International Journal of

Molecular Sciences
ArticleReg3α and Reg3β Expressions Followed by
JAK2/STAT3 Activation Play a Pivotal Role in the
Acceleration of Liver Hypertrophy in a Rat
ALPPS Model
Naohiko Otsuka 1 , Masato Yoshioka 2 , Yuki Abe 1 , Yasuhiko Nakagawa 1 , Hiroshi Uchinami 1
and Yuzo Yamamoto 1, *1

2*

Department of Gastroenterological Surgery, Akita University Graduate School of Medicine,
Akita 0108543, Japan; n.otsuka0719@gmail.com (N.O.); yuki-a@gipc.akita-u.ac.jp (Y.A.);
y-nakagawa@gipc.akita-u.ac.jp (Y.N.); huchi@gipc.akita-u.ac.jp (H.U.)
Department of Nursing, Akita University Graduate School of Health Science, Akita 0108543, Japan;
masato@gipc.akita-u.ac.jp
Correspondence: yamamoto-kyt@umin.ac.jp; Tel.: +81-18-884-6123



Received: 11 May 2020; Accepted: 5 June 2020; Published: 7 June 2020

Abstract: To explore the underlying mechanism of rapid liver hypertrophy by liver partition inassociating liver partition and portal vein ligation for staged hepatectomy (ALPPS), liver partition at
different sites was investigated. Increased inflammatory cytokines owing to the liver partition havebeen reportedly responsible. If this were true, rapid liver hypertrophy should be achieved regardlessof where the liver was split. A male Sprague-Dawley rat model was created, in which a liver split wasplaced inside the portal vein ligated lobe (PiLL), in addition to the ALPPS and portal vein ligation(PVL) models. Liver regeneration rate, inflammatory cytokine levels, activation status of the Janus
kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway and expressions ofregenerating islet-derived (Reg)3α and Reg3β were investigated. The liver regeneration rate wassignificantly higher in the ALPPS group than in the PiLL group, whereas inflammatory cytokine
levels were nearly equal. Additional volume increase in ALPPS group over PVL and PiLL groups
was JAK2/STAT3-dependent. Reg3α and Reg3β expressions were observed only in the ALPPS group.
An increase in inflammatory cytokines was not enough to describe the mechanism of rapid liver
hypertrophy in ALPPS. Expressions of Reg3α and Reg3β could play an important role in conjunction
with an activation of the JAK2/STAT3 pathway.
Keywords: ALPPS; Reg3α; Reg3β; JAK2/STAT3 pathway; liver hypertrophy

1. Introduction
Patients with large or multiple liver tumors require extended liver resection. However,
postoperative liver failure may occur in these patients due to insufficiency of remnant liver volume [1,2].
To solve this problem, portal vein ligation (PVL) or embolization is performed preoperatively, and thefuture liver remnant (FLR) increases by 10% to 45% with a waiting period from two to eight weeks [3–5].
However, tumor progression during this period is an issue, and the rate of liver resection after portal
vein occlusion remains at 70% [3,4].
As a new strategy, associating liver partition and portal vein ligation for staged hepatectomy(ALPPS) was introduced [5]. Although the ALPPS procedure consists of step 1 (PVL and split of theFLR) and step 2 (hepatectomy), a rapid liver regeneration during step 1 is characteristic in comparison to
PVL alone. This method enables the FLR to increase by 70% to 80% within only 10 days [5,6]. AlthoughInt. J. Mol. Sci. 2020, 21, 4077; doi:10.3390/ijms21114077

www.mdpi.com/journal/ijms

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a high mortality (90-day mortality of 9%) and morbidity (Grade ≥ IIIb of 40% in the Clavien-Dindo
classification) becomes a problem [7], technical modifications such as partial or mini-ALPPS have beenreported and contribute to fewer complications [8,9]. Although many studies on ALPPS have beenreported in clinical settings, there are not many works on basic research, and therefore the mechanismof rapid liver hypertrophy remains unclear. There are only several animal experiments trying to
explore the underlying mechanism by comparing the ALPPS model with the PVL model [10–13]. Whattheir findings suggested in common was that the increase in inflammatory cytokines, owing to theliver partition, was responsible. However, if the increased inflammatory cytokines alone were theelement that characterizes rapid liver hypertrophy in ALPPS, a liver partition placed at a different sitefrom the original ALPPS should also reproduce similar rapid hypertrophy, because wherever the liver
partition is placed, destruction of liver parenchyma itself would promote the release of inflammatory
cytokines [10–13]. In this study, we focused on the dramatic liver regeneration during step 1 of the
ALPPS procedure, and first investigated whether the liver partition at different sites had an influence on
the subsequent speed of liver hypertrophy and how inflammatory cytokines were involved. Secondly,
we explored a novel factor related to this mechanism.
2. Results
2.1. Effects of the Site of the Liver Partition on Liver Hypertrophy
Rats were allocated into three groups (Figure 1). As in previously published studies [10–13],
we prepared two groups to investigate the role of liver parenchymal partition over PVL: (a) PVL group,
all branches of the portal vein were ligated except the branch to the right median lobe (RML); and(b) ALPPS group, the portal vein branches were ligated as in the PVL group and the RML was split
along the right side of the demarcation line that emerged after ligating the left branch of the portal
vein [11]. To investigate whether the site of the liver partition placed in ALPPS has any significant
role, we created an additional experimental group in which the liver partition was intentionally placed
inside the lobe where the portal venous flow had been occluded: (c) Partition inside the Ligated Lobe
(PiLL) group, 80% portal vein ligation and liver partition inside the left lateral lobe (LLL). In all threegroups, arterial branches to all liver lobes were preserved so that the arterial blood perfusion of theportal vein ligated lobe could be maintained. Hematoxylin-eosin staining of the liver tissues from the
LLL on day 1 in every group showed neither ischemic changes nor apoptosis, affirming that the blood
perfusion was preserved in the portal vein ligated lobe as well.

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Figure 1. Illustrations of experimental models. (a) Portal vein ligation (PVL) group, portal branches to
Figure 1. Illustrations of experimental models. (a) Portal vein ligation (PVL) group, portal branches
the caudate lobe, left lateral and left median lobes and right lobe were ligated, which accounted for 80%
to the caudate lobe, left lateral and left median lobes and right lobe were ligated, which accounted for
PVL. (b) Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) group,
80% PVL. (b) Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS)
the right median lobe was split along the right side of the demarcation line that emerged after ligatinggroup, the right median lobe was split along the right side of the demarcation line that emerged after
the left branch of the portal vein in addition to PVL. (c) Partition inside the portal vein Ligated Lobeligating the left branch of the portal vein in addition to PVL. (c) Partition inside the portal vein Ligated(PiLL) group, liver partition was placed inside the left lateral lobe (portal vein ligated lobe) in addition
Lobe (PiLL) group, liver partition was placed inside the left lateral lobe (portal vein ligated lobe) into PVL. Arterial branches to all liver lobes were preserved. (d) Demonstrative snapshot of PVL at theaddition to PVL. Arterial branches to all liver lobes were preserved. (d) Demonstrative snapshot ofcommon branch to LML and LLL. Artery is conserved so that the perfusion is preserved even after
PVL at the common branch to LML and LLL. Artery is conserved so that the perfusion is preserved
PVL. Yellow arrows show the partitioning line. RML, right median lobe; LML, left median lobe; LLL,
even after PVL. Yellow arrows show the partitioning line. RML, right median lobe; LML, left medianleft lateral lobe; RL, right lobe; CL, caudate lobe; PV, portal vein.
lobe; LLL, left lateral lobe; RL, right lobe; CL, caudate lobe; PV, portal vein.

