Esculetin ameliorates hepatic fibrosis in high fat diet induced non-alcoholic fatty liver disease by regulation of FoxO1 mediated pathway

Abstract

Background:
Non-alcoholic fatty liver disease (NAFLD), a chronic metabolic disorder is associated with oxidative stress, inflammation and fibrotic cascades. In this study, we aimed to examine the effects of Esculetin, a well-known anti-oxidant on TGF-β1 mediated liver fibrosis and FoxO1 activity.

Methods:
A non-genetic murine model for NAFLD was developed by chronic high fat diet (HFD) (58% calories from fats) feeding in Wistar rats. The plasma biochemical parameters, liver function tests, oxidative stress, and histopathological alterations were assessed. The alterations in extracellular matrix (ECM) deposition and FoxO1 activity were assessed by immunohistochemistry.

Results:
The aberrations in plasma parameters, liver functioning, morphometric and microscopic changes in liver structure of HFD fed rats were significantly improved by treatment with Esculetin. Liver fibrosis, identified in the form of collagen deposition and expression of fibrotic proteins like TGF-β1 and fibronectin was also markedly controlled by Esculetin. The expression of phospho-FoxO1 was found to be reduced in HFD fed rats' liver, showing an increase in activation of FoxO1 under insulin resistant and hyperglycemic states. Esculetin treatment could improve phospho-FoxO1 expression, thus showing its ability to act on Akt/PI3K/FoxO1 pathway.

Conclusions:
As per the previous studies, a potential therapy for NAFLD may be the one with multi-faceted actions on insulin resistance, oxidative stress, inflammation and fibrosis. This study demonstrates the efficiency of Esculetin in improving liver fibrosis in HFD induced NAFLD.

