___________________________________________________ __________________________________________________ [600931]

___________________________________________________ __________________________________________________

*Corresponding author: E-mail: elsayedelsakka750@ya hoo.com, [anonimizat];

International Journal of Biochemistry Research
& Review
12(3): 1-10, 2016, Article no.IJBCRR.25627
ISSN: 2231-086X, NLM ID: 101654445

SCIENCEDOMAIN international
www.sciencedomain.org

Sulphasalazine Prevents Fibrosis; Relevance of
TGF βRI

Elsayed Gomaa Elsayed Elsakka 1* , Gamil Mohammed Abd-Allah 1,
Ahmed Ibrahim Abulsoud 1, Ahmed Mohammed Ibrahim Mansour 2
and Sayed Abdel Raheem 3

1Department of Biochemistry, Faculty of Pharmacy, Alazhar University, Cair o, 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 al l authors. Authors EGEE, GMAA and AMIM
designed the study, wrote the protocol and supervised the work. Authors EGEE and AMIM carried out
the animal modeling. Authors EGEE and AIA carried out laboratory work, performed the statistical
analysis, wrote the first draft of the manuscript, mana ged the literature searches and edited the
manuscript. Author SAR carried out the histopathologi cal assessment. All authors read and approved
the final manuscript.

Article Information

DOI: 10.9734/IJBCRR/2016/25627
Editor(s):
(1) Hector M. Mora Montes, Department of Biology, U niversity of Guanajuato, Mexico.
(2) Richard A. Manderville, Departments of Chemistry and Toxicology University of Guelph, Canada.
Reviewers:
(1) Anonymous, China Three Gorges University, China.
(2) Sahar S. Abd El-Rahman, Cairo University, Giza, Egypt.
(3) Anonymous, Sapienza University, Rome, Italy.
(4) Sura Wanessa Santos Rocha, Research Center Aggeu Maga lhaes, Brazil.
(5) Darko Nozic, Military Medical Academy, Belgrade , Serbia.
Complete Peer review History: http://sciencedomain.org/review-history/14979

Received 12 th March 2016
Accepted 3rd June 2016
Published 10 th June 2016

ABSTRACT

Aims: The aim of this study is to investigate the protecti ve effect of sulphasalazine on liver fibrosis.
Besides; investigation of the expression pattern of tr ansforming growth factor β receptor I (TGF βRI)
in liver fibrosis and the possible modulatory effect of sulphasalazine on it.
Study Design: Controlled experiment.
Place and Duration of Study: Department of Biochemistry and Department of Pharma cology and
Toxicology, Faculty of pharmacy (boys) Al-Azhar Unive rsity, between February 2015 and June
2015. Original Research Article

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no .IJBCRR.25627

2
Methodology: Five Sprague-Dawley rats groups were used for the e xperiment. Control group:
Receiving corn oil; DMSO group: Injected with DMSO; 6 weeks group: Injected with 50% CCl 4 in
corn oil; sulphasalazine group: Injected with sulphasalazi ne and CCl 4 and finally mesalazine group:
were given mesalazine and CCl 4. On the day after the last dose, rats were anestheti zed with diethyl
ether and blood samples were collected for measurement of blood chemistry. The animals then
were euthanized, and livers were harvested and divide d into 2 parts; one part was processed for
standard histology and immunofluorescence techniques an d the other was homogenized for
oxidative status assessment.
Results: TGF βRI has been shown to be upregulated in chronic liver injury; fibrosis stage; with
expression occurring mainly in cell membrane of lesion area. This expression was decreased
significantly with sulphasalazine treatment. Sulphasala zine has shown to have ability to diminish
fibrosis in chronic liver injury. This decrease in fi brosis observed with sulphasalazine was in parallel
with decrease in TGF βRI expression. Besides; this action of sulphasalazine ha s been suggested to
be due to the whole molecule not to its moiety; mes alazine.
Conclusion: TGF βRI may be used as a candidate marker for diagnosis an d prognosis of chronic
liver diseases. Besides; it may be used as a target thera py for chronic liver diseases. Moreover;
sulphasalazine might be a promising adjuvant therapy fo r chronic liver diseases.

Keywords: TGF βRI; fibrosis; sulphasalazine; mesalazine.

1. INTRODUCTION

Liver plays an unlimited 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].

Transforming growth factor β receptors
(TGF βRs) are of 2 types TGF- βRІ and TGF-βRІІ.
They mediate Hepatic stellate cells (HSCs)
activation [3] after binding to their ligand cytokine
TGF β, the profibrogenic cytokine that is
traditionally considered the key fibrogenic
stimulus to HSC [4].

