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*Corresponding author: E -mail:[anonimizat] ,[anonimizat] ;
International Journal of Biochemistry Research
& Review
12(3):1-10, 2016, Article no.IJBCRR .26031
ISSN: 2231 -086X, NLM ID: 101654445
SCIENCEDOMAIN international
www.sciencedomain.org
Growth Factor Receptors and Liver Injury
Elsayed Gomaa Elsayed Elsakka1*, Gamil Mohammed Abd -Allah1,
Ahmed Ibrahim El -Desouky Abulsoud1, Ahmed Mohammed Ibrahim Mansour2
and Sayed Abdel Raheem3
1Department of Biochemistry, Faculty of Pharmacy, Alazhar University, Cairo, Egypt.
2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alazhar University, Cairo, Egypt.
3Department of Pathology, Faculty of Medicine, Alazhar University, Cairo, Egypt.
Authors’ contributions
This work was carried out in collaboration between all authors. Authors EGEE, GMA Aand AMIM
designed the study, wrote the protocol and supervised the work. Author sEGEE and AMIM carried out
the animal modeling. Authors EGEE and AIE DA carried out laboratory work, performed the statistical
analysis, wrote the first draft of the manuscript, managed the literature searches and edited the
manuscript. Author SA Rcarried out the histopathological assessment. All authors read and approved
the final manuscript.
Article Information
DOI: 10.9734/IJBCRR/2016/26031
Editor(s):
(1)Yi-Ren Hong, College of Medicine , Kaohsiung Medical University, Taiwan .
(2)Luisa Di Paola, Chemical and Biochemical Engineering Fundamentals, Faculty of Engineering Università Campus
Biomedico, Via Alvaro del Portillo, Roma, Italy .
Reviewers:
(1)Shi Lei, China Three Gorges University, China .
(2)Jerzy Beltowski, Medical University, Lublin, Poland .
(3)Jungshan Chang, Graduate Institute of Medical Sciences, Taipei Medical University, Taiwan .
Complete Peer review History: http://sciencedomain.org/review -history/14889
Received 30thMarch 2016
Accepted 27thMay2016
Published 3rdJune2016
ABSTRACT
Aims:The aim of this study is to investigate the expression pattern of transforming growth factor β
receptor I(TGFβRI) and fibroblast growth factor receptor 3 (FGFR3) at the different stages of liver
injury including acute injury, fibrosis and cirrhosis.
Study Design: Controlled experiment.
Place and Duration of Study: Department of biochemistry and department of pharmacology and
toxicology, faculty of pharmacy (boys) Al -Azhar university, between February 20 15 and June 2015.
Methodology: Four Sprague -Dawley rats groups were used for the experiment. Control group: 9
rats received corn oil; Acute toxicity group :10 rats were injected 50% CCl 4in corn oil (4Original Research Article

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
2ml/kg/IP/single dose); 6 weeks group :12 rats were injected with 50% CCl 4in corn oil (4
ml/kg/IP/twice weekly/6 weeks); 11 weeks group :10 rats were injected with 50% CCl 4in corn oil
(4 ml/kg/IP/twice weekly/6 weeks) followed by 5 weeks of CCl 4treatment with the half previous
dose.
On theday after the last dose, rats were anesthetized with diethyl ether and blood samples were
collected for measurement of blood chemistry. The animals then were euthanized, and tissue
samples from the livers were harvested and divided into 2 parts; the first was processed for
standard histology and immunofluorescence techniques and the other was homogenized for
oxidative status assessment.
Results: TGFβRI and FGFR3 were shown to upregulate in chronic liver injury including stages of
fibrosis and cirrhosis. W hile TGFβRI was shown to be located mainly in the cell membrane, the
cytoplasm was shown to be the main site for FGFR3 localization.
Conclusion: TGFβRI and FGFR3 were suggested to be of critical importance in pathogenesis of
chronic liver injury so they ma y be used as a target for chronic liver injury therapy and/or candidate
marker for diagnosis and/or prognosis of chronic liver injury.
Keywords: TGFβRI; FGFR3; fibrosis; cirrhosis.
1. INTRODUCTION
Liver plays a limitless role in the preservation and
body homeostasis regulation. It is included in
most biochemical pathways to growth, protection
against disease, nutrient fund, energy facility and
reproduction [1].Hepatotoxicity can be generated
by certain common causes including therapeutic
agents, natural chemicals, laboratory and
manufacturing agents and herbal therapies
[2].