Full text

Original
article
Esculetin
ameliorates
hepatic
fibrosis
in
high
fat
diet
induced
non-
alcoholic
fatty
liver
disease
by
regulation
of
FoxO1
mediated
pathway
Anuradha
Pandey,
Priyank
Raj,
Santosh
Kumar
Goru,
Almesh
Kadakol,
Vajir
Malek,
Nisha
Sharma,
Anil
Bhanudas
Gaikwad*
Laboratory
of
Molecular
Pharmacology,
Department
of
Pharmacy,
Birla
Institute
of
Technology
and
Science
Pilani,
Pilani
Campus,
Rajasthan,
India
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
22
September
2016
Received
in
revised
form
20
January
2017
Accepted
3
February
2017
Available
online
4
February
2017
Keywords:
Non-alcoholic
fatty
liver
disease
Insulin
resistance
Fibrosis
FoxO1
Esculetin
A
B
S
T
R
A
C
T
Background:
Non-alcoholic
fatty
liver
disease
(NAFLD),
a
chronic
metabolic
disorder
is
associated
with
oxidative
stress,
inflammation
and
fibrotic
cascades.
In
this
study,
we
aimed
to
examine
the
effects
of
Esculetin,
a
well-known
anti-oxidant
on
TGF-
b
1
mediated
liver
fibrosis
and
FoxO1
activity.
Methods:
A
non-genetic
murine
model
for
NAFLD
was
developed
by
chronic
high
fat
diet
(HFD)
(58%
calories
from
fats)
feeding
in
Wistar
rats.
The
plasma
biochemical
parameters,
liver
function
tests,
oxidative
stress,
and
histopathological
alterations
were
assessed.
The
alterations
in
extracellular
matrix
(ECM)
deposition
and
FoxO1
activity
were
assessed
by
immunohistochemistry.
Results:
The
aberrations
in
plasma
parameters,
liver
functioning,
morphometric
and
microscopic
changes
in
liver
structure
of
HFD
fed
rats
were
significantly
improved
by
treatment
with
Esculetin.
Liver
fibrosis,
identified
in
the
form
of
collagen
deposition
and
expression
of
fibrotic
proteins
like
TGF-
b
1
and
fibronectin
was
also
markedly
controlled
by
Esculetin.
The
expression
of
phospho-FoxO1
was
found
to
be
reduced
in
HFD
fed
rats’
liver,
showing
an
increase
in
activation
of
FoxO1
under
insulin
resistant
and
hyperglycemic
states.
Esculetin
treatment
could
improve
phospho-FoxO1
expression,
thus
showing
its
ability
to
act
on
Akt/PI3
K/FoxO1
pathway.
Conclusions:
As
per
the
previous
studies,
a
potential
therapy
for
NAFLD
may
be
the
one
with
multi-faceted
actions
on
insulin
resistance,
oxidative
stress,
inflammation
and
fibrosis.
This
study
demonstrates
the
efficiency
of
Esculetin
in
improving
liver
fibrosis
in
HFD
induced
NAFLD.
©
2017
Institute
of
Pharmacology,
Polish
Academy
of
Sciences.
Published
by
Elsevier
Sp.
z
o.o.
All
rights
reserved.
Introduction
Non-alcoholic
fatty
liver
disease
(NAFLD),
characterized
by
hepatic
steatosis
without
any
other
causes
of
hepatic
fat
accumulation
such
as
significant
alcohol
consumption,
steatogenic
medication
or
hereditary
disorders
is
one
of
the
most
common
liver
diseases
globally
[1].
In
about
15%
of
all
NAFLD
cases,
steatosis
may
evolve
into
non-alcoholic
steatohepatitis
(NASH),
a
juncture
of
inflammation,
insulin
resistance
(IR),
hepatocellular
injury,
and
fibrosis,
often
resulting
in
cirrhosis
and
even
cancer
[2].
The
most
important
risk
factors
for
NAFLD
are
male
gender,
age,
obesity,
IR
and
metabolic
syndrome
[1].
Its
pathogenesis
involves
‘multiple
hits’-
‘first
hit’
includes
lipid
accumulation
in
the
liver,
followed
by
subsequent
hits
in
which
pro
inflammatory
mediators
induce
inflammation,
oxidative
stress,
hepatocellular
injury,
and
fibrosis.
Fibrosis,
excessive
accumula-
tion
of
extra
cellular
matrix
(ECM)
proteins
including
collagen
plays
a
significant
role
in
pathogenesis
of
liver
diseases
[3].
Advanced
liver
fibrosis
culminates
into
cirrhosis,
liver
failure,
portal
hypertension
and
often
requires
liver
transplantation.
Activated
hepatic
stellate
cells
(HSC),
portal
fibroblasts,
and
myofibroblasts
of
bone
marrow
origin
have
been
identified
as
major
collagen-producing
cells
in
the
injured
liver
[3].
Forkhead
box–containing
protein
O
subfamily-1
(FoxO1),
a
transcription
factor
controlling
the
expression
of
glucose-6-phosphatase,
catalytic
subunit
and
phosphoenolpyruvate
carboxykinase
also
plays
a
crucial
role
in
the
trans-differentiation
and
proliferation
of
HSCs
in
liver
fibrosis.
Hyperinsulinemia
has
been
known
to
inactivate
FoxO1
in
HSCs,
resulting
in
HSC
activation
and
may
result
in
the
fibrosis
in
NAFLD
[4].
Despite
vast
ongoing
research,
dietary
intervention
and
exercise
remain
the
first-line
therapy
and
there
are
no
surgical
or
pharmacological
interventions
approved
for
its
treatment.
*
Corresponding
author.
E-mail
address:
anil.gaikwad@pilani.bits-pilani.ac.in
(A.B.
Gaikwad).
http://dx.doi.org/10.1016/j.pharep.2017.02.005
1734-1140/©
2017
Institute
of
Pharmacology,
Polish
Academy
of
Sciences.
Published
by
Elsevier
Sp.
z
o.o.
All
rights
reserved.
Pharmacological
Reports
69
(2017)
666–672
Contents
lists
available
at
ScienceDirect
Pharmacological
Reports
journal
home
page
:
www.elsevier.com/locat
e/pharep

