Effect of Histone Deacetylase Inhibitor on Epithelial-mesenchymal Transition of Liver Fibrosis

1. Research Communication
Effect of Histone Deacetylase Inhibitor on
Epithelial-mesenchymal Transition of Liver
Fibrosis
Maggie M. Ramzy 1
*
Hend M Abdelghany1
Nagwa M. Zenhom1
Nashwa F. El-Tahawy2
1
Department of Biochemistry, Faculty of Medicine, Minia University, Egypt
2
Department of Histology and Cell Biology, Faculty of Medicine, Minia
University, Egypt
Abstract
Liver fibrosis is an excessively reversible wound healing pro-
cess and the fibrotic disorder is the activation of hepatic stel-
late cell that requires extensive alterations in gene expression.
As reversible deacetylation of histone proteins modulate gene
expression, we examined the effect of valproic acid (VPA) as
selective histone deacetylase inhibitor on CCl-4 induced liver
fibrosis. Thirty rats were divided into three equal groups; con-
trol group, fibrotic group and VPA-treated group. The rats
were sacrificed after 6 weeks of liver fibrosis induction. The
histopathological effect on liver tissue was examined. The
expression of a-SMA and Smad-4 mRNA and serum levels of
TGF-b1, alanine aminotransferase, and aspartate aminotrans-
ferase were determined. Treatment of rats with VPA attenu-
ated carbon tetrachloride-induced liver fibrosis. Moreover, a-
SMA and Smad-4 expression was repressed under VPA treat-
ment and both serum TGF-b1 and liver enzymes were signifi-
cantly decreased. The histone deacetylase inhibitor-1 VPA
inhibits the epithelial–mesenchymal transition and affects
hepatic stellate cell activation during liver fibrosis through
downregulation of Smad4 and a-SMA expression which may
serve as a promising agent in liver fibrosis treatment. VC 2018
IUBMB Life, 70(6):511–518, 2018
Keywords: liver fibrosis; histone deacetylase inhibitor; valproic acid;
TGF-b; a-SMA; and Smad-4
INTRODUCTION
Liver fibrosis is an important pathological wound healing pro-
cess in reaction to liver injury characterized by progressive
accumulation of extracellular matrix (ECM) components (1).
Although the etiologies of liver diseases may vary, fibrosis and
cirrhosis develop through common signaling pathways. Cas-
cades of reactions occurs leading to the accumulation of colla-
gen and other ECM components. Sustained stimulation and
accumulation of these materials lead to the liver cirrhosis,
destruction of liver structures, and decreased liver function
(2). ECM is deposited by a prominent population of myofibro-
blasts (3) which are absent from the normal liver and are
derived from hepatic stellate cells (HSCs) in the injured liver
(4). Consecutive rounds of epithelial–mesenchymal transition
(EMT) and mesenchymal–epithelial transition (MET) suggest
that EMT could be reversible and associated with epigenetic
modifications (5). Epigenetic modifications include DNA meth-
ylation, post-translational histone modifications, and RNA-
based mechanisms mediated by small non-coding microRNAs
(6). Histone modifications are regulated by histone acetyltrans-
ferases and histone deacetylases (HDACs) (7) through which
they have an effect on chromatin remodeling and have essen-
tial roles in regulating gene transcription (8).
HDACs are divided into four classes according to their
sequence: class I including HDAC1, 2, 3, and 8, class II includ-
ing HDAC4, 5, 6, 7, 9, and 10, class III including SIRT1
to SIRT7, and finally class IV of HDACs which consists of
HDAC11-related enzymes (9,10). Recently, much attention has
been focused on the ability of HDAC inhibitors to regulate
EMT (11). Valproic acid (VPA), a branched short-chain fatty
acid and a known treatment for several conditions including
VC 2018 International Union of Biochemistry and Molecular Biology
Volume 70, Number 6, June 2018, Pages 511–518
*Address correspondence to: Maggie M. Ramzy, Department of Biochem-
istry, Faculty of Medicine, Minia University, Egypt.