Carbon tetra chloride (CCl 4) is one of the most
communal models for induci ng hepatotoxicity. It
is transformed into a toxic CCl 3-radical by
hepatic cytochrome P4502E1 (CYP2E1). Thus; it
brings an acute Centro -lobular necrosis which
starts a wound healing response (fibrosis) [3].
However, Contact to these chemicals in humans
israre and generally occurs in the manufacturing
during fabrication and in places where these
chemicals is usually used [4].
Transforming growth factor β receptors (TGF –
βRs) are of 2 types TGF -βRІ and TGF -βRІІ. They
mediate hepatic stellate cell (HSC) activ ation[5]
after binding to their ligand cytokine TGFβ, the
profibrogenic cytokine that traditionally have
been considered the key fibrogenic stimulus to
HSC[6].
Fibroblast growth factor receptor 3 (FGFR3) is
involved in cell growth control, cell differentiation,
and migration[7].There are many reports that
indicated the involvement of FGF/ FGF receptor
(FGFR) signaling in the progression liver
diseases including fibrosis [8,9]and
hepatocellular carcinoma [10-13].The aim of this study was to i nvestigate the
expression pattern of TGFβRI and FGFR3 at
different stages of liver injury. This aspect of
study was verified by investigation of the
localization of these proteins in different liver
injury stages.
2.METHODOLOGY
2.1 Animal Model
Adult male Sprague -Dawley rats weighing 250 –
300 g and aging 70 days were used for the
current study. The animals were obtained from
the breeding colony maintained at the animal
house of the Nile Company for pharmaceuticals,
Cairo, Egypt. They were housed i n the animal
facility of Faculty of Pharmacy, Al -Azhar
University in 20 X 18 X 25 cm plastic cages with
stainless steel wire lids and mesh floor with 5
animals per cage. They were kept at 23±1°C, at
55% relative humidity, with 12:12 -h light: dark
cycle, an d maintained on a standard rodent chow
(El-Nasr Company, Abou -Zaabal, Egypt), and
allowed to food and water ad libitum . Animals
were randomly divided into four groups: control
group:9 rats: receiving corn oil (2 ml/kg/twice
weekly/IP); acute toxicity grou p:10 rats were
injected 50 % CCl 4in corn oil (4 ml/kg/IP/single
dose)[14];6weeks group :12rats were injected
with 50% CCl 4in corn oil (4 ml/kg/IP/twice
weekly/6weeks) and finally 11weeks group :10
rats were injected with 50% CCl 4in corn oil (4
ml/kg/IP/twice weekly/6 weeks) followed by 5
weeks of CCl 4treatment with the half previous
dose. At the end of experimental period; rats
were anesthetized with diethyl ether and blood
samples were withdrawn from ocular vein for

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
3measurement of blood chemistry. The animals
then were euthanized, liver tissue samples were
harvested and divided into 2 parts; the first
was processed by standard histology and
immunofluorescence techniques and the other
was homogenized in 0.15 M KCl for MDA and
SOD assay. All animal procedures were
performed in accordance with the international
guide for the care and use of laboratory animals
[15].
2.2 Antibodies and Chemicals
Rabbit polyclonal TGFβRI and FGFR3 antibodies
were purchased from Santa Cruz Biotechnology
(CA, USA). Cy3 -conjugated goat anti -rabbit
antibody was purchased from Jackson
Immunoresearch (PA, USA). 4,6 -Diamidino -2-
phenyl indol (DAPI). CCl 4was purchased fr om
Sigma-Aldrich (MO, USA).
2.3 Biochemical Analysis
Serum enzymatic activities of transaminases
(ALT and AST) were estimated by kinetic method
according to the method of international
federation of clinical chemistry (IFCC) [16,17].
