † 2 1.5
* 1
0.5 1 ** 0.5
0 0 0
HIRI + PH - + + + + HIRI + PH - + + + + HIRI + PH - + + + +
SFN - - + - + SFN - - + - + SFN - - + - +
Time (h) 0 3 3 6 6 Time (h) 0 3 3 6 6 Time (h) 0 3 3 6 6

D MCL1 E HGF
4 1.5
expression

expression

** †† **
Relative

Relative

* 1
2
0.5
0 0
HIRI + PH - + + + + HIRI + PH - + + + +
SFN - - + - + SFN - - + - +
Time (h) 0 3 3 6 6 Time (h) 0 3 3 6 6
Figure
Figure 6.
6. Effects
Effects of
of sulforaphane
sulforaphane (SFN)
(SFN) onon the
the mRNA
mRNA expression
expression of
of inflammatory
inflammatory and
and apoptosis-
apoptosis-
and liver regeneration-related genes in the livers of hepatic ischemia/reperfusion injury and partial
and liver regeneration-related genes in the livers of hepatic ischemia/reperfusion injury and partial
hepatectomy (HIRI + PH)−treated rats. (A) Tumor necrosis factor-alpha (TNF−α), (B) C−X−C motif
hepatectomy (HIRI + PH)−treated rats. (A) Tumor necrosis factor-alpha (TNF−α), (B) C−X−C motif
chemokine ligand 1 (CXCL1), (C) interleukin 10 (IL−10), (D) myeloid cell leukemia 1 (MCL1), and
chemokine
(E) ligand
hepatocyte 1 (CXCL1),
growth (C) interleukin
factor (HGF) 10 (IL−10),
mRNA expression was(D) myeloidbycell
analyzed leukemia
RT−PCR. 1 (MCL1),
The values inand
the
(E) hepatocyte
bar growththe
graphs represent factor
mean (HGF) mRNAdeviation
± standard expression
(SD;was
n =analyzed
3–4 rats by
perRT −PCR. The
time−point values
per in the
group). †p
<bar graphs
0.05 represent
and †† the mean
p < 0.01 versus ± standard
naïve rats. * p < deviation
0.05 and **(SD; n = 3–4
p < 0.01 ratsHIRI
versus per time −pointrats.
+ PH−only per group).
† p < 0.05 and †† p < 0.01 versus naïve rats. * p < 0.05 and ** p < 0.01 versus HIRI + PH−only rats.
2.4. SFN Decreases NF-κB Activation in HIRI + PH-Treated Rats
2.4. SFN Decreases NF-κB Activation in HIRI + PH-Treated Rats
NF-κB activation is an upstream factor of inflammatory cytokine transcription. The
NF-κB activation is an upstream factor of inflammatory cytokine transcription. The
electrophoretic mobility shift assay (EMSA) using the nuclear extract of the livers indi-
electrophoretic mobility shift assay (EMSA) using the nuclear extract of the livers indicated
cated that SFN suppressed the activation of NF-κB by HIRI + PH by 0.73-fold at 6 h with
that SFN suppressed the activation of NF-κB by HIRI + PH by 0.73-fold at 6 h with a p-value
a p-value of 0.024 (Figure 7).
of 0.024 (Figure 7).
Int. J. J.Mol.
Int. Mol.Sci. 2024,25,
Sci.2024, 25,579
x FOR PEER REVIEW 7 of 157 of 15

Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 15

Figure 7.
Figure 7. Effect
Effectof
ofsulforaphane
sulforaphane(SFN)(SFN)treatment
treatment onon
nuclear factor-kappa
nuclear factor-kappaB (NF−κB)
B (NF−activation in in
κB) activation
the livers
the livers of hepatic
of hepatic ischemia/reperfusion
ischemia/reperfusion injury and partial
injuryonand hepatectomy
partial hepatectomy(HIRI + PH)−treated
(HIRI + PH) rats.
−treated
Figure 7. Effect of sulforaphane (SFN) treatment nuclear factor-kappa B (NF−κB) activation in rats.
Binding capacities to the DNA probe harboring κB site in nuclear extracts of rat liver were analyzed
the liverscapacities
Binding of hepatic to
ischemia/reperfusion injury and
the DNA probe harboring κBpartial
site in hepatectomy (HIRI
nuclear extracts of +rat
PH)−treated
liver were rats.
analyzed
by an electrophoretic mobility shift assay (EMSA). The mean intensity of the bands from rats treated
Binding
by anHIRIcapacities to the
electrophoretic DNA probe
mobility shift harboring
assay*(EMSA).κB site in nuclear
The mean extracts of
of rat
theliver were analyzed
with + PH only was set to 1.0 (right). p < 0.05 versus HIRIintensity
+ PH−only rats.bands from rats treated
by an electrophoretic mobility shift assay (EMSA). The mean intensity of the bands from rats treated
with HIRI + PH only was set to 1.0 (right). * p < 0.05 versus HIRI + PH−only rats.
with HIRI + PH only was set to 1.0 (right). * p < 0.05 versus HIRI + PH−only rats.
2.5. SFN Decreases Mortality in Prolonged HIRI + PH-Treated Rats
2.5. SFN Decreases Mortality
TheDecreases
protective effect ofin in Prolonged
SFN in a lethal HIRI
model+ PH-Treated
with prolongedRats ischemic duration and
2.5. SFN Mortality Prolonged HIRI + PH-Treated Rats
PH wasThe protective
The investigated effect of
(Figure
protective effect SFN in
1C).inThe
of SFN a lethal
survival
a lethal model
ratewith
model with prolonged
wasprolonged ischemic
only 0.524ischemic
after HIRI duration
+ PH
duration and and
treat-
PH
ment.
PH was investigated
However,
was (Figure 1C).
1C). The
the administration
investigated (Figure The survival
ofsurvival rate was
was only
SFN significantly
rate only 0.524long-term
reduced
0.524 after HIRI
after HIRIischemia-in-
++ PH
PH treat-
treatment.
However,
duced the administration
mortality, with a survival of SFN
rate of significantly
0.927 (Figure reduced
8; p = long-term
0.0402).
ment. However, the administration of SFN significantly reduced long-term ischemia-in- ischemia-induced
mortality,
duced with awith
mortality, survival rate ofrate
a survival 0.927 (Figure
of 0.927 8; p =8;0.0402).
(Figure p = 0.0402).
1 HIRI+PH and SFN (0.927) *
1 HIRI+PH and SFN (0.927) *
0.8
0.8
0.6
0.6
0.4 HIRI+PH (0.524)
0.4
0.2 HIRI+PH (0.524)
0.20
0 0 24 48 72 96 120 144 168
0 Time
24 48after 96 120 144(h)
72Treatment 168
Figure 8.Time after
Effects Treatment(SFN)
of sulforaphane (h) on hepatic ischemia/reperfusion injury and partial hepatec-
tomy (HIRI + PH)−treated rat survival with a longer ischemic duration. Kaplan−Meier survival
Figure 8.8.Effects
Effectsofofsulforaphane
sulforaphane (SFN) ononhepatic ischemia/reperfusion injuryinjury
and partial hepatec-
Figure
curves of the cumulative survival of(SFN) hepatic
the following ischemia/reperfusion
treatment groups: HIRI + PH−only and partial
(filled hepate-
circles;
tomy (HIRI + PH)−treated rat survival with a longer ischemic duration. Kaplan−Meier survival
ctomy
n = 14) (HIRI
and HIRI + PH)
+ PH−treated
and SFN rat(open
survival withn a= longer
circles; 12). Theischemic Kaplanat−168
duration.survival
values represent Meier survival
h after
curves of the cumulative survival of the following treatment groups: HIRI + PH−only (filled circles;
HIRI + PH
curves of treatment.
the * p < survival
cumulative 0.05 versus
of HIRI
the + PH-only
following rats.
treatment groups: HIRI + PH −
n = 14) and HIRI + PH and SFN (open circles; n = 12). The values represent survival at 168 h after
only (filled circles;
n = 14) and HIRI + PH and SFN (open circles;
HIRI + PH treatment. * p < 0.05 versus HIRI + PH-only rats.n = 12). The values represent survival at 168 h after
2.6. SFN
HIRI + PH Inhibits Inflammation-Related
treatment. Gene +Expression
* p < 0.05 versus HIRI PH-only rats.and Blocks NF-κB Activation in
Cultured
2.6. Hepatocytes
SFN Inhibits Inflammation-Related Gene Expression and Blocks NF-κB Activation in
2.6. SFN
We Inhibitsassessed
further Inflammation-Related
Cultured Hepatocytes
Gene
the underlying Expression by
mechanisms andwhich
BlocksSFN
NF-κB Activation
suppresses theinin-
Cultured Hepatocytes
flammatory geneassessed
We further transcription using rat primary
the underlying cultured
mechanisms hepatocytes
by which [27,28]. First,
SFN suppresses we
the in-
found We further
that SFN assessed the underlying
dose–dependently mechanisms
suppressed nitric byproduction
oxide which SFN(Supplementary
suppresses the in-
flammatory gene transcription using rat primary cultured hepatocytes [27,28]. First, we
flammatory gene transcription using rat primary cultured hepatocytes [27,28]. First, we
found that SFN dose–dependently suppressed nitric oxide production (Supplementary
found that SFN dose–dependently suppressed nitric oxide production (Supplementary
Figure S2) without showing cytotoxicity up to 10 µM in IL-1β-stimulated hepatocytes. The
Int. J. Mol. Sci. 2024, 25,Int.
579J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of8 15
of 15