Recent
studies
show
that
insulin
sensitizers,
anti-oxidants,
and
statins
could
potentially
be
used
as
adjunctive
therapy
of
NAFLD
but
side
effects
and
skewed
risk
benefit
ratio
linked
with
synthetic
molecules
make
the
natural
products
or
their
derivatives
an
important
alternative
source
of
high
therapeutic
value
[5].
Ginger,
coffee,
moringa,
eucalyptus
leaf
extract,
liquorice
roots
and
sylibium
have
been
known
to
prevent
NAFLD
or
blunt
its
progression
[5].
Silymarin,
a
lipophilic
extract
derived
from
Silybum
marianum
has
anti-fibrotic,
insulin
sensitizing,
antioxi-
dant,
antiviral
and
anti-inflammatory
activities
which
in
turn
help
in
reduction
steatosis
severity,
liver
ballooning
and
fibrosis,
followed
by
lowered
aminotransferase
levels
in
both
short
and
long
lasting
therapies
[6].
Another
compound
from
nature’s
lap
with
profound
anti-inflammatory
and
insulin
sensitizing
proper-
ties
is
Esculetin,
a
dihydroxy
coumarin
derivative.
Esculetin
has
displayed
a
significant
hepatoprotective
effect
in
mice
fed
a
HFD
by
increasing
plasma
adiponectin
level
and
the
protein
and
mRNA
expression
of
hepatic
AdipoR2,
which
led
to
the
activation
of
5
0
AMP-activated
protein
kinase.
The
protein
and
mRNA
expression
of
hepatic
peroxisome
proliferator-activated
receptor
alpha
(PPARa)
were
up-regulated
by
Esculetin
and
those
of
Sterol
regulatory
element-binding
transcription
factor-
1c
were
down-regulated
[7].
Recently,
Choi
et
al.
that
Esculetin
supplementation
could
protect
against
development
of
NAFLD
in
diabetes
through
the
regulation
of
metabolism
of
glucose
and
lipids
and
also
through
reduction
of
inflammatory
makers
[8].
Recent
reports
suggest
that
chronic
HFD
feeding
induces
the
pathological
changes
including
visceral
obesity,
inflammation,
hyperlipidemia
and
increased
free
fatty
acids
which
are
quite
similar
to
that
of
the
human
NAFLD
[9,10].
A
study
reported
that
HFD
feeding
for
24
weeks
led
to
peri-sinusodial
fibrosis
which
progressed
from
36
to
48
weeks
with
insulin
resistance
[9].
These
reports
suggested
that
chronic
HFD
feeding
could
be
used
as
an
efficient
tool
to
simulate
clinical
NAFLD
and
associated
hepatic
fibrosis.
Based
on
the
above
literature
survey,
we
hypothesized
that
Esculetin
might
ameliorate
liver
fibrosis
associated
with
chronic
high
fat
diet
induced
NAFLD
in
Wistar
rats.
In
the
current
study,
we
aimed
to
study
the
possible
underlying
mechanism
through
which
Esculetin
ameliorates
NAFLD
and
the
associated
hepatic
fibrosis
in
a
chronic
HFD
fed
murine
model
for
NAFLD.
Materials
and
methods
Chemicals
Antibodies
against
phospho-FoxO1,
fibronectin
and
TGF-b1
were
obtained
from
Cell
Signalling
Technology
(Danvers,
MA,
USA).
For
biochemical
estimation,
spectrophotometric
kits
(Accurex
Biomedical
Pvt.
Ltd.,
Mumbai,
India)
and
ultra-sensitive
rat
insulin
kit
obtained
from
Crystal
Chem
(Downer’s
Grove,
IL,
USA)
were
used.
All
the
other
chemicals
were
purchased
from
Sigma
Aldrich
(St.
Louis,
MO,
USA),
unless
otherwise
mentioned.
Animal
studies
Adult
male
Wistar
rats
(160–180
g),
procured
from
the
Central
Animal
Facility
of
the
institute,
Birla
Institute
of
Technology
and
Science
Pilani
were
maintained
under
standard
environmental
conditions
and
were
provided
with
feed
and
water
ad
libitum.
All
the
animals
were
fed
on
normal
pellet
diet
(NPD)
(12%
calories
as
fat)
one
week
prior
to
the
start
of
experimental
protocol
which
is
in
accordance
with
the
guidelines
of
the
Committee
for
the
Purpose
of
Control
and
Supervision
of
Experiments
on
Animals,
Ministry
of
Social
Justice
and
Environment,
Government
of
India
based
on
the
guidelines
of
Institute
of
Laboratory
Animal
Resources,
(Wash-
ington,
DC,
USA).
A
prior
permission
was
sought
from
the
institutional
animal
ethics
committee,
BITS
Pilani,
for
conducting
the
study.
The
animal
model
was
developed
using
chronic
high
fat
diet
(HFD)
(58%
calories
as
fat)
and
the
normal
control
was
treated
with
vehicle
(0.5%
sodium
carboxy
methyl
cellulose)
as
described
previously
[11–13].
Based
on
the
previous
reports,
Esculetin
(50
and
100
mg/kg/day,
po)
was
administered
to
the
rats
fed
with
HFD
after
22
weeks
of
initiation
of
the
study.
The
number
of
animals
maintained
in
each
experimental
group
was
6
[11,14].
Assessment
of
plasma
biochemical
parameters
The
blood
samples
were
collected
and
plasma
was
analyzed
for
glucose
(PGL),
triglycerides
(TG)
and
total
cholesterol
(PTC).
Insulin
determination
was
made
by
ELISA
kit
using
rat
insulin
as
standard
(Rat
ELISA
kit,
Crystal
Chem,
USA)
[11,12].
Assessment
of
liver
function
The
plasma
parameters
which
are
potential
indicators
of
liver
function
include
alkaline
phosphatase
(ALP),
lactate
dehydroge-
nase
(LDH),
aspartate
aminotransferase
(AST),
and
alanine
aminotransferase
(ALT)
[15,16].
These
parameters
were
assessed
using
commercially
available
kits
(Accurex,
Mumbai,
India)
[12].
AST/ALT
ratio,
also
known
as
De
Ritis
ratio
was
calculated
to
determine
development
of
IR
[17].
Homeostasis
model
assessment
(HOMA)
HOMA
is
a
mathematical
model
to
estimate
an
individual’s
index
of
IR,
insulin
sensitivity
(%S)
and
steady
state
beta
cell
function
(%B)
based
on
simultaneous
measurements
of
fasting
plasma
glucose
and
insulin
or
C-peptide
concentrations
[18].
HOMA-IR,
%S
and%B
scores
were
calculated
at
the
end
of
the
experimental
period
[19].
Estimation
of
lipid
peroxide
and
reduced
glutathione
(GSH)
levels
in
liver
The
estimation
of
oxidative
stress
was
carried
out
by
assessing
the
lipid
peroxide
and
GSH
levels
in
the
liver.
Briefly,
after
sacrificing
the
animals,
the
liver
was
excised,
rinsed
with
normal
saline
and
weighed.
The
lipid
peroxide
level
in
animal
tissues
was
measured
according
to
method
described
by
Ohkawa
et
al..
After
weighing,
liver
tissue
was
minced
properly
and
the
homogenate
was
prepared
in
1
mL
of
cold
phosphate
buffered
saline
(pH
7.4)
and
centrifuged
at
700