Alkaline phosphatase (ALP) activity was assayed
according to the method of IFCC [18-20]and
serum albumin concentration according to the
method described by Gendler [21]. The liver
homogenate was used for the determination of
the oxidative stress parameters. The le vel
of thiobarbituric acid -reactive substances
(measured as malondialdehyde (MDA) was
determined as described by Mihara and
Uchiyama [22]. The activity of SOD was
determined using the method described by
Marklund [23].2.4Immunofluorescence Analysis
Slides containing liver tissue sections were
handled according to the method described by
Abdel-Bakky and his associates [24]. The
primary TGFβRI and FGFR3 antibodies were
diluted in blocking solution in the suitable dilution
(1:400) and left overnight in 4°C. Secondary
antibody (cyanine red conjugated) diluted in the
blocking solution was incubated for 30 min and
the nuclei were counterstained using DAPI.
Finally, all slides were mounted with the
fluoromount solution, covered by covering slips,
and allowed to stand for detection by
immunofluorescence microscope (Leica DM
5500B).
2.5 Statistical Analysis
Data were presented as the mean ± standard
error of mean (M±SEM). Statistical analysis was
performed using one -way analysis of variance
(ANOVA) followed by Tukey -Kramer as a post
test, according to the number of groups. The
0.05 level of probability was used as the criterion
of significance using GraphPad Prism software
version 5 (GraphPad Software Inc, CA, USA).
3. RESULTS
3.1Liver Functions and Oxidative Status
Assessment
To assess liver functions; we have performed
liver function tests including serum
transaminases, ALP, and albumin. The oxidative
status of liver tissues was determined through
MDA and SOD assay as indicated in Table 1.
Table 1. Liver functions and oxidative status tests
ALT(IU/L) AST(IU/L) ALP(IU/L) Albumin
(g/dl)MDA(nmol/g
tissue)SOD(U/mg
tissue)
Control 46.6±2.85 54.1±2.65 312±21.9 3.81±0.08 4.73±0.318 526±25.2
CCl4
single I.P.
dose294±11.9(a)(b)419±11.0(a)395±11.1(b)3.8±0.06(b)23.7±2.35(a)(b)308±8.05(a)(b)
CCl46
weeks I.P.349±13.4(a)436±15.4(a)570±24.5(a)3.39±0.13 31.9±1.44(a)223±14(a)
CCl411
weeks I.P.298±8.48(a)368±13.4(a)(b)790±24.9(a)(b)2.69±0.07(a)38.1±1.74(a)171±6.57(a)
Data are expressed as mean ± SEM
(a) Significantly different from control group
(b) Significantly different from 6 weeks CCl 4group using one -way ANOVA followed by
Tukey-Kramer test for multiple comparison test at P≤0.05

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
4Administration of I.P. CCl 4significantly elevated
serum transaminases activities compared to
control group whatever the duration of
administration. On the other hand; ALP showed
significant elevation only in chronic
administratio n of CCl 4with minimal change in
single dose CCl 4treatment. Supporting previous
results; albumin showed significant decrease
only with 11 weeks CCl 4treatment. On the other
hand; tissue MDA content was significantly
increased on CCl 4treated groups compared to
control group while SOD was shown to
significantly decrease in CCl 4treated groups
compared to control group.
3.2Expression and Localization of
TGFβRI
Fig. 1A shows that TGFβR1protein showed
minimal expression liver tissues o f control groupanimals and also animals treated with single
CCl4dose as a model of acute toxicity. The
expression was increased on conversion of
toxicity from acute to chronic with maximal
expression in 6 and 11 weeks groups. As
indicated in Fig.1B; itwas noted that the
expression increased by about 146% in 6 weeks
CCl4treatment group and by 55 % in 11 weeks
CCl4treatment group. Regarding localization; it
was observed that the expression is located in
the cell membrane of epithelial hepatic tissue. It
was also noted that the expression occurred in
areas that shows maximal lesions as necrosis
and steatosis. On the other hand; it appears as
intense area filling the cytoplasm extending along
the fibrous septa and surrounding the cirrhotic
nodule in areas that show high density of
fibroblastic cells as shown in 11 weeks group. On
the other hand there was also minimal
expression inside the cirrhotic nodule.