The weight of the RML of the liver to body weight (BW) ratio increased over time in all groups.
The weightof theRML of thedifference
liver to bodyweightratiotheincreased
overtime in all groups.
However,
there wasa significant
in thetrend(BW)betweengroups on
postoperativedays 1,
However,
there
was
a
significant
difference
in
the
trend
betweenthegroupson
postoperativedays
3 and 7 (Figure 2a). As expected, the RML/BW ratio (%) in the ALPPS group was significantly higher
1,
3
and
7
(Figure2a).
As
expected,the
RML/BW
ratio
(%)
in
the
ALPPS
group
was
significantly
than in the PVL group. In particular, the regeneration speed in the ALPPS group was significantly
higherthantheinPVL
the group
PVL group.
In 1,particular,
the regenerationin the ALPPS
group
was
faster thanuntil dayand after that,the difference speed
in the RML/BW
ratio that
occurredsignificantly
faster
than
the
PVL
group
until
day
1,
and
after
that,
the
difference
inthe
RML/BW
ratio
during this period was just maintained, suggesting that immediate response within one day afterthatoccurred
during thisserved
periodaswasjustofmaintained,
thatinimmediate
response withinone
the step
1 intervention
a key
rapidity ofsuggesting
regeneration
ALPPS. Interestingly,
the PiLLday
after
the step a1 significantlyintervention served
as a key ofrapidityof regenerationin andALPPS.
Interestingly,
groupdisplayed
lower RML/BW
ratio
than theALPPS groupnot different
from
the
PiLL
group
displayedasignificantly
lower
RML/BW
ratio
than
the
ALPPS
group
not
the PVL group. In agreement with this profile of the RML/BW ratio, the proliferating celland
nucleardifferent
from
the
PVL
group.
In
agreement
withthisprofileofthe
RML/BW
ratio,
theproliferating
antigen (PCNA) labeling index on day 1 was significantly higher in the ALPPS group than in thecellnuclearantigenshowing(PCNA)that
labeling
on dayentered
1 was into
significantly
higher inthe (PCNAALPPS mainly
group
other
two groups,
more index
hepatocytes
the cell-division
cycle
than
in
the
other
two
groups,showingthat
more
hepatocytes
enteredintothecell-division
cycle
detects the cells in late G1 and S phases) in the ALPPS group; there was no difference between
the
(PCNA
mainlydetects
the
cells
in
late
G1
and
S
phases)
in
the
ALPPS
group;
therewasnodifference
PVL and PiLL groups (Figure 2b). In addition to PCNA staining, we stained the liver tissue on days
betweenthephosphorylated
PVL and PiLL groups2b). aInmore
additionto PCNA
staining,
we stained
the
liver
1 and 3 forHistone(Figure
H3 (pH3),
specific
biomarker
that stains
cells inlate
G2
tissue
on
days
1
and
3
for
phosphorylated
Histone
H3(pH3),
amore
specific
biomarker
thatstainsand mitosis (Figure 2c). The ALPPS group displayed much more pH3-positive hepatocyte nuclei
cells in late G2 and mitosis (Figure 2c). The ALPPS group displayed much more pH3-positive
hepatocyte nuclei than the PVL and PiLL groups on day 1. On day 3, in the PVL and PiLL groups as

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than the PVL and PiLL groups on day 1. On day 3, in the PVL and PiLL groups as well, the number
well, the number of pH3-positive hepatocyte nuclei had shown a fair increase belatedly, catching up
of pH3-positive hepatocyte nuclei had shown a fair increase belatedly, catching up with that in thewith that in the ALPPS group. Although a delayed increase of the PCNA labelling index and pH3
ALPPS group. Although a delayed increase of the PCNA labelling index and pH3 staining in the PVL
staining in the PVL and PiLL groups eliminated the dominance in the ALPPS group by day 3,
and PiLL groups eliminated the dominance in the ALPPS group by day 3, considering the specificconsidering the specific feature of the ALPPS strategy—early rapid volume increase, like a boostfeature of the ALPPS strategy—early rapid volume increase, like a boost-start—these changes overstart—these changes over time seemed natural. It is very meaningful that more hepatocytes enter thetime seemed natural. It is very meaningful that more hepatocytes enter the cell cycle significantly
cell cycle significantly earlier after ALPPS than after PVL or PiLL.
earlier after ALPPS than after PVL or PiLL.

Figure 2. Chronological changes of liver hypertrophy, hepatocyte proliferation after surgery. (a) Weight
Figure 2. Chronological changes of liver hypertrophy, hepatocyte proliferation after surgery. (a)of the right median lobe of the liver to body weight (RML/BW) ratio. (b) Proliferating cell nuclear
Weight of the right median lobe of the liver to body weight (RML/BW) ratio. (b) Proliferating cell
antigen (PCNA) labeling index. (c) Immunohistochemistry for phosphorylated Histone H3 (pH3) on
nuclear antigen (PCNA) labeling index. (c) Immunohistochemistry for phosphorylated Histone H3days 1 and 3. Values are expressed as mean ± standard deviation (SD); n = 6 for each group at each(pH3) on days 1 and 3. Values are expressed as mean ± standard deviation (SD); n = 6 for each groupsacrifice time point; * p < 0.05 compared with the PVL and PiLL groups; ** p < 0.05.
at each sacrifice time point; * p < 0.05 compared with the PVL and PiLL groups; ** p < 0.05.

Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase
Aspartateaminotransferase(AST),
aminotransferase(ALT)
and lactate
dehydrogenase(LDH)
showed thehighest values on
day alanine
1 in all groups,
and gradually
decreased
thereafter(Figure 3).
(LDH)
showedthehighestvalues
on
day
1
in
all
groups,andgradually
decreased
thereafter(FigureThere were no significant differences among the three groups regarding serum levels of liver enzymes.
3). There were no significant differences among the three groups regarding serum levels of liver
enzymes.

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Figure 3. Chronological changes of serum liver enzymes after surgery. (a) Aspartate aminotransferase
Figure 3. Chronological changes of serum liver enzymes after surgery. (a) Aspartate aminotransferase(AST). (b) Alanine aminotransferase (ALT). (c) Lactate dehydrogenase (LDH). Values are expressed as(AST). (b) Alanine aminotransferase (ALT). (c) Lactate dehydrogenase (LDH). Values are expressedmean ± standard deviation (SD); n = 6 for each group at each sacrifice time point.
as mean ± standard deviation (SD); n = 6 for each group at each sacrifice time point.