Carbon tetra chloride (CCl 4) is one of the most
communal models for inducing hepatotoxicity. It
is transformed into a toxic CCl 3- radical by
hepatic cytochrome P 450 2E1 (CYP2E1). Thus;
it brings an acute Centro-lobular necrosis which
starts a wound healing response (fibrosis) [5].
However, Contact to these chemicals in humans
is rare and generally occurs in the manufacturing
during fabrication and in places where these
chemicals is usually used [6].

Hepatic stellate cells are the most important
player in hepatic injury. Quiescent HSCs are
characterized by significant desmin expression
and vitamin A storage. Following liver injury,
HSCs lose their vitamin A content, rise the
expression of α-smooth muscle actin ( α-SMA)
and gain a myofibroblast- like phenotype [7]. These events are primarily triggered by
mediators released by activated Kupffer cell and
injured hepatocytes [8].

Sulfasalazine is a synthetic drug obtained from
the grouping of sulfapyridine and 5-aminosalicylic
acid (mesalazine), an antibiotic and an anti-
inflammatory agent, respectively. This drug is
usually used in the inflammatory diseases of the
large intestine and rheumatoid arthritis [9]

Despite great knowledge about liver fibrosis,
there are little drugs approved for management
of it. So the aim of this work is to investigate the
possible protective effect of sulphasalazine on
liver fibrosis; an aspect that has gone side by
side with investigation of expression pattern of
TGF βRI in liver fibrosis and investigation of the
possible modulatory effect of sulphasalazine
on it.

2. METHODOLOGY

2.1 Animal Model

Adult male Sprague-Dawley (SD) rats weighing
250–300 g and aging 70 days were used in 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 in 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,
55% relative humidity with 12:12-h light: Dark
cycle. They were maintained on a standard

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no .IJBCRR.25627

3
rodent chow (El-Nasr Company, Abou-Zaabal,
Cairo, Egypt) and allowed to food and water ad
libitum . Animals were randomly divided into five
groups; (1) control group: 9 rats received
corn oil (2 mg/kg/twiceweekly/intraperitoneal(IP)/
repeated for 6 weeks); (2)DMSO group: 12 rats
were injected with DMSO, the vehicle of
sulphasalazine (1 ml/kg/day/IP/repeated for
6 weeks) and 50% CCl 4 in corn oil
(4 ml/kg/IP/twice weekly/repeated for 6 weeks);
(3) 6 weeks group:12 rats were injected with 50%
CCl4 in corn oil (4 ml/kg/IP/twice weekly/repeated
for 6 weeks); (4) sulphasalazine group: 12 rats
were injected with sulphasalazine (75
mg/kg/day/IP/repeated for 6 weeks) [10] and
CCl4 as previously indicated in 6 weeks group
and (5) mesalazine group: 12 rats were given
mesalazine (100 mg/kg/day/orally by oral
gavage/repeated for 6 weeks) [11] and CCl 4 as
previously indicated in 6 weeks group. At the
end of experimental period, rats were
anesthetized with diethyl ether and blood
samples have been drawn from retro orbital
plexus for measurement of blood chemistry. The
animals then were euthanized, and tissue
samples from the livers were harvested and
divided into 2 parts; one part was processed by
standard histology and immunofluorescence
techniques and the other was homogenized in
0.15 M KCl for oxidative stress assay. All animal
procedures were performed in accordance with
the international guide for the care and use of
laboratory animals [12].

2.2 Antibodies and Chemicals

Rabbit polyclonal TGF βR1 antibody was
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 indole
(DAPI). CCl 4 and DMSO were purchased from
Sigma-Aldrich (MO, USA). Sulphasalazine was
kindly supplied as yellowish powder by El-Kahera
Pharmaceuticals Company, Cairo, Egypt and
mesalazine was kindly supplied as white powder
by Pharopharm pharmaceuticals company,
Alexandria, Egypt.

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) [13,14].
Alkaline phosphatase (ALP) activity was assayed
according to the method of IFCC [15-17] and serum albumin concentration according to the
method described by Gendler [18]. The liver
homogenate was used for the determination of
the oxidative stress parameters. The level of
thiobarbituric acid-reactive substances was
assayed as malondialdehyde (MDA) as
described by Mihara and Uchiyama [19]. The
activity of SOD was determined using the
method described by Marklund [20].