Fig. 1A. Effect of CCl4 administration on hepatic expression of TGFβRI protein
Immunofluorescence staining of liver sections of CCl 4treated rats showing minimal TGFβR1 expression in
control group and also in single I.P. treated group. Six weeks CCl 4treated group showed maximal expression
located mainly in cell membrane (yellow rectangle).Eleven weeks treatment showed constitutive expression
(yellow rectangle) occurred mainly at area of fibrous septa or nodule border (white arrows) with minimal
expression in cirrh otic nodule (blue arrows)
Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
4Administration of I.P. CCl 4significantly elevated
serum transaminases activities compared to
control group whatever the duration of
administration. On the other hand; ALP showed
significant elevation only in chronic
administratio n of CCl 4with minimal change in
single dose CCl 4treatment. Supporting previous
results; albumin showed significant decrease
only with 11 weeks CCl 4treatment. On the other
hand; tissue MDA content was significantly
increased on CCl 4treated groups compared to
control group while SOD was shown to
significantly decrease in CCl 4treated groups
compared to control group.
3.2Expression and Localization of
TGFβRI
Fig. 1A shows that TGFβR1protein showed
minimal expression liver tissues o f control groupanimals and also animals treated with single
CCl4dose as a model of acute toxicity. The
expression was increased on conversion of
toxicity from acute to chronic with maximal
expression in 6 and 11 weeks groups. As
indicated in Fig.1B; itwas noted that the
expression increased by about 146% in 6 weeks
CCl4treatment group and by 55 % in 11 weeks
CCl4treatment group. Regarding localization; it
was observed that the expression is located in
the cell membrane of epithelial hepatic tissue. It
was also noted that the expression occurred in
areas that shows maximal lesions as necrosis
and steatosis. On the other hand; it appears as
intense area filling the cytoplasm extending along
the fibrous septa and surrounding the cirrhotic
nodule in areas that show high density of
fibroblastic cells as shown in 11 weeks group. On
the other hand there was also minimal
expression inside the cirrhotic nodule.
Fig. 1A. Effect of CCl4 administration on hepatic expression of TGFβRI protein
Immunofluorescence staining of liver sections of CCl 4treated rats showing minimal TGFβR1 expression in
control group and also in single I.P. treated group. Six weeks CCl 4treated group showed maximal expression
located mainly in cell membrane (yellow rectangle).Eleven weeks treatment showed constitutive expression
(yellow rectangle) occurred mainly at area of fibrous septa or nodule border (white arrows) with minimal
expression in cirrh otic nodule (blue arrows)
Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
4Administration of I.P. CCl 4significantly elevated
serum transaminases activities compared to
control group whatever the duration of
administration. On the other hand; ALP showed
significant elevation only in chronic
administratio n of CCl 4with minimal change in
single dose CCl 4treatment. Supporting previous
results; albumin showed significant decrease
only with 11 weeks CCl 4treatment. On the other
hand; tissue MDA content was significantly
increased on CCl 4treated groups compared to
control group while SOD was shown to
significantly decrease in CCl 4treated groups
compared to control group.
3.2Expression and Localization of
TGFβRI
Fig. 1A shows that TGFβR1protein showed
minimal expression liver tissues o f control groupanimals and also animals treated with single
CCl4dose as a model of acute toxicity. The
expression was increased on conversion of
toxicity from acute to chronic with maximal
expression in 6 and 11 weeks groups. As
indicated in Fig.1B; itwas noted that the
expression increased by about 146% in 6 weeks
CCl4treatment group and by 55 % in 11 weeks
CCl4treatment group. Regarding localization; it
was observed that the expression is located in
the cell membrane of epithelial hepatic tissue. It
was also noted that the expression occurred in
areas that shows maximal lesions as necrosis
and steatosis. On the other hand; it appears as
intense area filling the cytoplasm extending along
the fibrous septa and surrounding the cirrhotic
nodule in areas that show high density of
fibroblastic cells as shown in 11 weeks group. On
the other hand there was also minimal
expression inside the cirrhotic nodule.
Fig. 1A. Effect of CCl4 administration on hepatic expression of TGFβRI protein
Immunofluorescence staining of liver sections of CCl 4treated rats showing minimal TGFβR1 expression in
control group and also in single I.P. treated group. Six weeks CCl 4treated group showed maximal expression
located mainly in cell membrane (yellow rectangle).Eleven weeks treatment showed constitutive expression
(yellow rectangle) occurred mainly at area of fibrous septa or nodule border (white arrows) with minimal
expression in cirrh otic nodule (blue arrows)

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
5control4
single I.P. CCL4
6 weeks I.P.CCL4.