g.
Supernatant
was
collected
and
used
for
estimation
of
thiobarbituric
acid
reacting
substances
(TBARS)
by
using
spectrophotometric
method
at
532
nm
[20].
The
GSH
content
was
estimated
according
to
Ellman’s
method.
For
GSH,
liver
tissues
were
homogenized
in
10
mL
ice-cold
homogenizing
buffer
combined
with
sulphosalicylic
acid
and
the
homogenate
was
centrifuged.
Ellman’s
reagent
was
added
to
the
supernatant
to
produce
yellow
colour
of
5-thio-2-nitrobenzoate-SH.
The
absor-
bance
was
taken
at
412
nm
[21].
Histopathological
evaluation
Histopathology
was
performed
as
per
the
protocol
described
previously
[11].
Briefly,
the
liver
tissue
was
fixed
in
10%
(v/v)
formalin
in
phosphate
buffered
saline
and
embedded
in
paraffin.
5
mm
sections
were
stained
with
hematoxylin
and
eosin
(H&E)
to
study
glomerular
damage.
At
least
25
sections
from
each
group
(4-
5
sections
from
each
animal)
were
observed
for
any
histological
changes.
Histopathological
images
were
captured
by
using
Olympus
microscope
(Model
no.
BX41,
NY,
USA)
[22].
For
the
study
of
deposition
of
ECM
(collagen),
the
sections
were
stained
A.
Pandey
et
al.
/
Pharmacological
Reports
69
(2017)
666–672
667

with
Picro
Sirius
Red.
The
percentage
fibrotic
area
was
analyzed
using
ImageJ
software
and
the
data
analysis
was
carried
out
by
using
GraphPad
5
Prism
software
[23].
Immunohistochemistry
Immunohistochemistry
for
checking
the
expression
of
phos-
pho-FoxO1,
fibronectin
and
TGF-b1
was
performed
as
per
the
protocol
described
previously
[11,14].
Briefly,
liver
sections
(5
mm)
were
taken
from
paraffin
blocks
and
deparaffinized
with
xylene,
followed
by
antigen
retrieval
by
heating
in
citrate
buffer
(10
mmol/
L).
The
following
primary
antibodies
were
used:
anti-phospho
FoxO1,
fibronectin
and
TGF-b1
(rabbit,
1:200
dilution;
Cell
Signaling
Technology)
and
anti-rabbit
Horse
Radish
Peroxidase
conjugated
secondary
antibody
was
used,
followed
by
detection
with
diaminobenzidine
(DAB)
as
a
chromogen.
The
slides
were
counterstained
with
hematoxylin,
dehydrated
with
alcohol
and
xylene,
and
mounted
in
DPX
(Sigma-
Aldrich).
All
the
images
were
analyzed
using
ImageJ
software
for
calculating
DAB
positive
area.
Statistical
analysis
Experimental
values
were
expressed
as
means

