Therapeutic effect of olmesartan medoxomile alone and in combination with sulfasalazine in experimentally ulcerative colitis model in rats

Abstract

Background: Ulcerative colitis is a chronically recurrent inflammatory bowel disease of unknown origin. There is, as yet, no cure for this condition (other than colectomy in ulcerative colitis). Accumulating evidence has indicated the implication of angiotensinⅡ in the pathogenesis of inflammatory bowel diseases (IBD) via its proinflammatory features. The present study aimed to investigate the potential therapeutic effects of Omesartan Medoxomil (OLM-M) alone and in combination with sulfasalazine (SZ) on acetic acid induced- ulcerative colitis in rats. Methods: A total of 35 male wistar rats were included in the study. Animals were divided into 5 groups (n = 7): group (normal control group), groupⅡ (acetic acid group), groupⅢ (acetic acid+Sulfasalazine 360 mg/kg p.o used as reference), groupⅣ (acetic acid+ Omesartan medoxomil 5 mg/kg p.o), groupⅤ (acetic acid+Sulfasalazine 360 mg/kg+ Olmesartan medoxomil 3 mg/kg p.o). Rats received treatment for seven consecutive days after induction of colitis by intra-rectal acetic acid (2ml 4% v/v) administration. Rats were sacrificed under ether anesthesia for assessment of the colonic mucosal injury using body weight loss, colon weight / length ratio, macroscopic damage, histological study, as well as by biochemical measurement of reduced glutathione (GSH). Results: Our results showed that SZ, OLM-M and their combination decreased body weight loss, weight/length ratio, macroscopic and microscopic colonic damage scores caused by administration of acetic acid. Also significantly increased the levels of glutathione compared to acetic acid-induced colitis group. Conclusion: The results suggested that intracolonic instillation of acetic acid produced acute colitis. Both SZ and OLM-M exerted anti-inflammatory and antioxidant effects on acetic-acid-induced colitis. In addition, OLM-M potentiated the anti-inflammatory and antioxidant effect of SZ. © 2016, International Journal of Toxicological and Pharmacological Research. All rights reserved.