2.4 Immunofluorescence Analysis

Liver tissues sections were handled according to
method described by Abdel-Bakky et al. [21]. The
primary TGF βR1 antibody was 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 Histopathological Examination

Liver samples were taken from rats belonging to
the different groups and fixed in 10% neutral
buffered formalin for 24 hours. Washing was
done in tap water, and then serial dilutions of
alcohol (methyl, ethyl and absolute ethyl) were
used for dehydration. Specimens were cleared in
xylene and embedded in paraffin at 56°C in hot
air oven for 24 h. Paraffin wax tissue blocks were
prepared for sectioning at 4 microns thickness by
sledge microtome.

The obtained tissue sections were collected on
glass slides, deparaffinized, stained by
hematoxylin & eosin stain and then examination
was done through a light electric microscope
[22]. Histopathological grading was achieved by
subjective scoring approved by pathology
department, faculty of pharmacy, Al-Azhar
university, Cairo, Egypt with assistance of
attached clues.

2.6 Statistical Analysis

Data were presented as the mean ±SE.
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).

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no .IJBCRR.25627

4
3. RESULTS

3.1 Liver 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 has been determined
through MDA and SOD assay as indicated in
Table 1 and Table 2.

As indicated in Table 1 and Table 2; Serum ALT
and AST activity of control group rats were
46.6±2.85 and 54.1±2.65 respectively.
Administration of CCl 4 I.P. alone or combined
with DMSO for 6 weeks significantly increased
ALT and AST activity compared to control group.
Interestingly concomitant administration of CCl 4
with sulphasalazine reduced serum activity of
AST compared to DMSO group.

As shown in Table 1 and Table 2; ALP activity of
control group rats was 312±21.9. Administration
of CCl 4 I.P. for 6 weeks increased ALP
significantly by 83% if given only and by 86%
in combination with DMSO compared to
control group. Concomitant administration of
sulphasalazine with CCl 4 decreased ALP activity
by 10% compared to DMSO group.

Serum albumin of control group was 3.81±0.08
g/dl. A significant decrease was observed only in
mesalazine group as shown in Table 1 and
Table 2.
As shown in Table 1 and Table 2; the hepatic
MDA content of control group was 4.73±0.318
nmol/g tissue. This content was significantly
increased on CCl 4 treated groups to be
31.9±1.44 (nmol/g tissue) for 6 weeks group,
31.7±1.82 (nmol/g tissue) for DMSO group and
25.7±1.5 (nmol/g tissue) for sulphasalazine
group. On the other hand; combination of CCl 4
with mesalazine showed MDA content of
9.86±0.339 respectively; results that showed
significant decrease compared to 6 weeks CCl 4
treatment although they appeared non-
significantly changed from control group. Finally
sulphasalazine showed a significant decrease in
MDA content compared to DMSO group.

Table 1 and Table 2 show the M ± SEM for SOD
of studied groups. The control group showed
tissue SOD activity of 526±25.2 U/mg tissue.
This activity was significantly decreased in other
groups to become 242±15.4, 223±14, 281±16
and 225±9.65 for DMSO, 6 weeks,
sulphasalazine, and mesalazine groups.

3.2 Expression and Localization of
TGF ΒR1

Fig. 1 and Graph 1 show that TGF βR1 protein
showed minimal expression in normal liver
tissues. The tissue expression was showed to be
maximal in 6 weeks CCl 4 groups. It is noted that
the expression increased by about 146% in 6
weeks CCl 4 treatment group. It is also observed
that the expression was located in the cell
membrane of epithelial hepatic tissue in areas
that shows great lesions.

Table 1. Liver functions and oxidative status asses sment tests (samples were taken from all
animals belonging to each group)

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 ±0.32 526±25.2
CCl4 6 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)
CCl4 6 weeks
I.P. +DMSO I.P. 332±12.8 (a) 402±8.91 (a) 580±21.1 (a) 3.35±0.12 31.7±1.82 (a) 242±15.4 (a)
CCl4 6 weeks
I.P. +
sulphasalazine
I.P. 328±19 (a) 338±14.5 (a) (b)(c) 523±19.8 (a) 3.34±0.12 25.7±1.5 (a) 281±16 (a)
CCl4 6 weeks
I.P. + oral
mesalazine 315±19 (a) 486±24.2 (a) 701±44 (a)(b ) 3.18±0.16 (a) 9.86 ±0.34 (b) 225±9.65 (a)
Data are expressed as mean ± SEM
(a) Significantly different from control group
(b) Significantly different from 6 weeks CCl 4 group
(c) Significantly different from 6 weeks CCl 4 + DMSO (for sulphasalazine) using one-way ANOVA foll owed by Tukey-Kramer
test for multiple comparison test at P ≤0.05