2.2. Effects of the Site of the Liver Partition on Cytokine Production
2.2. Effects of the Site of the Liver Partition on Cytokine Production
Since the RML/BW ratio of the PiLL group did not increase much, remaining rather the same as the
Since
RML/BW
ratioofofthe
theliver
PiLL
group did not
much, remaining
the same as
PVL groupthe
despitesplittingparenchyma,
we increase
were prompted
to measurerather
the inflammatory
the
PVL group
despite(IL-6)
splittingof thenecrosis
liver parenchyma,
we were prompted to measure thecytokines
interleukin-6
and tumor
factor-α (TNF-α).
inflammatory
cytokines theinterleukin-6(IL-6) andtumor necrosis
factor-α
Figure 4a illustrates
relative increase
in messenger
ribonucleicacid(TNF-α).
(mRNA) expressions of IL-6
Figure4aillustrates
therelativeincreaseinmessenger
ribonucleic
acid(mRNA)
expressionsofand TNF-α on day 1 over their constitutive expressions. Even in the PVL group,
IL-6 gene
was strongly
IL-6
and TNF-αday
1 overtheirconstitutive
expressions.
Even
in the PVLingroup,
IL-6 genewas
up-regulated
by on
eight
times
(8.66
± 10.6).
However,the degree of
up-regulation
the ALPPS
and PiLL
strongly
up-regulated
eighttimes (8.66±10.6).
degreeof up-regulation
ALPPS
groups wastremendousbyandextended
to about 30 However,times (34.0the± 7.30in ALPPS,
27.8 ± 10.3 in thePiLL).
This
and
PiLL
groups wastremendousand extended
to about
30 times(34.0±7.30
in ALPPS,
in
result,as predicted,
confirms
that expressionof IL-6
gene wasvigorously
enhanced
also27.8±10.3
in the PiLL
PiLL).
result,predicted,that expression
gene
vigorously
enhancedgroup,Thisto thesameasextentas the confirms
ALPPS group.
Although oftheIL-6
scale
waswasdifferent,
the sametrend also
wasin
the PiLL group,to thesame
as theAlthoughofthescale
was different,
thesame
reproducedin TNF-α
gene.
In extentline with
this,ALPPS
serumgroup.
concentrations
thesecytokines
showedsimilartrend wasreproducedin comparing
TNF-α gene.
In linethis,production
serum concentrations
cytokines
profiles(Figure4b). When
profilesof withcytokinewith those of ofthethese
RML/BW
ratio,
showedprofiles(Figure4b). ofWhen
profilesof cytokine
production
withofa simple similar
deductionthat theincrease
IL-6 comparingor TNF-α owing
to theparenchymal
partitionis athosepivotalthe
RML/BW
ratio,
asimpledeductionthat
theincrease
of
IL-6
or
TNF-α
owing
to
the
parenchymal
element which characterizes the rapid volume increase in ALPPS seemed to be imprudent. Looking
partitionis differencesa pivotal element
whichcharacterizes
thegroups,
rapid volume
increasein ALPPS seemed
to beonly at the
betweenthe ALPPS
and PVL
the increaseof inflammatory
cytokines
imprudent.
Looking
onlyatthe
differences
between
the
ALPPS
and
PVL
groups,theincreaseofmight appear to be a specific feature of the ALPPS group, as was the case in previous reports. Whereas,
inflammatory
mightwithappearbe agroup,
specificof thegroup, ratio
as wasthe caseincomparing thecytokines
ALPPS groupthe toPiLL
thefeature
difference
inALPPS
the RML/BW
could
not beprevious
Whereas,comparingalone.
the ALPPS group with the PiLL group, the difference in the
explainedreports.
by cytokine
concentrations
RML/BW ratio could not be explained by cytokine concentrations alone.
2.3. Expression of Hepatocyte Growth Factor (HGF) mRNA in the Liver Tissue of FLR
2.3. Expression of Hepatocyte Growth Factor (HGF) mRNA in the Liver Tissue of FLR
Since it is well known that there is a close relationship between IL-6 and HGF expression during
the periodof thethat
ALPPS[13], we comparedlevelsof HGF mRNA
Since itofis step
well 1known
thereprocedure
is a close relationshipbetweenexpression
IL-6 and HGF
expression
duringamong
the threegroups(Figure5). Asexpected[13],fromwethecompared
serum concentration
IL-6, HGF
expression
theperiodof step
1 of the
ALPPS
procedure
expressionoflevelsof HGF
mRNA
was
more
in the (Figure
ALPPS 5).
andAsPiLL
groupsthaninserumthe PVL
group, and of
there
was
noexpression
difference
among
theintense
three groups
expectedfromtheconcentration
IL-6,
HGF
between
the
ALPPS
group
and
the
PiLL
group.
This
resultonce
again
convinced
us
that
the
orthodox
was more intense in the ALPPS and PiLL groups than in the PVL group, and there was no difference
regenerative
stimuli,grouplike a cascade
through
HGF once
to cellagain
division,
alone werenotthesufficienttobetweenthe ALPPS
and the from
PiLL IL-6
group.
This result
convinced
us thatorthodox
explain the boost-startmechanism
liverIL-6
regeneration
by day1 afterintervention.
regenerative
stimuli, likea cascadeof
fromthrough HGF
to celldivision,
alone were not sufficient
to explain the boost-start mechanism of liver regeneration by day 1 after intervention.

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Figure 4. The levels of inflammatory cytokines on day 1. (a) Messenger RNA expressions of interleukin
Figure4. Thelevels of inflammatory
cytokines
day 1.
(a) Messenger RNA
expressionsof Values
interleukin(IL)-6tumor
factor (TNF)-α.
(b) onSerumconcentrations
IL-6expressions
and TNF-α.
are
Figure and4. Thelevelsnecrosis
of inflammatory
cytokines
on day 1.
(a) MessengerofRNAof interleukin(IL)-6and tumor
necrosisfactor(TNF)-α.
(b) Serum
concentrationsof IL-6 and TNF-α. Values are
expressed
as
mean
±
SD;
n
=
6
for
each
group;
*
p
<
0.05;
N.S.,
not
significant.
(IL)-6 and tumor necrosis factor (TNF)-α. (b) Serum concentrations of IL-6 and TNF-α. Values are
expressed as mean ± SD; n = 6 for each group; * p < 0.05; N.S., not significant.
expressed as mean ± SD; n = 6 for each group; * p < 0.05; N.S., not significant.

Figurefactor(HGF)
in inthethefutureliver
remnant(rightmedianlobe)lobe)onFigure5.5.Expression
Expressionofofhepatocyte
hepatocytegrowth
growthfactor(HGF)
futureliver
remnant(rightmedianday
1.
Valuesareexpressed
as
mean
±
SD;
n
=
6
for
each
group;
*
p
<
0.05;
N.S.,
not
significant.
on day5.1.Expression
Values are of
expressed
asgrowthmean ±factor
SD; n(HGF)= 6 forineachgroup;liver* p < remnant
0.05; N.S.,(rightnot significant.
Figurehepatocyte
the future
median lobe)
2.4. Janus
Kinase(JAK)Transducerof Transcription
3 Pathway
on day1. Values
are2/Signal
expressed
as mean ±andSD;Activatorn = 6 for eachgroup; * p < (STAT)
0.05; N.S.,
not significant.

Kinase(JAK) 2/Signal Transducer and Activator of Transcription (STAT) 3 Pathway in thein2.4.
theJanus
ALPPS
Group
ALPPS
2.4.
JanusGroup
Kinase (JAK) 2/Signal Transducer and Activator of Transcription (STAT) 3 Pathway in the
Activation of the JAK2/STAT3 pathway is reportedly the main avenue from IL-6 signaling to cell
ALPPS
Group of the JAK2/STAT3 pathway is reportedly the main avenue from IL-6 signaling to cell
Activation
proliferation[14,15]. To gain insight into the regulatory commitment of the JAK2/STAT3 pathway inproliferation[14,15].
gain insightpathwaycommitment
ofthePiLL
JAK2/STAT3
pathwayinenhancingmoreregeneration
inintothe the
ALPPS
group thanPVLandgroups,weblockedActivation
ofliver
theTo
JAK2/STAT3
isregulatory
reportedly
the themain
avenuefrom
IL-6 signaling
to cellenhancing
more
liver
regeneration
in
the
ALPPS
group
than
the
PVL
and
PiLL
groups,weblockedthe
JAK2/STAT3
pathway
with
JAK2into
inhibitor
AG490. Rats
were sacrificed
on day 1, and
RML/BW
proliferation[14,15].
To gaininsight
the regulatorycommitmentof the JAK2/STAT3
pathwayinthe JAK2/STAT3
pathway
with JAK2
inhibitor
AG490.
Rats
weresacrificed
on day1, andratio,
mRNA
expressionof PCNAandlabeling
index
were
Unexpectedly,
inRML/BWthe
PVL
enhancing
more
liver
regeneration
inPCNA
the
ALPPS
group
than
theexamined.
PVL
and PiLL
groups,weblockedratio,mRNA expression
PCNA
and
PCNA AG490.
labelingRats
index
were
examined.
Unexpectedly,in thethe
JAK2/STAT3
pathwayofwith
JAK2
inhibitor
were
sacrificed
on day1, and RML/BW
ratio, mRNA expression of PCNA and PCNA labeling index were examined. Unexpectedly, in the