SEM.
Statisti-
cal
comparison
between
different
groups
was
performed
using
one
way
analysis
of
variance
to
detect
the
difference
in
observations
between
all
groups.
If
F
value
was
significant
then
multiple
comparisons
were
done
by
Tukey
test
using
Prism
software
(version
5.0;
GraphPad,
San
Diego,
CA,
USA)
for
Windows.
Data
was
considered
to
be
statistically
significant
if
p
<
0.05.
Results
Esculetin
improves
the
liver
function
tests
in
HFD
rats
The
plasma
parameters
are
the
hallmarks
of
liver
dysfunction.
The
HFD
fed
rats
were
found
to
show
a
marked
elevation
in
plasma
triglycerides,
cholesterol
and
insulin
but
the
glucose
levels
were
not
altered
significantly
as
compared
to
the
normal
control
(NC).
Esculetin
treatment
could
significantly
ameliorate
these
aberrant
alterations
at
both
the
doses.
HOMA-IR
is
an
independent
predictor
of
cardiovascular
disorders
in
type
2
diabetes.
Low
HOMA-IR
values
indicate
high
insulin
sensitivity,
whereas
high
HOMA-IR
values
indicate
low
insulin
sensitivity
(IR)
[24].
The
HOMA
model
showed
that
Esculetin
induced
a
reduction
in
IR
and
an
improvement
in
insulin
sensitivity
in
HFD
fed
rats
at
both
the
doses
(Table
1).
The
normal
functioning
of
liver
is
indicated
by
various
liver
specific
enzymes
which
act
as
hallmarks
of
liver
injury
or
dysfunction.
The
liver
function
parameters,
including
ALP,
LDH,
AST
and
ALT
also
found
to
be
elevated
by
chronic
HFD
feeding.
The
elevation
in
ALT
was
more
prominent
than
AST
in
the
HFD
fed
rats.
Esculetin
treatment
could
control
this
elevation
by
a
significant
margin,
thus
indicating
a
hepato-protective
aspect
of
Esculetin’s
therapeutic
effect.
De
Ritis
described
the
AST/ALT
ratio
as
being
a
useful
indicator
of
the
aetiology
of
hepatitis.
Both
serum
AST
and
ALT
increase
with
body
weight
but
this
is
more
prominent
for
ALT
rather
than
AST.
In
alcoholic
steatohepatitis
the
De
Ritis
ratio
is
always
over
1.0,
in
patients
with
NASH
the
AST/ALT
ratio
is
more
than
1.0
particularly
in
morbidly
obese
patients.
De
Ritis
value
of
less
than
1.0
indicates
progression
of
disease
towards
fibrosis
and
cirrhosis
[17].
In
this
study,
we
found
that
AST
and
ALT
levels
were
elevated
in
HFD
fed
rats,
but
AST/ALT
ratio
was
found
to
be
reduced
significantly
as
compared
with
the
normal
control
and
the
treatment
with
Esculetin
improved
the
De
Ritis
ratio.
This
indicates
the
ability
of
Esculetin
to
ameliorate
hepatic
fibrosis
in
chronic
high
fat
diet
rats
(Table
1).
Esculetin
reverses
microscopic
and
morphometric
alterations
in
HFD
fed
rat
liver
HFD
is
widely
used
to
induce
hepatic
steatosis
and
NASH
in
experimental
animals.
Chronic
HFD
feeding
for
24
weeks
led
to
a
significant
rise
in
the
body
weight
as
well
as
the
liver
weight.
Also,
there
was
a
notable
change
in
the
appearance
of
liver.
These
alterations
could
be
reversed
markedly
by
the
treatment
with
Esculetin
(both
at
50
and
100
mg/kg
doses).
The
relative
liver
weight
was
elevated
by
chronic
HFD
feeding
and
Esculetin
could
limit
this
aberration,
showing
its
ability
to
reduce
the
accumula-
tion
of
fats
in
the
liver
(Table
1).
The
H&E
staining
showed
that
liver
of
the
rats
fed
with
HFD
has
prominent
hypertrophy,
as
was
evidenced
by
the
reduction
in
Table
1
Morphometric
and
plasma
biochemical
parameters.
Normal
Control
(NC)
High
fat
diet
control
(HFD)
HFD+
Esculetin
(HFD
+
E1)
(50
mg/kg/
day,
po)
HFD+
Esculetin
(HFD
+
E2)
(100
mg/kg/day,
po)
Morphometric
parameters
Liver
weight
(gm)
6.675