E-mail: maggiemaher24@gmail.com
Received 6 February 2018; Accepted 5 March 2018
DOI 10.1002/iub.1742
Published online 30 March 2018 in Wiley Online Library
(wileyonlinelibrary.com)
IUBMB Life 511

2. epilepsy and bipolar disorder (12,13), is a selective class I- his-
tone deacetylase inhibitor which can prevent death in experi-
mental models following hemorrhagic shock (14). Recently, a
great number of the studies linking VPA with liver disease
have suggested an essential role for VPA and its sodium salt in
remodeling fibrosis (1) as inhibition of HDACs may suppress
EMT through their effects on TGF-b1 signaling (11). Previ-
ously, it has been reported that inhibition of HDAC activity
suppresses EMT induced by transforming growth factor-b
(TGF-b) in human renal epithelial cells (15).
TGF-b, mediates its action through binding to its serine/
threonine kinases-mediated receptors and subsequently acti-
vates signal transduction pathways via the downstream tran-
scription factors, SMADs (16).
Smad2 and/or Smad3 are two mediators of the Smad pro-
tein family. They are activated and associated with Smad4
then Smad protein complexes are translocated to the nucleus
to participate in transcriptional activation of target genes (17).
TGF-b/SMAD signaling plays a critical role in the EMT (18) so
this pathway could be the potential target for prevention and
treatment of fibrosis (19) and SMAD4 as a common mediator
can be a target for controlling HSCs activation during liver
fibrosis (16).
Resolution of liver fibrosis is associated with concomitant
reduction of HSCs activation and global cellular proliferation
(20) therefore, studying the underlying mechanisms of HSC
activation is an important step toward identification of molecu-
lar targets and the development of more effective therapies
(21). HSCs proliferate and differentiate into myofibroblast-like
cells with remarkable upregulation of a-smooth muscle actin
(a-SMA) which is referred to the activation of HSCs (22). Anti-
fibrotic drug research focuses on either the prevention/inhibi-
tion of the HSC activation and/or the stimulation of apoptosis
of the activated HSC (23).
The present study was designed to examine the effect of
VPA, a selective HDAC1 inhibitor on liver fibrosis and stellate
cell activation as this could have potential applications for new
therapeutic strategies against liver fibrosis.
MATERIAL AND METHODS
Animals
This study was carried out on 30 male Wistar albino rats. Rats
were kept in animal house under standard conditions of
boarding and feeding with free access to water.
The animals were divided into three groups: (i) control
group (10 rats) received vehicle by intraperitoneal injection
twice weekly for 6 weeks, (ii) fibrotic group (10 rats) was
received 0.5 lL/g body weight carbon tetrachloride (CCl4)
(Sigma–Aldrich, St. Louis, MO, USA) diluted 93 in corn oil
(Sigma–Aldrich, St. Louis, MO, USA) by intraperitoneal injec-
tion twice weekly for 6 weeks, (iii)treated group (10 rats); as
fibrotic group but rat received drinking water containing 0.4%
VPA twice a week, starting 2 days before the first CCl4
injection (24) which is considered clinically relevant dose in
vivo without significant toxicity (25). During the injection
period, animals were kept in their cages well ventilated, in
12 h day/night cycle. Ethical approval was obtained for the
study from Research Ethics Committee, Faculty of Medicine,
Minia University.
Experimental Procedures
Animals were sacrificed at the end of week six and the blood
was collected and centrifuged at 3,000 rpm for 15 min to
obtain serum which kept frozen at 2808C for determination of
serum TGF-b1, alanine aminotransferase (ALT), and amino-
transferase (AST). The liver tissue was removed and weighed.
Some specimens were immediately frozen in liquid nitrogen,
stored at 2808C for real-time polymerase chain reaction (RT-
PCR) and other parts were fixed in 10% buffered formalin for
histopathological studies.
Histological Procedures
Fixed liver specimens were processed for paraffin block prepa-
ration. Some sections of 5–7 lm thickness were cut, mounted
on glass slides and stained with hematoxylin and eosin (H&E)
and other sections were stained with Masson’s Trichrome (26)
Liver sections were examined and images were digitally cap-
tured using a high-resolution color digital camera mounted on
the microscope (Olympus, Japan), connected to a computer.