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no .IJBCRR.25627

5
Table 2. Liver functions and oxidative status asses sment tests (percentage change from
control) (samples were taken from all animals belon ging to each group)

ALT
(%change) AST
(%change) ALP
(%change) Albumin
(%change) MDA
(%change) SOD
(%change)
Control 100±6.12 100±4.9 100±7.02 100±2.1 100±6.77 100±4.79
CCl4 6 weeks
I.P. 749±28.8 (a) 806±28.47 (a) 183±7.85 (a) 89±3.41 674±30.4 (a) 42.4±2.66 (a)
CCl4 6 weeks
I.P. +DMSO
I.P. 712±27.5 (a) 743±16.5 (a) 186±6.76 (a) 87.9±3.15 670±38.5 (a) 46±2.93 (a)
CCl4 6 weeks
I.P. +
sulphasalazine
I.P. 704±40.8 (a) 625±26.8 (a)(b)(c) 168±6.35 (a) 87.7±3.15 543±31.7 (a) 53.4±3.04 (a)
CCl4 6 weeks
I.P. + oral
mesalazine 676±40.8 (a) 898±44.7 (a) 225±4.1 (a)(b) 83.5±4.2 (a) 208 ±7.19 (b) 42.8±1.83 (a)

Fig. 1. Immunofluorescence staining of liver sections of st udied groups showing minimal
TGF βR1 expression in control group. Six weeks CCl 4 treated group shows maximal expression
located mainly in cell membrane (yellow rectangle). It is noted also that there is non-significant
expression change between 6 weeks CCl 4 treatment alone or combined with DMSO.
Combination of CCl 4 with sulphasalazine or mesalazine show decrease in t he expression
compared to 6 weeks CCl 4 treatment with maximal decrease occurring with sulp hasalazine

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no .IJBCRR.25627

6
It is also noted that concomitant administration of
DMSO and CCl 4 for 6 weeks produced non-
significant change from CCl 4 only. On the
other hand; Concomitant administration of
sulphasalazine and CCl 4 decreased the
expression by about 46% compared to DMSO
group while concomitant administration of
mesalazine with CCl 4 reduced the expression by
about 36% compared to 6 weeks CCl 4 group.

TGF ββ ββR1
control 4
6 weeks I.P. CCL
+DMSO
4
6 weeks IP CCL
+sulphasalazine
4
6 weeks IP CCL + mesalazine
4
6 weeks IP CCL 050 100 150 200 250
aa
a,b
b,c Flourescence intensity
% of control

Graph 1. Fluorescence intensity was obtained
from 5 fields of each liver 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 4 group
(c) Significantly different from 6 weeks CCl 4 + DMSO
using one-way ANOVA followed by Tukey-Kramer test
for multiple comparison test at P ≤0.05

3.3 Histopathological Findings

Effects of CCl 4 treatment on histopathological
findings of liver tissue are represented in Table 3
and Fig. 2; the control group shows normal
hepatic architecture. On contrast, six weeks CCl 4
treatment shows expanded portal tract with
fibrous septa extending from a portal tract to
another (yellow arrows), marked micro- and
macro-vesicular steatosis. The hepatocytes
exhibit some aspects of single cell necrosis with
dilated central veins and mild interface activity.

It is noted that sulphasalazine had minimal
effect on steatosis. Interestingly; despite
sulphasalazine group animals show marked
steatosis, they also show completely diminished
underlying fibrosis in their liver tissues. Finally; combination of mesalazine with CCl 4
results in some alleviation in injury signs.
However; it seems not to have positive effects
regarding steatosis or fibrosis.

Histopathological micrograph of liver samples of
CCl4 treated groups combined with DMSO,
sulphasalazine and mesalazine ×235 using H
and E stain. Control: liver tissue showing
average PT (yellow arrows), average central vein
(red arrow), and average hepatocytes arranged
in cords. 6 weeks I.P. CCl 4: Liver tissue showing
expanded PT with underlying fibrosis and fibrous
septa extending from PT to PT (yellow arrows),
marked micro- and macro-vesicular steatosis. 6
weeks I.P. CCl 4 + I.P. DMSO: Liver tissue
showing average portal tract (yellow arrows),
fibrous septa extending from PT (blue arrows),
average hepatocytes and moderate steatosis.
6 weeks I.P. CCl 4 + I.P. sulphasalazine: Liver
tissue showing average PT (yellow arrow), and
marked steatosis. 6 weeks I.P. CCl 4 + oral
mesalazine: Liver tissue showing average PT
(yellow arrow), short fibrous septa (red arrow),
average hepatocytes and moderate steatosis.