11 weeks I.P. CCL050100150200250Flourescence intensity
% of controlTGFR1
a
a,b
b
Fig. 1B. Effect of CCl4 administration on
hepatic expression of TGFβRI protein
Fluorescence intensity was obtained from 5 fields
of each section (minimally 2 rats of each group)
using ImageJ software.
Data are expressed as mean ± SEM
a)Significantly different from control groupb)Significantly different from 6 weeks CCl 4group
one-way ANOVA followed by Tukey -Kramer
test for multiple comparison test at P ≤0.05
3.3Expression and Localization of
FGFR3
Fig. 2A and Fig. 2B show that FGFR3 protein
shows minimal expression in liver tissues of
control group animals and also animals treated
with single CCl 4dose as a model of acute
toxicity. The expression was increased on
conversion of toxicity from acute to chronic with
maximal expression in 6 and 11 weeks groups. It
was observed that FGFR3 expression occurs in
the cytoplasm of the cells that found in the
affected areas as areas of steatosis, underlying
fibrosis and fib rous septa. It was noted that the
expression increased by about 146% in 6 weeks
CCl4treatment group and. Intraperitoneal
administration of CCl 4for 11 weeks resulted in
maximal expression that increased by about
222% compared to control.
Fig. 2A. Effect of CCl 4administration on hepatic expression and localization of FGFR3 in CCl4
treated rats
Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
5control4
single I.P. CCL4
6 weeks I.P.CCL4.
11 weeks I.P. CCL050100150200250Flourescence intensity
% of controlTGFR1
a
a,b
b
Fig. 1B. Effect of CCl4 administration on
hepatic expression of TGFβRI protein
Fluorescence intensity was obtained from 5 fields
of each section (minimally 2 rats of each group)
using ImageJ software.
Data are expressed as mean ± SEM
a)Significantly different from control groupb)Significantly different from 6 weeks CCl 4group
one-way ANOVA followed by Tukey -Kramer
test for multiple comparison test at P ≤0.05
3.3Expression and Localization of
FGFR3
Fig. 2A and Fig. 2B show that FGFR3 protein
shows minimal expression in liver tissues of
control group animals and also animals treated
with single CCl 4dose as a model of acute
toxicity. The expression was increased on
conversion of toxicity from acute to chronic with
maximal expression in 6 and 11 weeks groups. It
was observed that FGFR3 expression occurs in
the cytoplasm of the cells that found in the
affected areas as areas of steatosis, underlying
fibrosis and fib rous septa. It was noted that the
expression increased by about 146% in 6 weeks
CCl4treatment group and. Intraperitoneal
administration of CCl 4for 11 weeks resulted in
maximal expression that increased by about
222% compared to control.
Fig. 2A. Effect of CCl 4administration on hepatic expression and localization of FGFR3 in CCl4
treated rats
Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
5control4
single I.P. CCL4
6 weeks I.P.CCL4.
11 weeks I.P. CCL050100150200250Flourescence intensity
% of controlTGFR1
a
a,b
b
Fig. 1B. Effect of CCl4 administration on
hepatic expression of TGFβRI protein
Fluorescence intensity was obtained from 5 fields
of each section (minimally 2 rats of each group)
using ImageJ software.
Data are expressed as mean ± SEM
a)Significantly different from control groupb)Significantly different from 6 weeks CCl 4group
one-way ANOVA followed by Tukey -Kramer
test for multiple comparison test at P ≤0.05
3.3Expression and Localization of
FGFR3
Fig. 2A and Fig. 2B show that FGFR3 protein
shows minimal expression in liver tissues of
control group animals and also animals treated
with single CCl 4dose as a model of acute
toxicity. The expression was increased on
conversion of toxicity from acute to chronic with
maximal expression in 6 and 11 weeks groups. It
was observed that FGFR3 expression occurs in
the cytoplasm of the cells that found in the
affected areas as areas of steatosis, underlying
fibrosis and fib rous septa. It was noted that the
expression increased by about 146% in 6 weeks
CCl4treatment group and. Intraperitoneal
administration of CCl 4for 11 weeks resulted in
maximal expression that increased by about
222% compared to control.
Fig. 2A. Effect of CCl 4administration on hepatic expression and localization of FGFR3 in CCl4
treated rats

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
6Fluorescence intensity was obtained from 5 fields of each section (minimally 2 rats of each group)
using ImageJ software.