Morphometric Study
1. Morphological changes were evaluated in H&E sections
according to Ishak-modified HAI system (27). In brief, the
necroinflammation scores were calculated as the sum of the
four categories: periportal or periseptal interface hepatitis
(0–4); confluent necrosis (0–6); focal (spotty) lytic necrosis,
apoptosis, and focal inflammation (0–4); portal inflammation
(0–4). The final numerical score was the result of summa-
tion of three separate fields per rat for six rats in each
group.
2. For scoring of hepatic fibrosis; the following were applied
according to Ishak-modified HAI system (27): 0, no fibrosis;
1, fibrous expansion of some portal areas with or without
short fibrous septa; 2, fibrous expansion of most portal
areas with or without short fibrous septa; 3, fibrous expan-
sion of most portal areas with occasional portal-to-portal
(P–P) bridging; 4, fibrous expansion of portal areas with
marked portal bridging [P–P as well as portal-to-central (P–
C)]; 5, marked bridging (P–P and/or P–C) with occasional
nodules (incomplete cirrhosis); 6, cirrhosis, probable, or
definite.
Biochemical Analysis
The levels of serum ALT, AST were estimated using Randox
Kits (UK) according to the method of Reitman and Frankel
(28). Determination of serum TGF-b1 was done by a commer-
cial ELISA kit according to the manufacturer’s instructions
(Abcam, UK).
IUBMB LIFE
512 EFFECT OF HDAC INHIBITOR ON EMT

3. Real-time Reverse Transcription Polymerase Chain
Reaction for the Relative Quantification of a-SMA and
Smad4
Total RNA was extracted from homogenized liver specimen
using ribozol RNA extraction reagent (Amresco, Solon, OH,
USA) following the manufacturer’s instructions.
cDNAs were synthesized using Revert Aid First Strand
cDNA Synthesis kit (Fermentas, Life Sciences, ON, Canada).
cDNA was reversely transcribed from 5 lg of mRNA in tran-
scription buffer, 200 U M-MuLV Reverse Transcriptase, 20 U
RNase inhibitor at 428C for 60 min followed by immediate
cooling on ice. RT-PCR was performed with 50 ng cDNA per
reaction using 25 lL of SYBR Green QPCR Mix (Solis BioDyne)
containing 20 lM of specific primers in the RT-PCR Detection
System. The SYBR green data were analyzed with a relative
quantification to GAPDH (Glyceraldehyde-3-phosphate dehy-
drogenase) as reference gene. The sets of primers used were
as follows: Smad4 sense primers, 50
-
GTTCAGGTAGGAGAGACGTTTAAGGT-30
, and Smad4 antisense,
50
-CCTTTACATTCCAACTGCACTCCT-3, a-SMA sense primers; 50
-
ACTGGGACGACATGGAAAAG-30
and antisense 50
-
CATCTCCAGAGTCCAGCACA-30
and GAPDH sense primers: 50
-
GTCGGTGTGAACGGATTTG-30
and antisense 50
-
CTTGCCGTGGGTAGAGTCAT-30
. The relative expression level
of each gene was calculated using the formula 2(–DDCt)
method
(29). They were scaled relative to controls where control sam-
ples were set at a value of 1. Thus, results for all experimental
samples were graphed as relative expression compared with
the control.
Statistical Analysis
Statistics were done using statistical package for the social sci-
ences (SPSS) program (SPSS Inc., version 20). All data were
expressed as mean 6 standard deviation (SD). One-way ANOVA
followed by Tukey’s post-hoc were used for assessment of the
statistical significance of differences between sample mean
values between more than two groups and between two
groups respectively. A P value of 0.05 was considered statisti-
cally significant.
RESULTS
Histopathological Studies
Liver sections from control group showed normal hepatic
structure. Hepatocytes were polygonal cells arranged in
A representative photomicrograph of rat liver from
the control group shows normal lobular architecture.
Hepatocytes (arrows) with acidophilic cytoplasm and
rounded vesicular nuclei arranged in plates (lines)
radiating from the central veins (CV) and separated
by blood sinusoids (S). Note that some hepatocytes
are binucleated (circle). Inset: portal tract containing
branches of the portal vein (PV), hepatic artery (HA),
and bile duct (D). H&E 3400.