Figure S2) without

half-maximal inhibitory showing cytotoxicity
concentration (IC50 ) valueupwas
to 10calculated
µM in IL-1β-stimulated
to be 1.20 µM.hepatocytes.
Since theThe
half-maximal inhibitory concentration (IC 50) value was calculated to be 1.20 µM. Since the
suppression of nitric oxide production is closely related to the suppression of inflammatory
suppression of nitric oxide production is closely related to the suppression of inflamma-
gene mRNA expression, not only by a variety of drugs but also by several natural phy-
tory gene mRNA expression, not only by a variety of drugs but also by several natural
tochemicals [29–32], SFN was
phytochemicals usedSFN
[29–32], at 10 µM
was foratfurther
used 10 µM foranalysis. SFN decreased
further analysis. TNF-α
SFN decreased TNF-
(0.02–0.05-fold, p IL-1β (0.06–0.19-fold, p < 0.001), IL-6 (0.07–0.32-fold, p
α (0.02–0.05-fold, p < 0.001), IL-1β (0.06–0.19-fold, p < 0.001), IL-6 (0.07–0.32-fold, 0.001),
< 0.001), < p < 0.001),
and CXCL1 mRNA and CXCL1expression (0.29–0.65-fold,
mRNA expression p < 0.005)
(0.29–0.65-fold, p < in IL-1β-treated
0.005) rat-cultured
in IL-1β-treated rat-cultured
hepatocytes (Figure 9A–D).
hepatocytes IL-1β9A–D).
(Figure stimulated NF-κB activation
IL-1β stimulated [32], whereas
NF-κB activation SFN inhibited
[32], whereas SFN inhib-
ited (Figure
NF-κB activation NF-κB activation
9E). (Figure 9E).

Figure
Figure 9. Effects 9. Effects of sulforaphane
of sulforaphane (SFN) on the(SFN) on the inflammatory
inflammatory gene expression
gene expression and nuclear
and nuclear factor-
factor-
kappa B (NF-κB) activation in interleukin 1β (IL−1β)−stimulated hepatocytes. Cells were treated
kappa B (NF-κB) activation in interleukin 1β (IL−1β)−stimulated hepatocytes. Cells were treated
with IL-1β (1 nM) in the presence or absence of SFN (10 µM) for the indicated time. The effect of
with IL-1β (1 nM)
SFNin the presence
treatment on the or absenceofof(A)
expression SFN (10necrosis
tumor µM) forfactor-alpha
the indicated time.(B)The
(TNF−α), effect
IL−1β, (C) of
IL−6,
SFN treatment on the expression of (A) tumor necrosis factor-alpha (TNF−α), (B) IL−1β, (C) IL−6,the
(D) C−X−C motif chemokine ligand 1 (CXCL1) mRNA. The values in the bar graphs represent
(D) C−X−C motif chemokine ligand 1 (CXCL1) mRNA. The values in the bar graphs represent the
mean ± standard deviation (SD; n = 3 experiments). The relative expression of each gene after 4 h of
IL−1β (+) and SFN (−) treatment was set as 100%. ** p < 0.01 versus IL−1β alone. (E) The effect of
SFN treatment on NF−κB activation analyzed by an electrophoretic mobility shift assay (EMSA). The
arbitrary units of NF−κB activation were determined by setting the band intensity at 4 h of IL−1β
(+) and SFN (−) treatment as 100 (lower).
Int. J. Mol. Sci. 2024, 25, 579 9 of 15