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PVL and PiLL groups, there was no influence of JAK2 inhibition on the RML/BW ratio. Whereas, inandALPPSPiLL groups,
was no influence
JAK2 inhibitionon theRML/BWtoratio.
Whereas,in thethegroup, there
the RML/BW
ratio was ofsignificantly
reduced,suppressed
the samelevel as
ALPPS
group,
RML/BW
ratio
significantly
reduced, suppressedto the samelevel asthe PVLPVL and
PiLL thegroups(Figure6a).wasIn otherwords, inhibitionof the JAK2/STAT3
pathwayabolished
and PiLL
groups (Figure6a).ofIntheotherwords,
inhibitionof the JAK2/STAT3
abolished
only anonly
an additional
increase
liver
volumein the ALPPS
group over pathway
the PVL and
PiLL groups.
additional increaseof the liver
volume andin thePCNA
ALPPSlabelinggroup over
thein
PVL
PiLL groups.
Parallelly,
Parallelly,
PCNA mRNA
expression
index
theandALPPS
group were
also
PCNA
mRNA
expression
andPCNAlabeling
index
in
the
ALPPS
group
were
also
suppressedtothesuppressed to the level observed in the PVL and PiLL groups (Figure 6b,c). These results suggested
leveltheobservedin thePVL and
PiLL groups(Figuregroup6b,c). was
Theseresults suggested
that the additionalthat
additional
volumeincreasein the ALPPS
JAK2/STAT3
pathway-dependentandvolumein the ALPPS
wasin
JAK2/STAT3
pathway-dependent
andnotthatdependent
the basal increasethat theincrease
basal increaseof thegroupvolumethe PVL and
PiLL groups wason theof the volumepathway,
in the PVL
andfully
PiLLatgroups
JAK2/STAT3
or notleast. was not dependent on the JAK2/STAT3 pathway, or not
fully at least.

Figure 6.
Blocking ofof thethe JAK2/STAT3
JAK2/STAT3 pathway(a) Weight
Weight ofof theFigure6. Blocking
pathway by
by JAK2
JAK2 inhibitor,
inhibitor, AG490.
AG490. (a)the right
right
median lobe
thetheliver
to body
weight(RML/BW)ratio. (b)Messenger
RNA expressionof proliferating
medianlobeofofliver
to body
weight(RML/BW)ratio.
(b) Messenger
RNA expressionofcell
nuclearantigen(PCNA).
(c)
PCNAlabeling
index.
Samples
were
taken
on
day
1
after
administration
proliferating cell nuclear antigen (PCNA). (c) PCNA labeling index. Samples were taken on day 1 afterof Janus kinaseof(JAK)
inhibitor
AG490;
valuesAG490;
are expressed
as mean± SD;asnmean= 6 foreachn group;
administration
Janus2 kinase(JAK)
2 inhibitor
values are
expressed± SD;= 6 for*eachp < 0.05.
group; * p < 0.05.

To confirm the JAK2/STAT3 pathway dependency of the ALPPS specific volume increase, activation
To confirm the JAK2/STAT3 pathway dependency of the ALPPS specific volume increase,of STAT3 was investigated by immunohistochemistry using anti-phosphorylated STAT3 (pSTAT3)
activation of STAT3 was investigated by immunohistochemistry using anti-phosphorylated STAT3
antibody. Of interest, pSTAT3 was not stained at all in the PVL and PiLL groups despite high IL-6
(pSTAT3) antibody. Of interest, pSTAT3 was not stained at all in the PVL and PiLL groups despite
concentrations, confirming that the JAK2/STAT3 pathway is not activated in these groups (Figure 7a,c).
high IL-6 concentrations, confirming that the JAK2/STAT3 pathway is not activated in these groups
On the other hand, the ALPPS group displayed a strong staining for pSTAT3 in the nuclei of hepatocytes(Figure 7a,c). On the other hand, the ALPPS group displayed a strong staining for pSTAT3 in the(Figure 7b). These results were in good agreement with the study using the JAK2 inhibitor. Even more
nuclei of hepatocytes (Figure 7b). These results were in good agreement with the study using theinterestingly, staining intensity of pSTAT3 was stronger in the area closer to the liver partition than the
JAK2 inhibitor. Even more interestingly, staining intensity of pSTAT3 was stronger in the area closer
area away from it (Figure 7d). Taken together, activation of the JAK2/STAT3 pathway plays a crucial roleto the liver partition than the area away from it (Figure 7d). Taken together, activation of thein an additional volume increase in the ALPPS group, and is independent of inflammatory cytokines.
JAK2/STAT3 pathway plays a crucial role in an additional volume increase in the ALPPS group, andis independent of inflammatory cytokines.

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Figure 7. Immunohistochemistry for phosphorylated signal transducer and activator of transcription 3(pSTAT3)
on day 1. (a) PVL group,for(b)phosphorylated
ALPPS group, (c)PiLLtransducergroup, (d)and
Theactivator
expression
was stronger
Figure 7. Immunohistochemistry
signalof transcriptionnear
the partition
site1.in(a)thePVL
ALPPS
group.
show
partition
3 (pSTAT3)
on daygroup,(b)Arrowheads
ALPPS group,(c)thePiLL
group,site.
(d) The expression wasstronger near the partition site in the ALPPS group. Arrowheads show the partition site.