4. DISCUSSION

Administration of I.P. CCl 4 for 6 weeks has
significantly elevated serum transaminases
enzymes activities suggesting hepatocellular
damage. These results agreed with previous
reports that CCl 4 significantly increases serum
transaminases [23-25]. This CCl 4 induced
hepatic damage has been reported to be
due to oxidative stress [26,27]. Concomitant
administration of sulphasalazine with CCl 4 has
reduced AST not ALT activity compared to
DMSO group suggesting the positive effect
toward liver injury. These effects might be due to
the anti-inflammatory action of sulphasalazine.
This disagrees with what has been reported by
Jennings and his coworkers a result that was
interpreted by the relationship of hepatic injury,
steatosis, fibrosis and cirrhosis to ulcerative
colitis not sulphasalazine medication [28].

ALP activity caused by IP CCl4 administration for
6 weeks and DMSO group has showed
significant increase compared to control which
was less than 3 times as control; a result that
might suggest hepatotoxicity and mild biliary
toxicity. This matches with that previously
reported by Posen and Doherty [29].
Concomitant administration of mesalazine and
CCl4 for 6 weeks has significantly elevated ALP
activity compared to 6 weeks CCl 4 treatment.

This result might sug gest the toxic effect of
mesalazine with the mentioned regimen on
hepatic tissue or synergistic effect of mesalazine
on CCl 4 toxicity; a suggestion that might be clear
upon ALP and albumin result.

Meanwhile; it was clarified that free radicals
production and oxidative stress are main players
in liver injury especially CCl 4 induced liver injury
[26,27]. In our study; administration of CCl
significantly elevated tissue content of MDA and
reduced serum SOD a ctivity. These results

Table 3. Histopathological findings of the studied groups

Control CCl 4
6 weeks
CV 0 +
Steatosis 0 +++
Hepatocyte 0 +
Spotty necrosis 0 0
Interface
activity 0 +
PT 0 +
Fibrosis 0 ++
Central vein (CV):
Steatosis, Spotty necrosis, Interface activity:
Hepatocytes: 0: within normal +: single cell necrosis++: confluent o
Portal tract: 0: within normal
+: fibrosis confined to enlarged portal zones
++: fibrosis of peri
+++: architectural distortion (septal or bridging fibrosis) without obvious cirrhosis

Fig. 2. Effect of CCl 4 administration on hepatic histopathological finding s with investigation of
the possi ble modulatory effect of sulphasalazine and mesalazi ne (minimally 2 rats of each
group with 5 fields of each rat at least) Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article

7
gest the toxic effect of
mesalazine with the mentioned regimen on
hepatic tissue or synergistic effect of mesalazine
toxicity; a suggestion that might be clear
Meanwhile; it was clarified that free radicals
and oxidative stress are main players
induced liver injury
. In our study; administration of CCl 4 has
significantly elevated tissue content of MDA and
ctivity. These results agreed with the previous reports that
demonstrated the great role of oxidative stress in
CCl4 induced liver injury [24,30,31 ]

Supporting the measured biochemical data: Our
histopatholo gical findings have demonstrated
that administration of CCl 4 for 6 weeks produced
fibrosis of peri-portal or portal- portal fibrosis with
intact architecture. This is similar to what has
been reported that continued administration of
CCl4 leads to hepatic f ibrosis, cirrhosis, and
hepatocellular carcinoma [32].
Histopathological findings of the studied groups (minimally 2 rats of each group with
5 fields of each rat at least)