Representative photomicrographs of rat liver from the fibrotic group showing: (a) disrupted lobular architecture with dilated
blood sinusoids (s), areas of degenerations (circles), and numerous inflammatory cell infiltrations (arrows). Inset shows a con-
fluent area of degeneration showing apoptotic hepatocytes with densely stained cytoplasm and pyknotic (arrows) or frag-
mented nuclei (arrowheads). Some degenerated hepatocytes with ill-defined cell boundaries and ghosts of nuclei (rectangle).
(b) & inset show hepatocytes around central vein (CV) with vacuolated cytoplasm and malformed nuclei (arrows). Notice the
scattered apoptotic cells (circles). H&E: (a) 340, (b) 3100, insets 3400.
FIG 1
FIG 2
RAMZY ET AL. 513

4. anastomosing plates and cords. The plates radiated from the
central vein (CV) and separated by blood sinusoids (Fig. 1).
The CCl4 group showed disturbed morphology characterized
by variable and patchy areas of degenerations with inflamma-
tory cells infiltration. Hepatocytes showed various degenera-
tive features, for example, numerous apoptotic hepatocytes
with densely stained cytoplasm and pyknotic or fragmented
nuclei; others were degenerated with ill-defined cell bound-
aries and ghosts of nuclei, and others with vacuolated cyto-
plasm and malformed nuclei (Fig. 2a, b). The VPA-treated
group showed preserved hepatocytes and lobular architecture
(Fig. 3a). However, few areas surrounding the CV showed
some hepatocytes with vacuolated cytoplasm (Fig. 3b). The
morphometric study confirmed these results as shown in Table 1.
Masson’s Trichrome Staining
Masson’s trichrome stained liver sections showed no signs of
collagen deposition in the control group; minimal amount sur-
rounding the CV and portal tract (Fig. 4a). Liver sections from
fibrotic group revealed increased deposition of collagen fibers
around CVs, portal tract, and extended between them with
both portal to portal (P–P) and portal to central (P–C) bridging
characterized by formation of nodules (Fig. 4b). Liver tissues
from VPA-treated group showed decreased collagen deposition
with more extension of normal hepatic parenchyma and
reduction of fibrous septa (Fig. 4c). Fibrosis scores (Table 2)
were significantly increased in CCL4 group compared to con-
trol group (P 5 0.0001), while it were significantly decreased in
VPA-treated group compared to fibrotic group (P 5 0.0001).
Regenerating hepatocytes were obviously present in rats of
VPA-treated group, which evidenced by appearance of paren-
chyma nodules (pseudonodules) that did not take the fibrosis
stain (Fig. 4c).
Liver Enzymes Assay and Serum TGF-b
Live enzymes assay showed that ALT and AST were highly sig-
nificantly increased in fibrotic group (P < 0.001) compared to
control group and markedly suppressed in VPA-treated group
(P < 0.001) as compared to fibrotic group but still significantly
higher than control group. Also Serum TGF-b1 was increased
significantly (P < 0.001) in the fibrotic group in comparison with
control group. Serum TGF-b was significantly reduced in VPA-
treated group compared with that in fibrotic group (Table 3).
Relative Expression of a-SMA and Smad-4 by RT-PCR
Liver a-SMA relative mRNA expression was highly significantly
elevated in the fibrotic livers compared to healthy livers
(P 5 0.0001) suggesting increase in HSC activation. VPA treat-
ment significantly suppresses the strong up-regulation of a-
SMA as its expression was highly significantly decreased in
VPA-treated group compared to fibrotic group (P 5 0.0001)
and was non-significant compared to control group (P 5 0.058)
(Fig. 5).
On assessment of Smad-4 mRNA expression, it was found
that VPA significantly reduced Smad-4 mRNA level in treated
group compared to fibrotic group (P 5 0.0001) but still signifi-
cantly higher than control group (P 5 0.008) (Fig. 5).