0.301
9.533
0.393
*
8.400
0.204
7.133

0.145
**
Body
weight
(gm)
241

4.409
318

14.79
*
266

3.59
**
249

2.459
**
Liver
weight/body
weight
ratio
(%)
2.769

0.068
2.997

0.026
*
3.15

0.056
2.864

0.060
**
Plasma
biochemical
parameters
Plasma
glucose
(mmol/L)
6.157

0.2271
7.190

0.4818
*
6.820

0.4787
6.885

0.5629
Plasma
triglyceride
(mg/dL)
53.60

9.541
245.4

19.20
*
177. 8

12.08
**
122.6

6.427
**
Plasma
total
cholesterol
(mmol/L)
1.134 
0.209
6.392

0.367
*
3.326

0.277
**
2.777

0.275
**
Plasma
insulin
(pmol/L)
125.3

5.494
267.2

17.90
*
178.7
11.45
**
155.4

8.599
**
HOMA-IR
2.42

0.538
5.1

1.10
*
3.48

0.769
**
3.02

1.30
**
HOMA-
b
113.9

8.072
159

10.68
*
120.8

5.923
**
97.8

9.01
**
HOMA-%
S
41.4

6.56
19.6

4.09
*
28.7

4.29
**
33.1

5.34
**
Alkaline
phosphatase
(IU/L)
120.3

13.57
583.6

49.62
*
375.6

28.18
**
243.1

14.88
**
Lactate
dehydrogenase
(IU/L)
301.0

33.33
644.3

46.79
*
506

23.44
396

25.1
**
Aspartate
aminotransferase-
AST
(IU/L)
24.13

5.875
70.06

5.934
*
49.23

6.856
**
49.19

2.926
**
Alanine
aminotransferase-
ALT
(IU/
L)
25.61

7.741
87.81

10.61
*
55.74

4.765
44.53
7.291
**
De
Ritis
ratio
(AST/ALT
ratio)
0.981

0.125
0.797

0.223
*
0.883

0.241
1.08

0.145
**
Note:
Data
represented
in
mean

SEM
(n
=
6).
*
p
<
0.001
vs.
NC,
**
p
<
0.001
vs.
HFD
668
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69
(2017)
666–672

number
of
nuclei
per
high
power
field
(hpf),
which
indicates
an
increase
in
individual
cell
size.
The
number
of
nuclei
per
hpf
was
found
to
be
improved
significantly
by
the
treatment
with
Esculetin.
There
was
no
significant
difference
observed
between
the
two
doses
of
Esculetin
(Fig.
1A).
Esculetin
ameliorates
oxidative
stress
in
liver
of
HFD
fed
rats
Oxidative
stress
is
one
of
the
major
inflammation
and
fibrosis
triggering
factors
for
development
of
NAFLD.
The
oxidative
stress
was
found
to
be
elevated
significantly
in
HFD
fed
rats,
owing
to
a
an
increase
in
lipid
peroxidation
elevated
thiobarbituric
acid
reactive
substrate
(TBARS)
and
reduced
anti
oxidants’
defence
mechanisms
(GSH
levels).
The
treatment
with
Esculetin
was
found
to
reduce
lipid
peroxidation
and
elevate
GSH
levels
in
the
liver
tissues
of
HFD
fed
rats.
This
result
is
in
line
with
the
previous
reports
which
had
demonstrated
anti-oxidant
potential
of
the
molecule
(Fig.
1B
and
C).
Esculetin
elevates
the
phosphorylation
of
FoxO1
in
liver
of
HFD
fed
rats
FoxO1
is
constitutively
located
in
the
nucleus
to
regulate
the
transcription
of
gluconeogenic
genes.
However,
under
fasting
state
its
phosphorylation
increases
leading
to
the
nucleo-cytoplasmic
transportation
which
results
in
activation
of
the
transcription
factor
[4].
The
phosphorylated
form
of
FoxO1
was
found
to
be
reduced
significantly
in
rat
liver
upon
chronic
HFD
feeding,
thus
showing
an
increase
in
the
activation
of
FoxO1.
The
treatment
with
Esculetin
could
revert
this
alteration
thus
indicating
its
ability
to
regulate
Akt/phosphatidylinositol
3-kinase
(PI3
K)/FoxO1
pathway
(Fig.
2).
Esculetin
reduces
liver
fibrosis
in
HFD
fed
rats
As
per
the
previous
reports,
chronic
HFD
feeding
leads
to
hepatic
peri-sinosodial
fibrosis
from
week
36
to
48
with
insulin
resistance
[9].
In
this
study,
we
found
that
accumulation
of
ECM
proteins
in
liver
was
found
to
be
significantly
elevated
by
long
term
HFD
feeding.
The
treatment
with
Esculetin
(both
50
and
100
mg/
kg)
could
significantly
reduce
the
elevated
deposition
of
ECM,
as
shown
by
the
amelioration
of
perivascular
fibrosis
in
Picro
Sirius
red
staining
and
deposition
of
fibrotic
proteins,
TGF-b1
and
fibronectin
in
hepatocytes.
However,
there
was
no
significant
difference
observed
between
the
effects
of
the
two
doses
of
Esculetin
(Fig.
3).
Discussion
The
current
study
demonstrates
the
role
of
Esculetin
in
improving
the
TGF-b1
mediated
hepatic
fibrosis
associated
with
advancement
of
NAFLD
induced
by
long
term
HFD
feeding
(Fig.
4).
This
anti-fibrotic
effect
may
be
due
to
its
ability
to
activate
Akt/
PI3
K/FoxO1
pathway
as
indicated
by
the
reduction
in
expression
of
phosphorylated
FoxO1.
Esculetin,
at
both
50
and
100
mg/kg/day
po
treatment
for
14
days
showed
a
marked
improvement
in
metabo-
lism
associated
biochemical
parameters,
liver
function
tests,
insulin
resistance
and
fibrosis.
However,
no
significant
difference
was
observed
between
the
effects
elicited
by
the
two
doses
of
Esculetin.
According
to
the
‘multiple
hit’
hypothesis,
multiple
factors
interact
for
the
pathogenesis
of
NAFLD.
The
first
hit
is
the
accumulation
of
fat
in
hepatocytes
mostly
due
to
IR
[3].
Chronic
HFD
feeding
leads
to
fat
accumulation
in
the
liver
which
is
closely
associated
with
metabolic
derangements
related
to
obesity
and
insulin
resistance
shown
by
metabolic
abnormalities
such
as
altered
plasma
biochemical
parameters
like
plasma
glucose,
triglyceride,
cholesterol
and
liver
function
tests.
Thus,
chronic
HFD
feeding
has
been
found
to
be
an
appropriate
method
for
induction
of
NAFLD
in
murine
model
[9].
In
this
study,
we
found
that
long
term
HFD
feeding
mimics
the
clinical
features
of
human
NAFLD.
These
pathological
features
could
be
attenuated
signifi-
cantly
by
the
treatment
with
Esculetin,
thus
confirming
its
potential
to
be
an
efficient
anti-hyperglycemic,
insulin
sensitizing
and
hepato-protective
agent.
The
subsequent
hits
for
progression
of
the
liver
disease
involve
a
combination
of
oxidative
stress,
lipid
peroxidation,
and
release
of
Fig
1.
(A)
Microscopic
changes
in
the
rat
liver
fed
with
high
fat
diet
and
effect
of
Esculetin,
using
hematoxylin
and
eosin
staining
(40).The
oxidative
stress
parameters:
(B)
GSH
and
(C)
TBARS.
Note:
Data
represented
in
mean