Data are expressed as mean ± SEM
(a) Significantly different from control group
(b) Significantly different from 6 weeks CCl 4group one -way ANOVA followed by Tukey -Kramer test for multiple
comparison test at P ≤0.05
FGFR3
control
single I.P. CCL46 weeks I.P.CCL4
11 weeks I.P. CCL4.0100200300400
baa,bFlourescence intensity
% of control
Fig.1B. Effect of CCl4 administration on
hepatic expression and localization of FGFR3
protein
Data are analysed using one -way ANOVA followed by
Tukey-Kramer test for multiple comparison at P ≤0.05.
a)Significantly different from control.
b)Significantly different from 6 weeks CCl 4group.
Fluorescence intensity was obtained from 5 fields
of each section (minimally 2 rats of each group)
using ImageJ software.3.4Histopathological Findings
Effects of CCl4treatment on histopathological
findings of liver tissue are represented in Table 2
and Fig. 3. The control group showed normal
hepatic architecture. On contrast, CCl 4treated
groups showed pathological findings changes
differed with the duration of tr eatment.
Single I.P. dose treated group showed average
portal tract without interface activity or underlying
fibrosis. On the other hand; it showed spotty
necrosis, and mild micro -vesicular steatosis and
dilated central vein. Six weeks treatment caused
expanded por tal tract with fibrous septa
extending from a portal tract to another, marked
micro-and macro -vesicular steatosis. The
hepatocytes exhibited some aspects of single
cell necrosis with dilated central veins and mild
interface activity.
Eleven weeks treat ment showed markedly
expanded portal tract with definite inflammatory
infiltrate, complete nodular formation (definite
cirrhosis) and marked micro -and macro –
vesicular steatosis. The hepatocytes exhibited
single cell necrosis with mild spotty necrosis.
Finally; the central veins were markedly dilated
with marked interface activity.
Table1. Histopathological findings of the studied groups
Control Single I.P. CCl 4CCl46 weeks CCl411 weeks
CV 0 + + ++
Steatosis 0 + +++ +++
Hepatocyte 0 ++ + +
Spotty necrosis 0 ++ 0 +
Interface activity 0 0 + ++
PT 0 0 + ++
Fibrosis 0 0 ++ ++++
Central vein (CV):
0: within normal +: dilated ++: markedly dilated
Steatosis, Spotty necrosis, Interface activity:
0: no +: mild ++: moderate to marked
Hepatocytes:
0: within normal +: single cell necrosis ++: confluent or diffuse necrosis
Portal tract (PT):
0: within normal +: expanded ++: expanded with i nflammatory infiltrate

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
7Fibrosis:
0: no fibrosis
+: fibrosis confined to enlarged portal zones
++: fibrosis of peri -portal or portal -portal septa with intact architecture
+++: architectural distortion (septal or bridging fibros is) without obvious cirrhosis
++++: probable or definite cirrhosis
Fig. 3.Histopathological micrograph of liver samples of CCl 4treated groups ×235 using H and
E stain. Control: liver tissue showing average portal tract (yellow arrows), average central vein
(red arrow), and average hepatocytes arranged in cords. Single I.P. CCl 4dose: liver tissue
showing average portal tract (yellow arrow), spotty necrosis (black arrow), and mi ld micro –
vesicular steatosis. 6 weeks I.P. CCl 4: liver tissue showing expanded portal tract with
underlying fibrosis and fibrous septa extending from a portal tract to another (yellow arrows),
marked micro -and macro -vesicular steatosis. 11 weeks I.P. CCl 4:liver tissue showing
markedly expanded portal tract (yellow arrows), complete nodular formation, marked micro –
and macro -vesicular steatosis
4. DISCUSSION
Administration of CCl 4either a single I.P. dose or
chronic I.P. doses for 6 or 11 weeks significantly
elevated serum transaminases enzymes
activities suggesting hepatocellular damage.
These results agreed with previous reports that
indicated that CCl 4significantly increases s erum
transaminases [25-27]. This CCl 4induced hepatic
damage was reported to be due to oxidative
stress[28,29]. This hepatocellular damage was
supported to some extent by serum ALP activity
that showed non -significant increase after single
I.P. CCl 4treatment compared to control group
suggesting minimal biliary injury detected upon
that model.This result opposes what was reported by Kālu’s
team[26]. This difference might be due
difference in animal strain or route of
administration that has not been men tioned. On
the other hand; ALP elevation in chronic models
including 6 weeks, 11 weeks groups showed
significant increase compared to control which is
less than 3 times as control; a result that might
suggest hepatotoxicity and mild biliary toxicity.