DISCUSSION
Hepatic fibrosis is a severe consequence of needless accumula-
tion of excessive connective tissues in the liver (30). HSCs are
implicated in chronic liver disease, as they are the most
important source of matrix and the main origin of myofibro-
blasts in the liver, which renders them an important target for
Representative photomicrographs of rat liver from VPA-treated group showing: (a) preserved hepatocytes and lobular architec-
ture with apparent normal hepatocytes (arrows) with acidophilic cytoplasm and rounded vesicular nuclei arranged in plates
(lines) radiating from the central veins (CV) and separated by blood sinusoids (S). (b) Few areas surrounding the central vein
(CV) show some hepatocytes with vacuolated cytoplasm (arrows). H&E: a &b 3400
Histopathological scoring of rat livers (H&E
sections) in the studied groups (n 5 10)
Groups
Histopathological score
P valueMean 6 SEM
Control group 0.2 6 0.1
Fibrotic group 8.9 6 0.6 0.0001c
*
VPA-treated group 3.1 6 0.9 0.0001c
*
0.0001cl
*
c: vs. the control group; cl: vs. fibrotic group.
*P 0.05 is significant.
FIG 3
TABLE 1
IUBMB LIFE
514 EFFECT OF HDAC INHIBITOR ON EMT

5. the treatment of liver fibrosis (31). HSC activation is an initial
event in liver fibrosis (24) and recent studies in animal models
have convincingly shown that liver fibrosis regression is in
part driven by the reversion of activated HSCs to quiescence-
like HSCs (32,33). The involvement of epigenetic regulation
during HSC activation was reported in a previous study by
Mann et al. (34). Lately, VPA was shown to have antifibrotic
effects in both experimental liver fibrosis and adriamycin-
induced nephropathy model (24,35). In the current study, VPA;
as HDAC inhibitor, reserved the histopathological changes in
the livers induced by CCl4. HE sections of the CCl4 group
showed disturbed lobular architecture with areas of focal
necrosis, which was in agreement with Mir et al. (36). While
administration of VPA, reduced the morphological changes
observed in the CCl4 group. This was in agreement with Aher
et al. (23) who described the beneficial effects of VPA in rats
with TAA-induced hepatic fibrosis in which the drug prevented
the infiltration of inflammatory cells and further induced-DNA
damage and apoptosis in activated HSC.
Indeed, Masson’s Trichrome-stained sections showed that
VPA-treated animals had less collagen deposition and decreased
fibrosis score as demonstrated by a decreased septal formation
and deposition of lower amounts of interstitial collagens than in
the CCl4-treated animals. This could be explained by Mannaerts
et al. (24) who mentioned that VPA hindered the process of fibro-
genesis induced by CCl4 through inhibition of HSCs activation.
Also our results showed that VPA has an effect on serolog-
ical marker for liver fibrosis (TGF-b1) and liver enzymes (ALT
and AST) suggesting that inhibition of HDACs may suppress
EMT through effects on TGF-b1 signaling as the level of TGF-
b1 started to decline in VPA-treated animals compared to
fibrotic animals. This confirms the fundamental role of histone
deacetylation in liver fibrosis and the contribution of the com-
plexity of gene regulation by the acetylation state of histone
molecules in HSCs (1).
To determine if resolution of liver fibrosis is associated
with concomitant reduction of HSCs activation, expression of
a-SMA was examined. Our results showed remarkable upregu-
lation of a-SMA mRNA which indicates the activation of
HSCs (22) and its expression was significantly declined by VPA
Representative Photomicrographs of rat liver from the studied groups showing: (a) normal liver structure from control group
with minimal amounts of collagen (arrows) surrounding central vein (CV) and portal tract (PT). (b) Extensive collagen deposi-
tion with portal to portal (P–P) bridging (arrows) forming nodules (n). Inset shows extensive collagen deposition around central
vein (CV) in the fibrotic group. (c) VPA-treated group shows decreased collagen deposition (arrow). Notice the pseudonodules
(pn) that not surrounded by collagen fibers (thick arrows). Masson’s Trichrome 3100
Fibrosis scoring of rat livers (Masson’s Trichrome
sections) in the studied groups (n 5 10)
Groups
Fibrosis score
P valueMean 6 SEM
Control group 0.0 6 0.1
Fibrotic group 4.7 6 0.2 0.0001c
*
VPA-treated group 1.9 6 0.1 0.0001c
*
0.0001cl
*
c: vs. the control group; cl: vs. fibrotic group.
*P 0.05 is significant.