3. Discussion
In this study, the hepatoprotective effects of SFN were investigated in the in vivo HIRI
+ PH model and the in vitro liver injury model. The results suggested several possible
mechanisms for the effects. In this HIRI + PH rat model, SFN reduced the frequency of
pathological liver injuries (Figure 2), including apoptosis (Figure 3) and the infiltration of
neutrophils (Figure 4), and attenuated changes in serum levels of AST and ALT (Figure 5).
TNF-α contributes to neutrophil recruitment in the liver, which is ultimately involved in
the later phases of injury [33]. SFN significantly decreased the levels of TNF-α and CXCL1
mRNA and increased the levels of IL-10 mRNA in the livers of HIRI + PH-treated rats
(Figure 6A–C). SFN treatment upregulated MCL1 mRNA (Figure 6D), a negative regulator
of apoptosis, thereby suppressing HIRI + PH-induced apoptosis. NF-κB functions as an
important regulator of hepatocyte regeneration and anti-apoptosis [33]. In the EMSA of nu-
clear extracts from the liver, SFN inhibited the activation of NF-κB by HIRI + PH (Figure 7).
The cytoprotective and anti-inflammatory effects of SFN may be partially attributed to
its ability to modulate the transcription factor NF-κB. Moreover, the administration of
SFN improved survival in a more severe HIRI model of 30 min of ischemia followed by
70% hepatectomy (Figure 8). SFN may improve survival rates by suppressing the expres-
sion of the inflammatory cytokines and chemokines and enhancing the expression of the
anti-inflammatory cytokine and anti-apoptotic genes.
The two main types of liver cells affected by HIRI are hepatocytes and sinusoidal
endothelial cells, the cells differ in their sensitivity to different types of warm or cold
ischemia, with hepatocytes being more sensitive to warm ischemia [34]. Hepatocyte
apoptosis is a feature of liver inflammation and is associated with the production of a
variety of inflammatory cytokines. The suppression of the inflammatory response with
SFN administration may contribute to the marked reduction in hepatocyte apoptosis. After
identifying the efficacy of SFN in the near-clinical setting of hepatic ischemia/reperfusion
and partial hepatectomy, we next confirmed the mechanisms using primary cultured
hepatocytes. In rat models of acute liver injury induced by ischemia/reperfusion or
partial hepatectomy and in primary rat hepatocytes stimulated by IL-1β, several drugs
and bioactive compounds with hepatoprotective effects have been shown to decrease the
production of inflammatory mediators, such as TNF-α, through the suppression of NF-
κB [35–38]. TNF-α increases the release of other molecules, such as IL-6 [39] and numerous
chemokines (including CXCL1) [40]. SFN reportedly interacts with the NF-κB subunits,
directly reducing their ability to bind DNA [24]. SFN decreased levels of inflammatory
cytokines and chemokine mRNA (Figure 9A–D), the end products of NF-κB, indicating
that the hepatoprotective effects were achieved through the inhibition of the expression
of these inflammatory genes. As with in vivo experiments, SFN also suppressed NF-κB
activation in cultured hepatocytes (Figure 9E). Several intracellular signaling pathways are
known to be involved in NF-κB activation in primary cultured rat hepatocytes, including
IκBα and Akt upon stimulation with IL-1β. However, SFN did not affect the induction of
IκBα degradation or Akt activation. It remains to be elucidated whether SFN suppresses
NF-κB activity through other signaling pathways or upstream factors.
Promoting liver regeneration may prevent liver failure after partial hepatectomy [41].
In rats, the process of liver regeneration in rats begins with an increase in the levels of
various signaling molecules 3 h after hepatectomy, peaks at two to three days, and ends
with the enlargement of the remnant lobe to the original liver size at five to seven days [42].
The prime phase of liver regeneration occurs 24 h after partial hepatectomy, as assessed by
the liver-to-body weight ratio and immunohistochemical markers of proliferative events,
such as Ki-67 [43]. The hepatoprotective effects of SFN may involve liver regeneration after
HIRI + PH treatment. SFN significantly enhanced HGF expression at 6 h of HIRI + PH
treatment (Figure 6E), suggesting that SFN may affect liver regeneration via HGF. The
activation of the Nrf2 transcription factor, an intracellular target of SFN, has been reported
to promote liver regeneration [44]. These results suggest that SFN may promote liver
regeneration, even in HIRI + PH; whether SFN promotes liver regeneration through these
Int. J. Mol. Sci. 2024, 25, 579 10 of 15