Thismatches with that previously reported by
Posen and Doherty [30].Continuous CCl 4for 11
weeks showed significant elevation in ALP
activity compared to 6 weeks CCl 4treatment
suggesting that chronic hepatocellular
intoxication might lead to biliary injury.
Supporting previous data; Serum albumin
Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
7Fibrosis:
0: no fibrosis
+: fibrosis confined to enlarged portal zones
++: fibrosis of peri -portal or portal -portal septa with intact architecture
+++: architectural distortion (septal or bridging fibros is) without obvious cirrhosis
++++: probable or definite cirrhosis
Fig. 3.Histopathological micrograph of liver samples of CCl 4treated groups ×235 using H and
E stain. Control: liver tissue showing average portal tract (yellow arrows), average central vein
(red arrow), and average hepatocytes arranged in cords. Single I.P. CCl 4dose: liver tissue
showing average portal tract (yellow arrow), spotty necrosis (black arrow), and mi ld micro –
vesicular steatosis. 6 weeks I.P. CCl 4: liver tissue showing expanded portal tract with
underlying fibrosis and fibrous septa extending from a portal tract to another (yellow arrows),
marked micro -and macro -vesicular steatosis. 11 weeks I.P. CCl 4:liver tissue showing
markedly expanded portal tract (yellow arrows), complete nodular formation, marked micro –
and macro -vesicular steatosis
4. DISCUSSION
Administration of CCl 4either a single I.P. dose or
chronic I.P. doses for 6 or 11 weeks significantly
elevated serum transaminases enzymes
activities suggesting hepatocellular damage.
These results agreed with previous reports that
indicated that CCl 4significantly increases s erum
transaminases [25-27]. This CCl 4induced hepatic
damage was reported to be due to oxidative
stress[28,29]. This hepatocellular damage was
supported to some extent by serum ALP activity
that showed non -significant increase after single
I.P. CCl 4treatment compared to control group
suggesting minimal biliary injury detected upon
that model.This result opposes what was reported by Kālu’s
team[26]. This difference might be due
difference in animal strain or route of
administration that has not been men tioned. On
the other hand; ALP elevation in chronic models
including 6 weeks, 11 weeks groups showed
significant increase compared to control which is
less than 3 times as control; a result that might
suggest hepatotoxicity and mild biliary toxicity.
Thismatches with that previously reported by
Posen and Doherty [30].Continuous CCl 4for 11
weeks showed significant elevation in ALP
activity compared to 6 weeks CCl 4treatment
suggesting that chronic hepatocellular
intoxication might lead to biliary injury.
Supporting previous data; Serum albumin
Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
7Fibrosis:
0: no fibrosis
+: fibrosis confined to enlarged portal zones
++: fibrosis of peri -portal or portal -portal septa with intact architecture
+++: architectural distortion (septal or bridging fibros is) without obvious cirrhosis
++++: probable or definite cirrhosis
Fig. 3.Histopathological micrograph of liver samples of CCl 4treated groups ×235 using H and
E stain. Control: liver tissue showing average portal tract (yellow arrows), average central vein
(red arrow), and average hepatocytes arranged in cords. Single I.P. CCl 4dose: liver tissue
showing average portal tract (yellow arrow), spotty necrosis (black arrow), and mi ld micro –
vesicular steatosis. 6 weeks I.P. CCl 4: liver tissue showing expanded portal tract with
underlying fibrosis and fibrous septa extending from a portal tract to another (yellow arrows),
marked micro -and macro -vesicular steatosis. 11 weeks I.P. CCl 4:liver tissue showing
markedly expanded portal tract (yellow arrows), complete nodular formation, marked micro –
and macro -vesicular steatosis
4. DISCUSSION
Administration of CCl 4either a single I.P. dose or
chronic I.P. doses for 6 or 11 weeks significantly
elevated serum transaminases enzymes
activities suggesting hepatocellular damage.