FIG 4
TABLE 2
RAMZY ET AL. 515

6. treatment, this in agree with Mannaerts et al. (24) which sug-
gested that a-SMA is one of the strongest VPA-dependent gene
expression changes during HSC activation as VPA treatment in
vitro resulted in decreased a-SMA protein. On the other hand,
Trichostatin A (TSA), the broad spectrum inhibitor has been
shown to inhibit HSC activation (37) but because of TSA’s lim-
ited use in vivo (38), it was important to test the effect of the
more selective class I HDI as VPA on HSC activation.
Previous studies have revealed that therapeutic interven-
tions of hepatocyte growth factor (HGF) as remarkable anti-
fibrotic effects in induced-liver fibrosis by CCl4 is associated
with decreased a-SMA expression, attenuated deposition of
collagen and reduced serum level of ALT and AST (39,40) con-
firming that inhibition of HSC activation are pivotal goals for
intervention in the hepatic fibrogenesis cascade (1).
Since Smads are involved in the regulation of collagen
expression (41), the effect of VPA on Smad4, which is involved
in the fibrogenic pathway, was examined. Our results showed
that expression of Smad4 in fibrotic liver tissue is significantly
increased while VPA was able to exert significant decrease in
its expression. SMAD4 could potentially be used as a target for
the EMT so previously Lan et al. (42) suggested that VPA
inhibits Smad4 expression in prostatic carcinoma cells and it
was very important as disruption of the of Smad4 pathway
leads to the inhibition of TGF-b signals (42). Also recently it
was demonstrated that VPA is an effective inhibitor of type I
collagen expression in conjunctival fibroblasts via the selective
regulation of Smads (41).
On the other hand, previous study has shown that TSA
(broad spectrum HDAC inhibitor) exerted anti-EMT effects and
these effects rely on epigenetic modulation of type I collagen
by deactivating the homolog 3 (Smad3)/Smad4 transcription
complex (43). As VPA is known to have higher efficiency with
lower toxicity than TSA (44), it might be a suitable alternative to
TSA in epigenetic reprogramming of liver fibrosis remodeling.
To our knowledge, this is the first study to demonstrate
that VPA downregulates Smad4 expression in CCl4-induecd
fibrosis model and this may be considered as one of the critical
therapeutic strategy for the treatment of liver fibrosis as it has
prominent role in TGF-b signaling in chronic liver disease.
Previously it was shown that the early TGF-b responders
Smad6 and Smad7 were not affected by VPA treatment (24)
but our study showed that some late responses to TGF-b1
stimulation through smad4 are affected, this may suggest that
class I HDACs are not the only VPA-targets in HSCs involved in
the fibrotic process.
The promising findings of HDAC inhibition in liver fibrosis
are recently highlighted and a growing body of evidence now
supports the safety and efficacy of HDAC inhibitors in experi-
mental models of liver disease (45). Hence, it may be helpful to
show that the VPA as histone deacetylase inhibitor-1 has an
effect on EMT and HSC activation of liver fibrosis and further
evaluation of VPA cellular effects and mechanisms, coupled
Level of liver enzymes and serum TGF-b in different
groups of the study
Control
group
(mean 6 SD)
Fibrotic
group
(mean 6 SD)
VPA-treated
group
(mean 6 SD)
ALT (U/mL) 29.1 6 3.8 161.9 6 15.9a
68.6 6 8.1a,b
AST (U/mL) 59.6 6 6.8 127.8 6 19.2a
90.8 6 4.4a,b
TGF-b (pg/mL) 173.3 6 10.89 286.2 6 28.29a
214.2 6 24.66a,b
a
Statistically significant compared to control group.
b
Statistically significant compared to fibrotic group.
(a) Relative expression of a-SMA mRNA in liver tissues in control, fibrotic group, and VPA-treated group. (b) Relative expres-
sion of Smad4 mRNA in liver tissues in control, fibrotic group and VPA-treated group. (**, P 0.001): highly significant, NS:
non-significant.
TABLE 3
FIG 5
IUBMB LIFE
516 EFFECT OF HDAC INHIBITOR ON EMT

7. with regulated drug uptake, may help towards optimizing the
clinical benefits of VPA while minimizing possible side effects.
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IUBMB LIFE
518 EFFECT OF HDAC INHIBITOR ON EMT