4
6 weeks CCl 4 6 weeks
+ DMSO CCl 4 6 weeks +
sulphasalazine CCl
mesalazine
0 0 0
++ +++ ++
0 0 0
+ 0 0
0 0 0
++ 0 0
++ 0 ++
Central vein (CV): 0: within normal +: dilated ++: markedly dilated
Steatosis, Spotty necrosis, Interface activity: 0: no +: mild ++: moderate to marked
0: within normal +: single cell necrosis++: confluent o r diffuse necrosis
0: within normal +: expanded ++: expanded with inflammatory infiltrate
Fibrosis: 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 fibrosis) without obvio us cirrhosis
++++: probable or definite cirrhosis

administration on hepatic histopathological finding s with investigation of
ble modulatory effect of sulphasalazine and mesalazi ne (minimally 2 rats of each
group with 5 fields of each rat at least)

no.IJBCRR.25627

agreed with the previous reports that
demonstrated the great role of oxidative stress in
].
Supporting the measured biochemical data: Our
gical findings have demonstrated
for 6 weeks produced
portal fibrosis with
intact architecture. This is similar to what has
been reported that continued administration of
ibrosis, cirrhosis, and
(minimally 2 rats of each group with
CCl 4 6 weeks +
mesalazine

++

++

administration on hepatic histopathological finding s with investigation of
ble modulatory effect of sulphasalazine and mesalaz ine (minimally 2 rats of each

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no.IJBCRR.25627

8
It has been found that concomitant administration
of sulphasalazine and CCl 4 had markedly cleared
fibrosis; major findings seen in CCl 4. However,
there is no positive effect on steatosis. This may
be an expected result according to in-vitro study
reports of Oakley et al. [33] concerning the
inhibitory effect of sulphasalazine on the
machinery and machinery of fibrous tissue
synthesis.

As an interesting matter; the decrease in
histopathological scores observed in mesalazine
group didn’t agree with sulphasalazine group
although it was documented that sulphasalazine
is a prodrug for mesalazine in some
pharmacological actions [34]. This might suggest
that the action of sulphasalazine previously
reported by Oakley et al. [33] may be related to
the sulphasalazine molecule at all not related to
its moiety, mesalazine.

The TGF-β is the principal regulator in chronic
liver injury sharing in all stages of disease
progression [35]. Its action begins by binding to
its receptor TGF βRI and TGF βRII [36]. It has
been observed that TGF βR1expression occurred
in the cell membrane. This agrees with
Massague and Chen reports [37]. It has been
also shown that TGF βR1 expression was
upregulated only in chronic liver toxicity;
specifically phases that showed some extent of
tissue remodeling prescribed by Devaux et al.
[38] suggesting involvement of TGF βR1in
cellular processes involved in chronic not acute
liver injury. This was similar to the previous
reports in myocardial infarction (MI) by Devaux et
al. [38]. Besides; they also showed that the
maximal expression occurs in lesion areas
suggesting the direct relationship between
TGF βR1expression and lesions grade; a result
that also is quite similar to Devaux et al. [38]. I t
was also observed that TGF βR1expression was
nearly diminished in sulphasalazine group
suggesting the tight linkage between TGF βR1
expression and fibrosis but not steatosis. This
result can be explained by pro apoptotic effect of
sulphasalazine toward activated HSC, the major
cellular promoter of fibrogenesis [39].

5. CONCLUSION

Our findings indicate, for the first time, that
TGF βR1 content is increased in chronic liver
injury. TGF βR1 is upregulated in injury combined
with underlying fibrosis; a result that may suggest
usage of TGF βR1 as a candidate marker for
diagnosis and prognosis of chronic liver diseases and a target for liver disease therapy. Moreover,
sulphasalazine exhibits a fibroprotective effect in
experimental liver fibrosis. This suggests a
possible use of sulphasalazine as adjuvant in
therapy of chronic liver diseases.

ETHICAL APPROVAL

All authors hereby declare that all experiments
have been examined and approved by the
appropriate ethics committee of Alazhar
University.

COMPETING INTERESTS

Authors have declared that no competing
interests exist.

REFERENCES

1. Sharma A, Chakraborti KK, Handa SS.
Antihepatotoxic activity of some Indian
herbal formulations as compared to
silymarin. Fitoterapia. 1991;62:229-35.
2. Ostapowicz G, Fontana RJ, Schiodt FV,
Larson A, Davern TJ, Han SH, et al.
Results of a prospective study of acute
liver failure at 17 tertiary care centers in
the United States. Ann Intern Med.
2002;137(12):947-54.
3. Roberts AB. Tgf-beta signaling from
receptors to the nucleus. Microbes Infect.
1999;1(15):1265-73.
4. Tsukamoto H. Cytokine regulation of
hepatic stellate cells in liver fibrosis.
Alcohol Clin Exp Res. 1999;23(5):911-6.
5. Domenicali M, Caraceni P, Giannone F,
Baldassarre M, Lucchetti G, Quarta C, et
al. A novel model of CCl 4-induced cirrhosis
with ascites in the mouse. J Hepatol.
2009;51(6):991-9.
6. Mormone E, George J, Nieto N. Molecular
pathogenesis of hepatic fibrosis and
current therapeutic approaches. Chem Biol
Interact. 2011;193(3):225-31.
7. Gressner AM. Transdifferentiation of
hepatic stellate cells (ITO cells) to
myofibroblasts: A key event in hepatic
fibrogenesis. Kidney Int Suppl.
1996;54:s39-s45.
8. Nieto N. Oxidative-stress and il-6 mediate
the fibrogenic effects of [corrected] kupffer
cells on stellate cells. Hepatology.
2006;44(6):1487-501.
9. Ardizzone S, Bianchi PG. A practical guide
to the management of distal ulcerative
colitis. Drugs. 1998;55(4):519-42.