factors or other mechanisms should be analyzed after assessing liver regeneration by the
liver-to-body weight ratio and immunohistochemical markers.
These findings suggest that the preoperative administration of SFN may be a potential
therapeutic option to prevent liver injury after hepatic resection in humans. Although the
dose of SFN used in this study (5 mg/kg) is above the dietary dose, this dose is similar to
a dose (3 mg/kg) that has been used in previous studies using rat models [20]. Adverse
effects were observed with SFN administration at 25 mg/kg. However, the dosages utilized
in the in vivo and the in vitro experiments in this study are relatively higher than those
used in the human diet [45], thus serving as a limitation in these models.

4. Materials and Methods

4.1. Materials
SFN (molecular weight, 177.29) was purchased from Cayman Chemical (≥99.0%, Ann
Arbor, MI, USA) and LKT Laboratories, Inc. (≥99.0%, St. Paul, MN, USA). Recombinant
human IL-1β (2 × 107 U/mg protein) was obtained from MyBioSource, Inc. (San Diego, CA,
USA). Male Sprague–Dawley (240–260 g; 6 weeks old) and Wistar rats (180–220 g; 5 weeks
old) were obtained from the Jackson Laboratory, Inc. (Yokohama, Japan). Sprague–Dawley
rats were used in the previous in vivo rat model of HIRI [35]. On the other hand, the
in vitro model of liver injury has been established using the hepatocytes prepared from
Wister rat livers [36,46–49]. In this study, the most appropriate strain was applied to each
model. The rats were housed at 22 ◦ C with a 12 h light/dark cycle for ≥7 days to allow
acclimatization before experiments. Food and water were provided ad libitum. Animal
care and experiments were performed in accordance with the standards outlined in the
ARRIVE [50] and PREPARE [51] guidelines. The study protocol was approved by the
Animal Care Committee of Kansai Medical University (Osaka, Japan) (approval No. 22-039
and No. 22-040).

4.2. Induction of Hepatic Warm Ischemia/Reperfusion Injury and Partial Hepatectomy in Rats
For the in vivo HIRI + PH, male Sprague–Dawley rats (272.5 ± 29.17 g; 7 weeks old
after 7-day acclimatization) were anesthetized with 3% isoflurane during 15 min PM by
clamping the hepatoduodenal ligament. After 70% hepatectomy, the inflow occlusion was
released by declamping. Liver and blood samples were collected from rats after 3 and
6 h (Figure 1B) or from untreated rats as a naïve control. In the survival experiment, the
hepatoduodenal ligament was clamped for 30 min before PH. After 70% PH, the remnant
liver was reperfused, and all rats were observed for 7 days to determine their survival
(Figure 1C) [52]. The rats were randomly assigned to receive intraperitoneal injections
of either SFN (5 mg/kg) or saline 1 and 24 h before PM. The route and dose of SFN
administration were modified from a previous in vivo study [20]. We followed the NIH
Office of Animal Care and Use [53] score and severity assessment to assess the animals
following liver resection [54]. The rats were euthanized when they appeared weak and
moribund due to the progression of liver failure, congestion, and multi-organ failure.