2.5. Regenerating Islet-Derived (Reg)3α and Reg3β Are Related to Rapid Liver Hypertrophy in ALPPS

2.5. Regenerating(Reg)3α andAre Related
to Rapid
Liver
Hypertrophy
ALPPS.
To explore aIslet-Derived
factor that activates
the Reg3β
JAK2/STAT3
pathwayin the
ALPPS
group,in
we
performed
comprehensive gene analysis by a complementary RNA (cRNA) microarray system. Of 20,000 genes
To explore a factor that activates the JAK2/STAT3 pathway in the ALPPS group, we performed
analyzed, approximately 100 genes were up-regulated in the ALPPS group relative to the PVL group
comprehensive gene analysis by a complementary RNA (cRNA) microarray system. Of 20,000 genes(Table 1). Among them, we focused on Reg3α and Reg3β genes for further investigation because
analyzed, approximately 100 genes were up-regulated in the ALPPS group relative to the PVL groupthey are reportedly strongly related to cell proliferation and differentiation through the JAK2/STAT3(Table 1). Among them, we focused on Reg3α and Reg3β genes for further investigation because they
pathway [16,17].
are reportedly strongly related to cell proliferation and differentiation through the JAK2/STAT3
Figure 8a shows mRNA expressions of Reg3α and Reg3β in the liver tissue from the sham operation
pathway [16,17].
group. Reg3α mRNA was constitutively expressed, but Reg3β mRNA was negative or at least very
Figure 8a shows mRNA expressions of Reg3α and Reg3β in the liver tissue from the sham
faint. We evaluated Reg3α and Reg3β mRNAs as a relative expression to glyceraldehyde 3-phosphate
operation group. Reg3α mRNA was constitutively expressed, but Reg3β mRNA was negative or at
dehydrogenase (GAPDH) on day 1. Actually, expression was more prominent in the ALPPS group thanleast very faint. We evaluated Reg3α and Reg3β mRNAs as a relative expression to glyceraldehyde 3in the PVL group (Figure 8b,c). More importantly, the Reg3β gene was barely expressed in either thephosphate dehydrogenase (GAPDH) on day 1. Actually, expression was more prominent in the ALPPSPVL or PiLL group. The ALPPS group alone displayed an up-regulation of Reg3β gene by more thangroup than in the PVL group (Figure 8b,c). More importantly, the Reg3β gene was barely expressed
two times (Figure 8c). This means that the expression profile of the Reg3β gene is parallel to that ofin either the PVL or PiLL group. The ALPPS group alone displayed an up-regulation of Reg3β gene
the RML/BW ratio, the PCNA labeling index, as well as the activation of the JAK2/STAT3 pathway.
by more than two times (Figure 8c). This means that the expression profile of the Reg3β gene is parallelItnoteworthy
the increase
IL-6, aslittleexpressionof the Reg3β
could betowas
thatalsoof theRML/BW thatratio,despite
the PCNA
labeling ofindex,
well
as the activationof thegene
JAK2/STAT3
observed
in
the
PiLL
group.
Asfor
Reg3α,
althoughthedifference
did
not
reach
statisticalsignificance,
pathway. It was also noteworthy that despite the increase of IL-6, little expression of the Reg3β geneit
was belessobserved
expressed
PiLL
groupAs
than
the ALPPS
group,
demonstrating
a similar
profile to
could
in in
thethePiLL
group.
for inReg3α,
althoughthe difference
did not
reach statistical
Reg3β(Figure8b).
These
features
of
Reg3α
and
Reg3β
gene
expression,
along
with
activationof theasignificance, it was less expressed in the PiLL group than in the ALPPS group, demonstrating
JAK2/STAT3
pathway,
more8b).
convincing
as an element
ALPPS
than an along
increaseinsimilar profile
to Reg3βwere(FigureThese featuresof Reg3αcharacterizing
and Reg3β gene
expression,withinflammatory
cytokines.
activation of the JAK2/STAT3 pathway, were more convincing as an element characterizing ALPPS
than an increase in inflammatory cytokines.

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Table 1. Statistically up-regulated 104 genes in the ALPPS group compared to the PVL group.
Gene

Classification

Ratio

Gene

Classification

Ratio

Akr1c2
Adh4
XAF1
Procr
Pla2g7Itgb8Siglec10
Ppdpf
Reg3a
Reg3b
Pla2g2aEch1
Acsm5
Acsm2aPltp
Slc13a5
Cxcl13Il1bSiglec5
Clec4a2
Il7r
Cxcl1
Cish
Ccl7
Il10
Ccl3
Adgre1
Clec4a3
Slamf6
Il1a
Csf2rbTimd4
Siglec8
Pilra
Cd22
Selp
Cd7
Cd14
Cxcr5
Emr4
Il2rg
Cd44
Samsn1
Cd72
Igsf6
Cebpd
Csf1r
Fcgr2aSlamf7
Cd37
Cd84
Tcp11l2

Alcohol metabolism
Alcohol metabolism
Apoptosis regulatory protein
Blood coagulation
Blood coagulation
Cell adhesion
Cell adhesion
Cell growth/differentiation
Cell growth/differentiation
Cell growth/differentiation
Cell growth/differentiation
Energy metabolism
Energy metabolism
Energy metabolism
Energy metabolism
Energy metabolism
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response
Immune response

14.32
2.596
2.259
2.425
2.395
4.272
2.108
2.021
5.619
3.748
3.803
2.998
2.990
2.847
2.688
2.649
7.468
6.861
6.649
5.488
3.849
3.438
3.361
3.189
3.134
3.113
3.051
2.985
2.972
2.917
2.714
2.630
2.618
2.592
2.530
2.462
2.396
2.372
2.329
2.306
2.283
2.275
2.250
2.226
2.180
2.163
2.160
2.118
2.093
2.090
2.083
2.080

Il7
Il1r1
Dhrs7
St6galnac4
Akr1c12
Ldhb
Dcps
Ndnf
Gfra1
Slc1a2
Ptprt
Sarm1
Adora2b
Kcnn2
Asrgl1
Asb15
Slc7a8
Glul
Pcsk5
Pbsn
Pbsn
Rhbdf2
Sectm1b
Inhbe
Cyp2j4Cyp3a9Cybb
Cyp4b1
Ephx2
Serpina3m
Ubd
Hspb1
Cyp8b1
Rac2
Tbxas1
Steap4
Nos1apReg3g
Sult1c2
Naip5
Cyp7b1
Clec4a1
Rnd2
Fxyd2
Slco1a1
Slc43a3
Slc13a3
Snx10
Slc22a8
Fam169b
RT1-N2
Klra7

Immune response
Immune response
Metabolism
Metabolism
Metabolism
Metabolism
mRNA metabolism
Neuronal factor
Neuronal factor
Neuronal factor
Neuronal factor
Neuronal factor
Neuronal factor
Neuronal factor
Neuronal factor
Protein metabolism
Protein metabolism
Protein metabolism
Protein metabolism
Purin metabolism
Purin metabolism
Signal transduction
Signal transduction
Signal transduction
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Stress response
Transport
Transport
Transport
Transport
Transport
Transport
Unknown
Unknown
Unknown

2.044
2.025
7.958
2.505
2.338
2.057
2.187
6.066
2.839
2.757
2.313
2.278
2.276
2.201
2.026
10.38
2.374
2.059
2.037
2.098
2.015
2.248
2.185
2.093
2.553
2.521
2.140
5.069
3.787
3.509
3.229
3.008
2.840
2.576
2.479
2.474
2.437
2.430
2.427
2.342
2.096
2.079
2.052
2.290
2.139
2.137
2.110
2.110
2.009
3.655
2.759
2.749

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Figure 8. Messenger RNA expressions of regenerating islet-derived (Reg)3α and Reg3β. (a) Expressions inFigure 8. Messenger RNA expressions of regenerating islet-derived (Reg)3α and Reg3β. (a) Expressions
sham-operated liver. (b) Quantitative expressions of Reg3α messenger RNA on day 1. (c) Quantitativein sham-operated liver. (b) Quantitative expressions of Reg3α messenger RNA on day 1. (c)expressions of Reg3β messenger RNA on day 1. Values are expressed as mean ± SD; n = 6 for each
Quantitative expressions of Reg3β messenger RNA on day 1. Values are expressed as mean ± SD; n =group; * p < 0.05; N.S., not significant.
6 for each group; * p < 0.05; N.S., not significant.