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International Journal of Toxicological and Pharmacological Research 2016; 8(2); 97-106
ISSN: 0975-5160
Research Article
*Author for Correspondence
Therapeutic Effect of Olmesartan Medoxomile Alone and in
Combination with Sulfasalazine in Experimentally Ulcerative Colitis
Model in Rats
Darwish Fatima, Al laham Shaza*
Pharmacology and Toxicology department - Faculty of Pharmacy - Damascus University – Damascus- Syria.
Available Online:1st May, 2016
ABSTRACT
Background: Ulcerative colitis is a chronically recurrent inflammatory bowel disease of unknown origin. There is, as yet,
no cure for this condition (other than colectomy in ulcerative colitis). Accumulating evidence has indicated the implication
of angiotensinⅡ in the pathogenesis of inflammatory bowel diseases (IBD) via its proinflammatory features. The present
study aimed to investigate the potential therapeutic effects of Omesartan Medoxomil (OLM-M) alone and in combination
with sulfasalazine (SZ) on acetic acid induced- ulcerative colitis in rats. Methods: A total of 35 male wistar rats were
included in the study. Animals were divided into 5 groups (n = 7): group (normal control group), groupⅡ (acetic acid
group), groupⅢ (acetic acid+Sulfasalazine 360 mg/kg p.o used as reference), groupⅣ (acetic acid+ Omesartan medoxomil
5 mg/kg p.o), groupⅤ (acetic acid+Sulfasalazine 360 mg/kg+ Olmesartan medoxomil 3 mg/kg p.o). Rats received
treatment for seven consecutive days after induction of colitis by intra-rectal acetic acid (2ml 4% v/v) administration. Rats
were sacrificed under ether anesthesia for assessment of the colonic mucosal injury using body weight loss, colon weight
/ length ratio, macroscopic damage, histological study, as well as by biochemical measurement of reduced glutathione
(GSH). Results: Our results showed that SZ, OLM-M and their combination decreased body weight loss, weight/length
ratio, macroscopic and microscopic colonic damage scores caused by administration of acetic acid. Also significantly
increased the levels of glutathione compared to acetic acid-induced colitis group.
Conclusion: The results suggested that intracolonic instillation of acetic acid produced acute colitis. Both SZ and OLM-M
exerted anti-inflammatory and antioxidant effects on acetic-acid-induced colitis. In addition, OLM-M potentiated the anti-
inflammatory and antioxidant effect of SZ.
Keywords: colitis, Sulfasalazine, Olmesartan, Angiotensin, reduced glutathione.
INTRODUCTION
Ulcerative colitis (UC) and Crohn’s disease (CD) are
chronic, relapsing, immunologically mediated disorders
that are collectively referred to as inflammatory bowel
diseases (IBD)1. UC primarily affects the mucosal lining
of the colon and rectum, whereas CD can involve any
segment of the gastrointestinal tract2. Patients with
ulcerative colitis are at high risk of developing colorectal
carcinoma3. The main clinical manifestations are
abdominal pain, diarrhea, mucous, bloody, and purulent
stools, recurrent attacks and relapse4. Although the
etiology of IBD remains largely unknown, it involves a
complex interaction between the genetic, environmental or
microbial factors and the immune responses5. Activation
of intestinal immune system is associated with excessive
generation of inflammatory cytokines such as tumor
necrosis factor- α (TNF- α) which amplifies the
inflammatory cascade by triggering the generation of other
proinflammatory cytokines and enhancing the recruitment
of macrophages and neutrophils6. IL-1β appears to be a
primary stimulator of diarrhea the main symptom of
intestinal inflammation. Infiltration of neutrophils result in
the production of cytotoxic reactive oxygen species (ROS)
that are destructive on intestinal cell macromolecules,
ultimately leading to mucosal disruption and ulceration2.
While a number of medical strategies are available, many
of these have substantial side-effects including immune
suppression; thus, newer approaches are greatly needed7.
Sulfasalazine is one of the standard and orally used drugs
for the conservative management of IBD8. The benefits of
sulfasalazine generally are dose related. Therefore, high
doses of sulfasalazine may be necessary to induce
remission. Some patients cannot tolerate high dose
because of nausea and stomach upset9. It is necessary to
search for special target of inflammatory cascades in UC
so as to improve the therapeutic effects. The renin-
angiotensin system (RAS) is well known to have various
physiologic roles10, including effects on vascular tone,
hormone secretion, tissue growth, and neuronal
activities11. The role of RAS in inflammatory process is
gaining wide spread interest in recent times12 . Angiotensin
Ⅱ (AngⅡ), the main effector peptide of the renin
angiotensin system, has potent proinflammatory features

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linked with the pathogenesis of several chronic
inflammatory disorders including IBD13. Indeed,
(Hirasawa et al., 2002) reported colonic localization of
RAS14 .via its actions on angiotensin Ⅱ type1(AT1)
receptors Angiotensin Ⅱ promotes tissue inflammation
through upregulation of adhesion molecules, increasing
vascular permeability, and thus, enhancing neutrophil
infiltration, which contributes to gut ulceration15. It also
increases the release of proinflammatory cytokines such as
TNF-a probably, through activation of NF-κB.
Additionally, Ang Ⅱ triggers oxidative stress via
activation of NADH/NADPH oxidase with consequent
generation of superoxide anions13. Moreover, Ang Ⅱ is
known to regulate motility in the intestine, as well as ions
and water absorption via receptors in the mucosa and
muscle16. The expression of angiotensin converting
enzyme (ACE) and its associated receptors have been
identified within the gastrointestinal mucosa, and their
abundance has been shown to increase in states of intestinal
injury and colitis in both animal models and humans7.
Accumulating evidence has indicated the efficacy of the
drugs that affect the angiotensin Ⅱ such as angiotensin
converting enzyme inhibitors (ACE-I)17,18 , Angiotensin Ⅱ
receptor antagonists (ARBs)19,20 and renin inhibitor
(Aliskiren)12 in the attenuation of colon injury in
experimental colitis. Because combination treatment is the
preferred strategy for most patients of ulcerative colitis21.
These findings encouraged us to investigate the potential
therapeutic effects of OLM-M alone and in combination
with sulfasalazine on acetic acid induced- ulcerative colitis
in rats, an experimental model of human UC.
Acetic acid (AA) induced UC model is an easily inducible
model of UC in rats bears similarities to acute human UC
in terms of histopathological appearances22
MATERIAL & METHOD
Animals
Adult male Wistar rats weighing (250-315 g) were
procured from the Scientific Research Center, Damascus,
Syria. The animals were kept at controlled environmental
conditions (temperature 23 ± 2°C, humidity 55 ± 15%,
lighting regimen of 12-h light: 12-h dark). They were
acclimatized for one week before any experimental
Table 1: The effects of SZ, OLM-M and their combination on the body weight in AA induced ulcerative colitis in rats.
Parametric data were expressed as mean ± S.E.M (n =7). AA; Acetic Acid, (NC); normal control, (CC); colitis control,
SZ; Sulfasalazine, OLM-M; Olmesartan Medoxomile
Parameter
Group
Initial body weight
Final body weight
Body weight change
%
Group Ⅰ(NC)
251.9±10.94
267.0±10.03
+6.18±1.32
Group Ⅱ(CC)
262.4±11.93
224.1±8.73
-14.15±3.01
Group Ⅲ(SZ)
267.1±8.34
270.9±9.47
+1.3±1.59
Group Ⅳ(OLM-M)
315.0±15.84
304.3±18.86
-3.51±2.59
Group Ⅴ(SZ+OLM-M)
298.4±11.35
303±13.39
+1.44±1.69
NC
CC
SZ
OLM-M
SZ+OLM-M
-20
-10
0
10
+++
^^^
^
^^^
groups
Change of body weight(g)
%
Figure 1: The effects of SZ, OLM-M and their combination on the body weight in AA induced ulcerative colitis in
rats. Data are presented as means ± SEM (n =7).
+++ Significant difference as compared to normal control group at p<0.001. ^^^ Significant difference as compared to
colitis group at p <0.001.
^ Significant difference as compared to colitis control group at p <0.05.