4.3. Culture of Primary Rat Hepatocytes

Perfusion of collagenase (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) was
used to isolate hepatocytes from male Wistar rats (200–250 g; 6–7 weeks old) [47,55]. The
isolated hepatocytes were suspended in Williams’ medium E at 6 × 105 cells/mL, seeded
into 35 mm plastic dishes (2 mL/dish; Falcon Plastics, Oxnard, CA, USA), and incubated
at 37 ◦ C in a humidified chamber (5% CO2 ). The medium contained 10% fetal calf serum,
N-(2-hydroxyethyl)piperazine-N′ -2-ethanesulfonic acid (5 mmol/L), penicillin (100 U/mL),
streptomycin (100 µg/mL), amphotericin B (0.25 µg/mL), aprotinin (0.1 µg/mL; Roche,
Basel, Switzerland), dexamethasone (10 nmol/L), and insulin (10 nmol/L). After 2 h, the
medium was replaced with a fresh serum-free medium (1.5 mL/dish). After another
3 h, the medium was again replaced with a fresh serum- and hormone-free medium
(1.5 mL/dish), and the cells were cultured overnight before use. SFN was first dissolved in
Int. J. Mol. Sci. 2024, 25, 579 11 of 15

dimethylsulfoxide at 10 mM. On the day after cell culture, the hepatocytes were washed
with a fresh serum- and hormone-free medium and incubated with IL-1β (1 nmol/L). SFN
was diluted 1000-fold in the same medium (final concentration, 10 µM).

4.4. Histopathological Analysis of the In Vivo Rat Model

The excised liver specimens were fixed in 4% paraformaldehyde in a phosphate
buffer and embedded in paraffin. Three to four animals per group and three to five sites
per animal were used for slide preparation. Sections of 3–5 µm in thickness were cut.
After staining with H&E, the severity of lesions was graded on a 4-point scale (0 = no
change, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked) for sinusoidal congestion,
cytoplasmic vacuolation of hepatocytes, and parenchymal necrosis, as described by Suzuki
et al. [56]. The toxicologic pathologist (K.Y.) certified by the International Federation of
Societies of Toxicologic Pathologists performed histopathological evaluations according to
previously defined histopathological terminology and diagnostic criteria. Apoptotic bodies
in the hepatocyte nuclei were detected by TUNEL staining using an in situ Apoptosis
Detection Kit (MK500; Takara Bio Inc., Kusatsu, Shiga, Japan). Neutrophil infiltration was
evaluated by immunostaining with MPO using rabbit polyclonal anti-MPO antibodies
(Dako, Carpinteria, CA, USA) before counterstaining with hematoxylin. The number of
TUNEL- and MPO-positive cells per square millimeter was counted by analysts blinded to
the treatment.

4.5. Serum Biochemical Analysis of the In Vivo Rat Model

Serum AST and ALT levels were quantified using a Transaminase CII Test WAKO (code
431-30901, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan, range 3.9~500 Karman units
for AST and 2.4~500 Karman units for ALT, sensitivity OD555 = 0.240~0.320 for 100 Karman
units of AST and OD555 = 0.290~0.370 for 100 Karman units of ALT, inter-assay ≤ ±30% for
AST and ALT, and intra-assay CV ≤ 3% for AST and ≤8% for ALT). The sera were diluted
by distilled water before measurement.

4.6. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) of the Cultured Hepatocytes and
the In Vivo Rat Model
Total RNA was extracted from liver samples or the cultured hepatocytes using Sepasol
I Super G (Nacalai Tesque Inc., Kyoto, Japan), which contains a guanidinium thiocyanate–
phenol–chloroform mixture [57]. For strand-specific RT-PCR, cDNA was first synthesized
from total RNA with an oligo (dT) primer for mRNAs. Touchdown quantitative PCR was
then performed using the primer pairs listed in Supplementary Table S1 with Rotor-Gene Q
(Qiagen, Valencia, CA, USA). The values of the mRNA levels obtained were normalized to
those of elongation factor-1α (EF) mRNA. The normalized value of each gene in HIRI + PH
for 3 h or IL-1β only for 4 h was set as 1.0 or 100%, respectively.