2.6. Location of Reg3α and Reg3β Protein Expression
2.6. Location of Reg3α and Reg3β Protein Expression
Since mRNA expression is not always correlated with protein expression, we confirmed
SincemRNA expression
is not always correlated
withproteinexpression,
we
confirmed
proteinproteinexpression
by immunohistochemistryon day-1
tissues.
Consistent
with
mRNA
expression,
immunohistochemistryon day-1
aexpression
constitutivebyweak
staining of Reg3α protein
was tissues.
detectedConsistentin the shamwith
livermRNA(Figureexpression,9a), and thea
constitutive
weakinstaining
Reg3α
in thesham9a), andthestaining
intensity
the PVLofand
PiLLproteingroups was
was detected
not different
fromthe liversham(Figureliver (Figure9b,d).
staining
intensityin thethe intensity
PVL and was
PiLLstronger
groups than
was not
different
the9c),sham9b,d).
InIn
the ALPPS
group,
thesegroupsfrom(Figureandliverthe (Figure
expression
was
the
ALPPS
group,
theintensity
wasstrongerthanthesegroups(Figure
9c),
and
the
expression
wasdiffuse throughout
liver (Figure 9e). Regarding the Reg3β protein, the difference in expression
diffusethethroughout
the
liver
(Figure 9e).
Regardingthe Reg3β
protein,
theALPPS
difference
expression
amonggroups wasvery
distinctive.
It wasvery strongly
expressedin thegroupin(Figure
9h),among thegroupswas very
distinctive.
was very
expressedin the ALPPS
group (Figure
whereasit wascompletely
negativein theItsham,
PVLstrongly
and PiLL
groups (Figure
9f,g,i). Furthermore,9h),induction
whereasof itReg3β
was protein
completely
negativein thein sham,
PVL
and lobules
PiLL groups
9f,g,i).
thewas moreprominentzone 3 ofhepatic
closer to(Figure
the site oftheFurthermore,partition,
the inductionof Reg3β
protein was
more prominentzone
3 of hepatic
lobules
closerparenchymal
just likethe expressionof pSTAT3(Figure 9j).inThis
strongexpressionof Reg3β
to theof the parenchymal
partition,justonlylikeintheof pSTAT3(Figure9j). Thisnear
thesiteparenchymal
partition was
realized
theexpression
liver lobe where
the portal
venousflowstrongwasexpression(RMLof Reg3β
the group).
parenchymal
partition
onlyveinin theliver like
lobeLLL
where
thepreserved
in thenear
ALPPS
However,in thewas
loberealizedwith portal
ligationin theportalvenousflow wasofpreserved(RML
in the
ALPPS
group).
However,
lobe
with
vein
PiLL
group,
inductions
Reg3α andReg3β
proteins
were
not observed
at in
all,theeven
near
theportal
partition
ligation
like9k,l).
LLL in the PiLL group, inductions of Reg3α and Reg3β proteins were not observed at
site(Figureall, even near the partition site (Figure 9k,l).

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Figure 9. Immunohistochemistry for regenerating islet-derived (Reg)3α and Reg3β on day 1.
Figure9. Immunohistochemistry
regenerating
islet-derived(Reg)3α and Reg3β
on day 1.
(a–d)
(a–d)
Reg3α
in the right medianfor
lobe(RML)—future
liver remnant—of
each group.
Although
Reg3α
in
the
right
medianlobe(RML)—futureliver
remnant—of
eachgroup.
Although
Reg3α
Reg3α staining was observed in every group, the staining intensity was enhanced only in the ALPPS
staining
observed
theinstaining
intensity
wasReg3αenhancedin theALPPS acrossgroup.thegroup.
(e) wasLoupe
image inof everyReg3αgroup,
staining
the ALPPS
group.
was only
evenlyexpressed
(e)
Loupe
image
of
Reg3α
staining
in
the
ALPPS
group.
Reg3α
wasevenlyexpressed
acrossthe
RML.
RML. (f–i) Reg3β in the RML of each group. Reg3β staining was negative in the sham, PVL and
PiLL(f–i) Reg3β in the RML of each group. Reg3β staining was negative in the sham, PVL and PiLL groups.
groups. In the ALPPS group, Reg3β was very strongly expressed especially around the central vein.
In the ALPPS group, Reg3β was very strongly expressed especially around the central vein. (j) Loupe(j) Loupe image of Reg3β staining in the ALPPS group. The staining was more intense in the tissue
image of Reg3β staining in the ALPPS group. The staining was more intense in the tissue closer to thecloser to the site of parenchymal partition. (k,l) Reg3α and Reg3β staining in the left lateral lobe, wheresite of parenchymal partition. (k–l) Reg3α and Reg3β staining in the left lateral lobe, where the portal
the portal vein was ligated and the liver partition was placed, in the PiLL group. Neither Reg3α norvein was ligated and the liver partition was placed, in the PiLL group. Neither Reg3α nor Reg3β wasReg3β was expressed. PVL, portal vein ligation group. ALPPS, associating liver partition and portal
expressed. PVL, portal vein ligation group. ALPPS, associating liver partition and portal vein ligationvein ligation for staged hepatectomy group. PiLL, partition inside the portal vein ligated lobe group.
for staged hepatectomy group. PiLL, partition inside the portal vein ligated lobe group. Loupe images
Loupe images are low magnification pictures of the liver tissue where the parenchymal partition was
are low magnification pictures of the liver tissue where the parenchymal partition was placed.
placed. Arrowheads show the partition site.
Arrowheads show the partition site.

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3. Discussion
The basic research related to ALPPS was first published by Schlegel et al. [10]. They and others
using animal models have stated that rapid liver hypertrophy in ALPPS is attributable to the increaseof inflammatory cytokines—such as IL-6 and TNF-α—resulting from liver partition [10–13]. In linewith those reports, production of IL-6 and TNF-α in the ALPPS group was significantly higher than in
the PVL group. However, one noticeable result was that despite the RML/BW ratio being significantly
less increased in the PiLL group than in the ALPPS group, IL-6 and TNF-α increased to as much asthat in the ALPPS group. If rapid liver hypertrophy in ALPPS were simply attributable to the increaseof inflammatory cytokines, the RML/BW ratio in the PiLL group should be comparable to that in the
ALPPS group. Of course, we agree with the importance of paying notice to the tremendous increaseof IL-6 in the ALPPS and PiLL groups, but a 30-fold increase in IL-6 over steady state might notnecessarily be more important in initiating or accelerating liver regeneration than an 8-fold increase.
There is even a possibility that the effect of an increase in IL-6 on liver regenration is already saturated
with an 8-fold increase produced by PVL alone. We also confirmed the expression of HGF on day 1 as a
key molecule in the lower stream of the IL-6 cascade, and its expression pattern was completely similar
to that of IL-6. This fact again suggests that the excessive increase of HGF on day 1 in response to a
massive IL-6 storm generated in the PiLL group does not take effect on additionally facilitating liver
regeneration over the PVL group. The discrepancy between non-acceleration of liver hypertrophyin the PiLL group over the PVL group and the increased inflammatory cytokines as much as in the
ALPPS group—including resulting HGF induction—strongly implies that there must be somethingelse intervening in the secret mechanism underlying ALPPS.
Activation of the JAK/STAT3 pathway by IL-6 is known to accelerate cell proliferation. STAT3
plays an important role in liver regeneration after partial hepatectomy [18], and IL-6 deficient mice
do not realize normal regeneration [19,20]. JAKs are activated through autophosphorylation, andin turn, they phosphorylate and activate STATs, which translocate into the nucleus to regulate genetranscription. Suppression of the JAK2/STAT3 pathway by JAK2 inhibitor resulted in the abolishment of
the additional portion of liver hypertrophy in the ALPPS group, and the magnitude of volume increase
reduced to basal hypertrophy, as obtained by the PVL or PiLL group. This result definitely indicatesthat the additional portion of liver hypertrophy in the ALPPS group is JAK2/STAT3-dependent. What isstrange here is that the basal hypertrophy in the PVL or PiLL group was not affected by the JAK2/STAT3
pathway. Regarding this question, multiple studies with solid as well as blood tumors indicated thatSTAT3 and STAT5 were constitutively activated by tyrosine kinases other than JAK [21,22], and the
JAK family protein consisted of four proteins, JAK1, JAK2, JAK3 and tyrosine kinase 2. Therefore,basal hypertrophy seen in the PVL or PiLL group might be dependent on STAT activation through a
non-JAK pathway, or at least other than the JAK2/STAT3 pathway. In other words, our study happened
to exhume that the molecular mechanism underlying liver regeneration by PVL also has not been
fully elucidated. Further elucidation of a mechanistic pathway around IL-6/STAT in relation to PVL
is intriguing.
Reg family proteins were discovered from the pancreatic juice of rats with chemically induced
experimental pancreatitis [23,24], and afterwards classified into four subtypes [25]. Growing evidence
links Reg3α and Reg3β proteins to regeneration of exocrine and endocrine tissues [26]. The beneficial
effect of Reg3α on acute liver failure was reported in mice [27]. Lieu et al. investigated the role of thehuman hepatocarcinoma-intestine-pancreas/pancreas-associated protein (HIP/PAP) (another name forReg3β) in liver regeneration using HIP/PAP-transgenic mice [28]. They showed that Reg3β accelerated
the accumulation/degradation of nuclear pSTAT3. Conformity of Reg3 expression and STAT3 activationin the liver tissue strongly suggests that expression of Reg3 family protein is attributable to theactivation of the JAK2/STAT3 pathway in our ALPPS model as well.
Unlike the contradiction observed between IL-6 and the regeneration rate of the liver, expression
profiles of Reg3 genes were parallel to the regeneration rate and also to the PCNA labeling index.
According to Loncle et al., Reg3β induced by IL-17 promoted cell growth through activation of