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no.IJBCRR.25627

9
10. Tugcu V, Ozbek E, Tasci AI, Kemahli E,
Somay A, Bas M, et al. Selective nuclear
factor kappa-b inhibitors, pyrolidium
dithiocarbamate and sulfasalazine, prevent
the nephrotoxicity induced by gentamicin.
Bju Int. 2006;98(3):680-6.
11. Hayashi Y, Aoyagi K, Morita I, Yamamoto
C, Sakisaka S. Oral administration of
mesalazine protects against mucosal injury
and permeation in dextran sulfate sodium-
induced colitis in rats. Scand J
Gastroenterol. 2009;44(11):1323-31.
12. Institute of Laboratory Animal Resources
(US). Use of Laboratory Animals, National
Institutes of Health (US). Guide for the
care and use of laboratory animals.
National Academies; 1985.
13. Bergmeyer HU, Horder M, Rej R.
International Federation of Clinical
Chemistry (IFCC) Scientific Committee,
analytical section: Approved
recommendation (1985) on IFCC methods
for the measurement of catalytic
concentration of enzymes. Part 2. IFCC
method for aspartate aminotransferase (l-
aspartate: 2-oxoglutarate aminotransfer-
ase, ec 2.6.1.1). J Clin Chem Clin
Biochem. 1986;24(7):497-510.
14. Bergmeyer HU, Horder M, Rej R.
International Federation of Clinical
Chemistry (IFCC) Scientific Committee,
analytical section: approved
recommendation (1985) on IFCC methods
for the measurement of catalytic
concentration of enzymes. Part 3. IFCC
method for alanine aminotransferase
(l-alanine:2-oxoglutarate aminotransferase,
ec 2.6.1.2). J Clin Chem Clin Biochem.
1986;24(7):481-95.
15. Tietz NW, Rinker AD, Shaw LM.
International Federation of Clinical
Chemistry. IFCC methods for the
measurement of catalytic concentration of
enzymes. Part 5. IFCC method for alkaline
phosphatase (orthophosphoric-monoester
phosphohydrolase, alkaline optimum, ec
3.1.3.1). IFCC document stage 2, draft 1,
1983-03 with a view to an IFCC
recommendation. Clin Chim Acta. 1983;
135(3):339f-67f.
16. Tietz NW, Rinker AD, Shaw LM. IFCC
methods for the measurement of catalytic
concentration of enzymes part 5. IFCC
method for alkaline phosphatase
(orthophosphoric-monoester phosphohy-
drolase, alkaline optimum, ec 3.1.3.1). J Clin Chem Clin Biochem. 1983;21(11):
731-48.
17. Tietz NW, Burtis CA, Duncan P, Ervin K,
Petitclerc CJ, Rinker AD, et al. A reference
method for measurement of alkaline
phosphatase activity in human serum. Clin
Chem. 1983;29(5):751-61.
18. Gendler S. Uric acid. Kaplan A et al. Clin
chem the CV Mosby Co St Louis Toronto
Princeton. 1984;1268-73.
19. Mihara M, Uchiyama M. Determination of
malonaldehyde precursor in tissues by
thiobarbituric acid test. Anal Biochem.
1978;86(1):271-8.
20. Marklund SL. Superoxide dismutase
isoenzymes in tissues and plasma from
New Zealand black mice, nude mice and
normal balb/c mice. Mutat Res. 1985;
148(1-2):129-34.
21. Abdel-bakky MS, Hammad MA, Walker LA,
Ashfaq MK. Tissue factor dependent liver
injury causes release of retinoid receptors
(RXR-alpha and RAR-alpha) as lipid
droplets. Biochem Biophys Res Commun.
2011;410(1):146-51.
22. Banchroft JD, Stevens A, Turner DR.
Theory and practice of histoloicl
techniques. Churchil Livingstone, New
York, London, San Francisco, Tokyo;
1996.
23. Ahsan R, Islam KM, Musaddik A, Haque E.
Hepatoprotective activity of methanol
extract of some medicinal plants against
carbon tetrachloride induced hepatotoxicity
in albino rats. Global J Pharmacol.
2009;3(3):116-22.
24. Kalu FN, Ogugua VN, Ujowundu CO,
Nwaoguikpe RN. Aqueous extract of
Combretum dolichopentalum leaf-A potent
inhibitor of carbon tetrachloride induced
hepatotoxicity in rats. Journal of Applied
Pharmaceutical Science. 2011;1(10):114.
25. Dalton SR, Lee SM, King RN, Nanji AA,
Kharbanda KK, Casey CA, et al. Carbon
tetrachloride-induced liver damage in
asialoglycoprotein receptor-deficient mice.
Biochemical Pharmacology. 2009;77(7):
1283-90.
26. Manibusan MK, Odin M, Eastmond DA.
Postulated carbon tetrachloride mode of
action: A review. J Environ Sci Health C
Environ Carcinog Ecotoxicol Rev. 2007;
25(3):185-209.
27. Weber LW, Boll M, Stampfl A.
Hepatotoxicity and mechanism of action of
haloalkanes: Carbon tetrachloride as a