These results agreed with previous reports that
indicated that CCl 4significantly increases s erum
transaminases [25-27]. This CCl 4induced hepatic
damage was reported to be due to oxidative
stress[28,29]. This hepatocellular damage was
supported to some extent by serum ALP activity
that showed non -significant increase after single
I.P. CCl 4treatment compared to control group
suggesting minimal biliary injury detected upon
that model.This result opposes what was reported by Kālu’s
team[26]. This difference might be due
difference in animal strain or route of
administration that has not been men tioned. On
the other hand; ALP elevation in chronic models
including 6 weeks, 11 weeks groups showed
significant increase compared to control which is
less than 3 times as control; a result that might
suggest hepatotoxicity and mild biliary toxicity.
Thismatches with that previously reported by
Posen and Doherty [30].Continuous CCl 4for 11
weeks showed significant elevation in ALP
activity compared to 6 weeks CCl 4treatment
suggesting that chronic hepatocellular
intoxication might lead to biliary injury.
Supporting previous data; Serum albumin

Elsakka et al.; IJBCRR, 12(3):1-10, 2016; Article no. IJBCRR.26031
8concentration showed only significant decrease
from control at 11 weeks CCl 4treatment, a group
that showed cirrhotic nodule formation
suggesting decreased functioning tissue mass as
reported by Giannini and his team [31].
Meanwhile; it was clarified that free radicals
production and oxidative stress are main players
in liver injury especially CCl 4induced liver injury
[28,29,32-34]; reports that agreed with our
results that showed that administration of CCl 4
significantly elevated tissue content of MDA and
reduced serum SOD activity.
Supporting the measured biochemical data: Our
findings demonstrated that single I.P. dose of
CCl4resulted in marked spotty necrosis and
confluent hepatocytes necrosis. On the other
hand prolonged administration of CCl 4to 6
weeks produced fibrosis with intact architecture
while prolonged CCl 4to 11 weeks produced
definite cirrhosis with clear cirrhotic nodule. This
agreed with what has been reported that
histopathological chang es occur according to
CCl4administration period. A single dose of CCl 4
leads to centrizonal necrosis and steatosis [35],
while continued administration leads to hepatic
fibrosis, cirrhosis, and HCC [36].
The TGF -β is the principal regulator in chronic
liver injury sharing in all stages of disease
progression [37]. Its action begins by binding to
its receptor TGFβRI and TGFβRII [38]. It was
observed that TGFβR1expression occurs
intracellularly in the cell membrane. This agrees
with Massague and Chen repo rts[39]. It was
also shown that TGFβR1 expression was only
upregulated in chronic liver toxicity; specifically
phases that showed some extent of tissue
remodeling prescribed by Devaux and his team
[40]suggesting involvement of TGFβR1in
cellular processes involved in chronic not acute
liver injury. This was similar to the previous
reports in myocardial infarction (MI) by Devaux
and his team [40]. Besides; they also showed
that the maximal expression occ urs in lesion
areas suggesting the direct relationship between
TGFβR1expression and lesions grade; a result
that also is quite similar to Devaux and his team
reports[40].
The FGF/FGFR signaling axis has been reported
to be a contributor to fibro sis in the liver [8,9]. On
the other hand FGFR3 was reported to be
activated by FGF2 [41]; a cytokine that has been
believed to be involved in fibrotic disorders
[42,43].Our results showed that FGFR3 expression
increased in chronically intoxicated groups
suggesting involvement of FGFR3 in chronic liver
injury processes. A result that is quite similar to
reports of Lee’s team [44]. Lee and his team
have also reported that expression of all FGFR
seem to be cytoplasmic. This is quite similar to
our results. O ur results showed that the
expression of FGFR3 appeared as large spread
spot that may indicate that the expression was
cytoplasmic.
5. CONCLUSION
In conclusion, our findings indicate, for the first
time, that TGFβR1 and FGFR3 are upregulated
in chronic not acute liver injury. TGFβR1 is
upregulated in injury combined with underlying
fibrosis or cirrhosis while FGFR3 is upregulated
in injury combined with steatosis, fibrosis and
cirrhosis; results that may suggest usage of
TGFβR1 and FGFR3 as a candidate markers for
diagnosis and prognosis of chronic liver diseases
and a target for liver disease therapy.
COMPETING INTERESTS
Authors have declared tha t no competing
interests exist.
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