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the JAK2/STAT3-dependent pathway in pancreatitis [17]. Furthermore, they showed that adding
recombinant Reg3β promoted cell proliferation in the pancreas cancer cell line. The mitosis-facilitatingrole of Reg3β was reported in other experiments as well. Simon et al. reported that Reg3β stimulated
liver regeneration after partial hepatectomy through the protein kinase A signaling pathway [29].
These observations reinforce our hypothesis that Reg3β induced in the ALPPS group activated the
JAK2/STAT3 pathway and resulted in rapid liver regeneration.
The limitation of the present study is that we could not fully elucidate the secret why the
up-regulation of Reg3 family genes was triggered in the ALPPS group only. However, we showed thatthe expression of Reg3β was limited in the liver tissue where the portal venous flow was maintained.
As stated by Starzl, insulin is a principal, although not the only, portal hepatotrophic factor playing apivotal role in liver regeneration [30]. Even under cytokine stimulation, in order to trigger the Reg3
up-regulation, adding some factor like insulin supply through portal venous flow might be required.
In addition, deviations of the expression location closer to the partition site may suggest that thecommitment of some autocrine or paracrine factors other than humoral factors might be involved.
The reason for more intense expression of Reg3β in zone 3 remains elusive. Recently, however, there arereports showing the existence of central-vein-associated lineage-restricted progenitor cells responsible
for liver regeneration [31,32]. The relationship with them is intriguing.
Finally, the anatomy—and probably some physiological and/or immunological aspects as well—of
the liver are different between rats and humans. In addition, step 1 of the original ALPPS procedureincludes complete devasculization—affecting not only the portal vein but also the hepatic artery—ofsegment IV. These differences may have additional effects on liver regeneration in a human setting.
However, given that a clinically often-used modification of ALPPS in which the parenchymal partitionis laid along the Cantlie line (our rat model is more compatible to this modified one than the original
ALPPS) also induces faster liver regeneration than PVL, the mechanism of rapid liver regeneration
newly elucidated by the present study will help further clarify the details of multifactorial mechanism
underlying the ALPPS method.
4. Materials and Methods
4.1. Animals
Male Sprague-Dawley rats, 8 weeks old and weighing 250–300 g, were used (CLEA Japan, Tokyo,
Japan). The animals were housed in chip-bedded cages in an air-conditioned room (24 ± 1 ◦ C) with
controlled 12-h light/dark cycles. They were allowed free access to water and standard rat chow, andreceived humane care under the protocol approved by Animal Research Committee of Akita University(No. a-1-3011).
4.2. Surgical Procedures and Study Design
Under anesthesia by intraperitoneal injection of medetomidine hydrochloride, midazolam andbutorphanol tartrate, a midline laparotomy was laid. For PVL, portal vein branches to the caudate,left lateral, left median and right lobes were ligated with 7-0 nylon under an operating microscope(TOPCON, Tokyo, Japan). During this procedure, portal branches were carefully isolated and arterial
branches were untouched so that the blood perfusion of the portal vein ligated lobes could bemaintained. In the ALPPS group, a partition of the liver was placed from the liver surface up to justanterior to the inferior vena cava using a bipolar coagulation forceps. In the PiLL group, the liver
partition was placed in the center of left lateral lobe. In the study by Schlegel et al., they created aremote injury model (kidney, spleen and lung injuries) to assess the effect of systemic inflammatory
response syndrome-like conditions on ALPPS liver hypertrophy [10]. However, they did not measure
the strength of induced inflammation by remote organ injury, including blood IL-6 or TNF-α level; andtheir model may fail to detect potentially responsible factors which might be produced by the partitionof the liver itself. Therefore, we newly developed a PiLL model in which only the partition site wasInt. J. Mol. Sci. 2020, 21, 4077

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altered from ALPPS to avoid the flaw derived from injuring organs other than the liver. The abdomen
was closed in layers. Rats were sacrificed to collect blood samples and liver tissues from RML on
days 1, 3 and 7 (n = 6 for each group per each time point). Blood samples were collected from the heart
and they were centrifuged at 4000 rpm for 5 min. Serum was stored at −80 ◦ C until use. Aliquots ofliver tissues of RML were frozen in liquid nitrogen and stored at −80 ◦ C. The remaining liver tissues
were fixed in 20% formaldehyde. The weight of the RML of the liver and BW were measured on days 1,
3 and 7. RML/BW ratio (%) was denoted as liver hypertrophy index. In addition to these groups,
a sham operation group (n = 6) was created, in which only laparotomy was performed, and sacrificed
the next day (day 1) to obtain the blood and liver tissue samples as a control. Another 6 rats were used
to ascertain the RML/BW ratio without intervention.
4.3. Serum Liver Enzymes
Serum on days 1, 3 and 7 was analyzed for AST, ALT and LDH levels using Transaminase CII Test
Wako and Lactate dehydrogenase CII Test Wako (Wako Pure Chemical Industries, Osaka, Japan).
4.4. Enzyme-Linked Immunosorbent Assay (ELISA) for Serum Inflammatory Cytokines
IL-6 and TNF-α were measured in the serum on day 1 with ELISA kits (Rat IL-6; Immuno-Biological
Laboratories, Gunma, Japan and Rat TNF-α; R&D Systems, Minneapolis, MN, USA).
4.5. Immunohistochemistry
Liver tissues fixed in formaldehyde were embedded in paraffin and sectioned into 3µm thickness.
After deparaffinization, antigen retrieval and the removal of endogenous peroxidase, the sections wereincubated overnight at 4 ◦ C with a mouse monoclonal anti-PCNA antibody (1:200 dilution; Dako,
Glostrup, Denmark), anti-phospho histone H3 antibody (1:500 dilution; Proteintech Group, Chicago,
IL, USA), anti-Reg3α antibody (1:50 dilution; R&D Systems), anti-Reg3β antibody (1:50 dilution; R&D
Systems) or a rabbit monoclonal anti-phosphorylated STAT3 antibody (1:200 dilution; Cell Signaling
Technology, Danvers, MA, USA), followed by an incubation for 30 min at room temperature with
TaKaRa POD Conjugate For Tissue (Takara Bio, Shiga, Japan) for visualization. The sections were
immersed in diaminobenzidine solution and counterstained with hematoxylin.
To evaluate hepatocyte proliferation on days 1 and 3, the average percentage of PCNA-positive
cells to total hepatocytes in 10 random high-power fields was established as the PCNA labeling index.
4.6. JAK2 Inhibitor in ALPPS Model
To explore the role of the JAK2/STAT3 pathway, selective JAK2 inhibitor (AG490, Chemscene,
Monmouth Junction, NJ, USA) was used. Another set of animals was used for JAK2 inhibition
experiments. We prepared again three groups of PVL, ALPPS and PiLL for AG490 administration as
well as their corresponding control groups (n = 6 for each group). AG490 (10 mg/kg) was administeredintraperitoneally 2 h before the operation with 0.5 mL/kg of dimethylsulfoxide (DMSO) as a vehicle.
In the control groups, only DMSO was administered. All rats of this experiment were sacrificed on
day 1 to obtain liver samples.
4.7. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total ribonucleic acid (RNA) was extracted from the liver tissues on day 1 using RNeasy
Mini Kit (QIAGEN, Venlo, the Netherlands). Total RNA of 1 µg was reverse-transcribed into
complementary deoxyribonucleic acid (cDNA) using PrimeScript 1st strand cDNA Synthesis Kit(Takara Bio). To quantify mRNA expressions of IL-6, TNF-α, HGF, Reg3α, Reg3β and PCNA, quantitative
real-time PCR (RT-PCR) was performed with LightCycler 480 SYBR Green I Master (Roche, Basel,
Switzerland). Results of RT-PCR were expressed as the relative expression to GAPDH. The primer
sequences for real-time RT-PCR are shown in Table 2.