SEM
(n
=
6).
a
p<0.001
vs.
NC,
b
p
<
0.001
vs.
HFD.
(For
interpretation
of
the
references
to
colour
in
this
figure,
the
reader
is
referred
to
the
web
version
of
this
article.)
A.
Pandey
et
al.
/
Pharmacological
Reports
69
(2017)
666–672
669

inflammatory
mediators
[25].
The
initiation
and
perpetuation
of
cell
injury
in
NAFLD
is
associated
with
the
increase
of
free
radicals
and
the
depletion
of
endogenous
anti-oxidant
defence
both
in
human
and
rodents.
In
the
current
study
we
found
that
Esculetin
could
control
the
abrupt
rise
in
free
radical
stress
by
limiting
lipid
peroxidation
and
improving
GSH
in
the
liver
of
the
HFD
fed
rats.
Increased
oxidative
stress
leads
to
the
activation
of
stress
kinases
that
induce
insulin
resistance
by
serine
phosphorylation
of
IRS
proteins
and
Akt
pathway
[26].
Activated
Akt
plays
an
essential
role
in
regulating
renal
cells
proliferation,
fibroblast
activation
and
matrix
production
culminating
into
fibrosis
in
chronic
kidney
diseases
[27].
NAFLD
encompasses
a
spectrum
of
histopathological
condi-
tions,
ranging
from
simple
steatosis
to
NASH
and
cirrhosis,
with
risk
of
hepatocellular
carcinoma.
NASH
is
considered
the
more
aggressive
part
of
NAFLD
spectrum,
associated
with
progressive
fibrosis
as
well
as
higher
risk
of
cardiovascular
disease
[28].
Fibrosis
characterized
by
derangements
in
synthesis
and
degradation
of
ECM,
macrophage/lymphocyte
infiltration,
mesan-
gial
cells
and
podocyte
apoptosis,
accumulation
of
activated
fibroblasts,
epithelial-mesenchymal
transition
[27].
If
fibrosis
continues
unopposed,
it
disrupts
the
normal
architecture
of
the
liver
and
alters
its
normal
functioning
[29].
Recently,
fibrosis
stage
has
been
suggested
to
predict
liver-specific
mortality
more
reliably
than
NAFLD
activity
score
[30].
Previous
studies
demonstrated
that
PI3
K/Akt/mammalian
target
of
Rapamycin
(mTOR),
a
non-Smad
TGF-b
pathway,
plays
an
important
role
in
the
fibrosis
of
different
organs
such
as
the
lung,
kidney,
skin
and
liver.
The
activation
of
PI3
K/Akt/mTOR
pathway
also
promotes
collagen
production
and
scar
formation
in
the
acute
contused
skeletal
muscle
[31].
Akt
phosphorylates
FoxO1,
one
of
the
most
widely
expressed
sub-
families
of
the
winged
helix
forkhead
factors,
at
Ser-253
and
enhances
FoxO1
export
into
the
cytoplasm
[32,33].
The
protein
14-
3-3
changes
the
subcellular
localisation
by
affecting
the
structural
properties
of
the
target
protein
or
by
obstructing
the
scaffolding
[34–36].
This
prompts
an
interaction
between
Exportin
and
the
Fig.
2.
Immunostaining
for
phospho-FoxO1
expression
in
high
fat
diet
rat
liver
and
its
evaluation
(40).
Note:
Data
represented
in
mean