Elsakka et al.; IJBCRR, 12(3): 1-10, 2016; Article no.IJBCRR.25627

10
toxicological model. Crit Rev Toxicol.
2003;33(2):105-36.
28. Jennings PE, Blandford RL, Rosenthal FD.
Acute sulphasalazine hepatotoxicity.
Postgrad Med J. 1986;62(726):305-6.
29. Posen S, Doherty E. The measurement of
serum alkaline phosphatase in clinical
medicine. Adv Clin Chem. 1981;22:
163-245.
30. Dalton SR, Lee SM, King RN, Nanji AA,
Kharbanda KK, Casey CA, et al. Carbon
tetrachloride-induced liver damage in
asialoglycoprotein receptor-deficient mice.
Biochem Pharmacol. 2009;77(7):1283-90.
31. Noori S, Rehman N, Qureshi M, Mahboob
T. Reduction of carbon tetrachloride-
induced rat liver injury by coffee and green
tea. Pakistan Journal of Nutrition. 2009;
8(4):452-8.
32. Tamayo RP. Is cirrhosis of the liver
experimentally produced by cc14 an
adequate model of human cirrhosis?
Hepatology. 1983;3(1):112-20.
33. Oakley F, Meso M, Iredale JP, Green K,
Marek CJ, Zhou X, et al. Inhibition of
inhibitor of kappa B kinases stimulates
hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis.
Gastroenterology. 2005;128(1):108-20.
34. Azad Khan AK, Piris J, Truelove SC. An
experiment to determine the active
therapeutic moiety of sulphasalazine.
Lancet. 1977;2(8044):892-5.
35. Dooley S, Ten DP. Tgf-beta in progression
of liver disease. Cell Tissue Res.
2012;347(1):245-56.
36. Devaux Y, Bousquenaud M, Rodius S,
Marie PY, Maskali F, Zhang L, et al.
Transforming growth factor beta receptor 1
is a new candidate prognostic biomarker
after acute myocardial infarction. BMC
Med Genomics. 2011;4:83.
37. Massague J, Chen YG. Controlling TGF-
beta signaling. Genes Dev. 2000;14(6):
627-44.
38. Devaux Y, Bousquenaud M, Rodius S,
Marie PY, Maskali F, Zhang L, et al.
Transforming growth factor beta receptor 1
is a new candidate prognostic biomarker
after acute myocardial infarction. Bmc Med
Genomics. 2011;4:83.
39. Friedman SL. Evolving challenges in
hepatic fibrosis. Nat Rev Gastroenterol
Hepatol. 2010;7(8):425-36.
___________________________________________________ ______________________________
© 2016 Elsakka et al.; This is an Open Access artic le distributed under the terms of the Creative Comm ons Attribution License
(http://creativecommons.org/licenses/by/4.0 ), which permits unrestricted use, distribution, an d reproduction in any medium,
provided the original work is properly cited.

Peer-review history:
The peer review history for this paper can be accessed her e:
http://sciencedomain.org/review-history/14979