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Table 2. Primer sequences for real-time RT-PCR.
Genes

Forward

ReverseSize (bp)

IL-6
TNF-α
HGF
Reg3α
Reg3β
PCNA
GAPDH

TGCTCTGGTCTTCTGGAGTTCATGTACCTGGGAGGAGTCTTCAAGAGTGGCATCAAGTGCCAG
TGACTGAAGCTGAATGAAAGG
CTCCCTCACAGTTAAGATGTTGC
TATTTGGCTCCCAAGATCGAAGACATCAAGAAGGTGGTGAAGCTGTTGCTCAGACTCTCCCTTCAGAGTAATGGGGGTCAGAGTCCTGGATTGCTTGTGAAACACC
CAAGCAAGTACAGCCTTGTCATGCCTAACTGCCACATGAGACTTTC
TTGGTGACAGAAAAGACCTCAG
ATGGGAGTTGCTGTTGAAGTC

249
188
145
236
165
121
104

IL, interleukin; TNF, tumor necrosis factor; HGF, hepatocyte growth factor; Reg, regenerating islet-derived; PCNA,
proliferating cell nuclear antigen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

4.8. cRNA Microarray for Gene Expression Profiling
In the PVL and ALPPS groups, gene expression was profiled by a cRNA microarray systemusing liver tissues of RML (a hypothetical part for FLR) obtained from three rats on day 1. Total
RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified by SV Total
RNA Isolation System (Promega, Madison, WI, USA). RNA samples were quantified by an ND-1000
spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the quality was confirmedwith an Experion System (Bio-Rad, Hercules, CA, USA). The cRNA was generated from total RNAof 100 ng and labeled, using GeneChip™ WT PLUS Kit and hybridized to GeneChip™ Rat Gene 2.0
ST Array (Applied Biosystems, Waltham, MA, USA). All hybridized microarrays were scanned by
an Affymetrix scanner. Relative hybridization intensities and background hybridization values were
calculated using Affymetrix Expression Console™ (Affymetrix, Santa Clara, CA, USA).
Raw signal intensities of all samples were normalized by quantile algorithm with Affymetrix
Power Tool version 1.15.0 software (Affymetrix), then were applied to Linear Models for Microarray
Analysis (limma) package of Bioconductor software [33,34]. We established the criteria for regulated
genes: (up-regulated genes) limma p-value < 0.05 and ratio ≥ 2.0-fold, (down-regulated genes) limma
p-value < 0.05 and ratio ≤ 0.5-fold.
4.9. Statistical Analysis
Data were expressed as mean ± standard deviation. Statistical analysis was performed by unpaired
Student’s t-test. Statistical significance was defined as p < 0.05.
5. Conclusions
An increase in inflammatory cytokines like IL-6 due to liver parenchymal partition alone was notenough to produce the ALPPS effect. The JAK2/STAT3 pathway seems to play a crucial role in the
additional increase of the liver volume in ALPPS over PVL, but not in the basal hypertrophy produced
by PVL alone. Activation of the JAK2/STAT3 pathway in ALPPS was independent of inflammatory
cytokines. Expression of Reg3α and Reg3β in the liver tissue remnant was specific to ALPPS and could
play a significant role in rapid liver hypertrophy in ALPPS in conjunction with an activation of the
JAK2/STAT3 pathway.
Author Contributions: Conceptualization, Y.Y. and N.O.; methodology, Y.Y., M.Y. and N.O.; experiments, N.O.,
Y.A., and Y.N.; analysis, N.O., Y.Y. and H.U.; data curation, Y.Y. and H.U.; writing—original draft preparation, N.O.
and M.Y.; writing—review and editing, Y.Y.; visualization, N.O.; project administration, Y.Y.; funding acquisition,
Y.Y. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Research Grant Program from Eli Lilly Japan K.K., Research Support
Program from Merck Biopharma Co., Ltd., Research Support Program from Chugai Pharmaceutical Co., Ltd., andResearch-in-aid from Yakult Honsha Co., Ltd., as well as Taiho Pharmaceutical Co., Ltd.
Conflicts of Interest: Y.Y. has received Research Grant Program from Eli Lilly Japan K.K., Research Support
Program from Merck Biopharma Co., Ltd., Research Support Program from Chugai Pharmaceutical Co., Ltd.,and Research-in-aid from Yakult Honsha Co., Ltd., as well as Taiho Pharmaceutical Co., Ltd. For the remaining

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authors none were declared. The funders had no role in the design of the study; in the collection, analyses, orinterpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations
ALPPS
PiLL
PVL
JAK2
STAT3
Reg3
FLR
RML
LLL
BW
Day
PCNA
pH3
AST
ALT
LDH
IL-6
TNF-α
HGF
RNA
mRNA
HIP/PAP
cRNA
GAPDH
ELISA
cDNA
DMSO
RT-PCRRNA
limma

associating liver partition and portal vein ligation for staged hepatectomy
partition inside the ligated lobe
portal vein ligation
Janus kinase 2
signal transducer and activator of transcription 3
regenerating islet-derived 3
future liver remnant
right median lobe
left lateral lobe
body weight
postoperative day
proliferating cell nuclear antigen
phosphorylated Histone H3
aspartate aminotransferase
alanine aminotransferase
lactate dehydrogenase
interleukin-6
tumor necrosis factor-α
hepatocyte growth factor
ribonucleic acidmessenger ribonucleic acidhuman hepatocarcinoma-intestine-pancreas/pancreas-associated protein
complementary ribonucleic acidglyceraldehyde 3-phosphate dehydrogenase
enzyme-linked immunosorbent assay
complementary deoxyribonucleic acid
dimethylsulfoxide
reverse transcription polymerase chain reaction
ribonucleic acidLinear Models for Microarray Analysis

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