4.7. EMSA of the Nuclear Extracts

The EMSA was performed using a previously described method [49]. Nuclear extracts
were prepared from the liver samples or the cultured hepatocytes. Protein concentrations of
nuclear extracts were measured using the Bradford method. A double-stranded DNA probe
containing a κB site (sense strand: 5′ -AGTTGAGGGGACTTTCCCAGGC) was labeled with
[γ-32 P]-ATP (PerkinElmer Inc., Waltham, MA, USA). The nuclear extracts (4 µg of nuclear
protein per lane) incubated with the labeled DNA probes (40,000 dpm) were resolved by
polyacrylamide gel electrophoresis. Autoradiography was used to analyze the dried gels.
The gel-shift band intensities were quantified using ImageJ 1.53k (National Institutes of
Health).

4.8. Determination of Nitric Oxide Concentration and LDH Activity in the Cultured Hepatocytes
After the hepatocytes were incubated for 8 h with IL-1β and SFN, the medium was
collected. Nitrite, which is a stable metabolite of nitric oxide, in the medium was measured
Int. J. Mol. Sci. 2024, 25, 579 12 of 15

in triplicate by the Griess method [47,58]. Cell viability was evaluated based on the activity
of LDH released into the medium using a Cytotoxicity LDH Assay Kit-WST (code CK12,
Dojindo Laboratories, Kumamoto, Japan).

4.9. Statistical Analyses

Quantitative results were obtained from three or four rats per group or from three
independent experiments, and the means and standard deviations (SDs) were calculated. A
one-way analysis of variance (ANOVA) followed by a Tukey–Kramer multiple comparisons
test was performed to analyze differences. Survival curves were obtained using the Kaplan–
Meier method and statistically analyzed using a log-rank test. Statistical analyses were
performed using the JMP 16.2 Statistics software (SAS Institute Inc., Carly, NC, USA).
p-values of <0.05 and p < 0.01 were considered significant versus the naïve rats, HIRI +
PH-only rats, or IL-1β alone.

5. Conclusions
SFN suppressed liver injury after HIRI + PH administration, which may be due, in part,
to the suppression of inflammatory cytokine and chemokine expression and the increased
expression of anti-inflammatory cytokine and anti-apoptotic genes. The suppression of the
expression of these inflammation-related genes may be associated with the inactivation of
NF-κB in hepatocytes. Thus, SFN has powerful hepatoprotective effects against hepatic
ischemia/reperfusion injury induced after occlusion of blood flow performed for partial
hepatectomy. Further in-depth research is needed to investigate its clinical potential in the
development of novel therapeutic agents.

Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/ijms25010579/s1.
Author Contributions: Conceptualization, R.N.; data curation, M.I., H.Y. and H.K.; formal analysis,
R.N. and T.O.; funding acquisition, R.N. and T.O.; investigation, R.N., T.O. and Y.H.; methodology,
R.N. and M.N.; project administration, R.N.; supervision, M.N. and M.S.; validation, M.I., H.Y. and
H.K.; visualization, R.N., T.O. and K.Y.; writing—original draft, R.N.; writing—review and editing,
T.O. and K.Y. All authors will be informed about each step of manuscript processing including
submission, revision, revision reminder, etc. via emails from our system or assigned Assistant Editor.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by JSPS (Japan Society for the Promotion of Science) KAKENHI
Grant-in-Aid for Scientific Research (C) numbers 19K18370, 19KK0413, 21K09032, and 21K08697.
Institutional Review Board Statement: The animal study protocol was approved by the Animal
Care Committee of Kansai Medical University (Osaka, Japan) (approval No. 22-039(1), 2022.5.30 and
No. 22-040(1), 2022.5.30).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Acknowledgments: We would like to thank Kyodo Byori (Kobe, Japan) for staining the liver spec-
imens. We also would like to thank Tadayoshi Okumura (Department of Surgery, Kansai Medical
University) for his valuable advice and technical support, and the consultation on statistical methods
for Tomoki Kitawaki (Department of Mathematics, Kansai Medical University).
Conflicts of Interest: The authors declare no conflicts of interest.

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