SEM
(n
=
6).
a
p
<
0.001
vs.
NC,
b
p
<
0.001
vs.
HFD.
(For
interpretation
of
the
references
to
colour
in
this
figure,
the
reader
is
referred
to
the
web
version
of
this
article.)
Fig.
3.
Effect
of
Esculetin
on
fibrosis
in
HFD
fed
rats’
liver
(A)
Picro
Sirius
red
staining
(B)
TGF-
b
1
immunostaining
and
evaluation
(C)
Fibronectin
immunostaining
and
evaluation
(40).
Note:
Data
represented
in
mean

SEM
(n
=
6).
a
p
<
0.001
vs.
NC,
b
p
<
0.001
vs.
HFD.
(For
interpretation
of
the
references
to
colour
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
this
article.)
670
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Pandey
et
al.
/
Pharmacological
Reports
69
(2017)
666–672

nuclear
export
signal
and
thereby
prevents
re-entry
of
FoxO
factors
into
the
nucleus
by
masking
the
nuclear
localisation
sequence
[36,37].
FoxO1,
is
biologically
‘omni-functional’
owing
to
its
far-
flung
roles
in
metabolism,
cell
cycle,
tissue
differentiation
and
development
and
oxidative
stress
response
[38].
Existing
reports
indicate
an
important
link
between
oxidative
stress,
FoxO1
activity
and
TGF-b1
mediated
fibrotic
cascade.
A
study
showed
that
FoxO1
facilitates
reduction
of
oxidative
stress
in
keratinocytes
to
maintain
cell
migration
and
prevent
cell
death
in
a
TGF-b1–
independent
manner
and
also
up
regulates
TGF-b1
to
promote
wound
healing
[39,40].
In
a
study
of
HSCs
and
liver
fibrosis,
inactivation
of
FoxO1,
prompted
by
hyperinsulinemia
was
found
to
promote
proliferation
and
trans-differentiation
of
HSCs
resulting
in
the
increase
in
susceptibility
of
FoxO1+/
mice
to
experimentally
induced
liver
fibrosis
[4].
The
current
study
demonstrated
that
Esculetin
significantly
reduces
fibrosis
in
chronic
HFD
fed
rats
by
regulating
TGF-b1
expression,
which
may
be
orchestrated
through
its
effect
on
Akt/PI3
K/FoxO1
pathway.
As
per
the
previous
studies,
a
potential
therapy
for
NAFLD
may
be
the
one
with
multi-faceted
actions
on
insulin
resistance,
oxidative
stress,
inflammation
and
fibrosis.
To
the
best
of
our
knowledge,
this
is
the
first
report
showing
the
ability
of
Esculetin
to
ameliorate
hepatic
fibrosis
in
NAFLD
and
its
effect
on
FoxO1
activity.
Thus,
Esculetin,
having
a
prominent
anti-oxidant
and
insulin
sensitizing
effect
appears
to
be
a
drug
of
choice
for
the
treatment
of
NAFLD
and
this
applicability
is
further
promoted
by
the
results
of
this
study
which
prove
it
to
be
an
anti-fibrotic
and
hepato-protective
agent.
Further
studies
need
to
be
done
to
elaborate
the
detailed
mechanism
through
which
Esculetin
elicits
hepato-protection
in
HFD
induced
NAFLD.
Conflict
of
interests
We
declare
that
we
have
no
conflict
of
interest.
Acknowledgements
A.P.
acknowledges
a
Ph.D.
fellowship
from
DST
-
INSPIRE
[IF131160].
A.B.G
sincerely
acknowledges
the
financial
support
obtained
from
the
Science
&
Engineering
Research
Board
–
Department
of
Science
&
Technology
(SERB-DST),
Govt.
of
India
(SB/EMEQ-053/2013)
and
University
Grant
Commission
–
Major
Research
Project(UGC-MRP)
[F.NO.
42-702/2013
(SR)]
for
the
research
work.
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S,
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Marino
M,
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Dis
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Marrero
JA,
Fontana
RJ,
Su
GL,
Conjeevaram
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