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procedures and were fed with standard commercial rat
pellets and allowed water ad libitum.
Drugs and chemicals
Olmesartan medoxomil was obtained from Nutre
Specialities private limited.
Sulfasalazine was obtained from Kanawati Medical
Products.
Acetic acid (AA) (99-100%) was purchased from Merck
company. All other chemicals and solvents were of highest
grade commercially available.
Glutathione Assay Kit for direct assay of reduced
glutathione in colonic tissue was provided by Abnova
Chemical Company (Taiwan).
Experimental design and treatment protocol
animals were randomly divided into five groups (7 rats per
group):
- GroupⅠ (Normal control group): received physiological
saline intra rectally instead of 4% acetic acid + oral vehicle
(10ml/kg/day sodium CMC, 0.5% w/v).
- Group Ⅱ(AA control group) : received single dose of
acetic acid (AA) intrarectally (2ml 4% v/v in saline, i.r. )
+ oral vehicle( 10ml/kg/day sodium CMC, 0.5%) .
- GroupⅢ (reference group): received (AA) intrarectally +
SZ (360 mg/kg/day, p.o.) 8, 23
- GroupⅣ (OLM-M group ) : received (AA) intrarectally
+ OLM-M(5 mg/kg/day, p.o.)24,25
- Group Ⅴ(combination group): received (AA)
intrarectally+ SZ(360 mg/kg/day, p.o.) + OLM-M (3
mg/kg/day, p.o.).
All drugs were given by oral gavage syringe once daily for
seven consecutive days, starting 24 h after the induction of
colitis, and were suspended in sodium carboxy methyl
cellulose solution (vehicle) (sodium CMC 0.5%w/v ),
which does not affect the severity of acetic acid-induced
ulcerative colitis26.
Induction of experimental colitis in ratsrats were fasted for
24 h with free access to water before induction of colitis.
Colitis was induced in rats using 2 ml acetic acid 4% or
saline alone (normal control group) via intra-colonic
Table 2: The effects of SZ, OLM-M and their combination on colon weight / length in AA induced ulcerative colitis in
rats. Parametric data were expressed as mean ±S.E.M (n =7). AA; Acetic Acid, (NC); normal control, (CC); colitis
control, SZ; sulfasalazine, OLM-M; Olmesartan Medoxomile.
Parameter
Group
Colon weight (g)
colon length(cm)
colon weight / length
(g/cm)
Group Ⅰ(NC)
1.82±0.052
16.50±0.393
0.110±0.0021
Group Ⅱ(CC)
2.75±0.099
14.00±0.293
0.196±0.0051
Group Ⅲ(SZ)
2.24±0.106
15.59±0.556
0.145±0.0098
Group Ⅳ(OLM-M)
2.34±0.108
15.43±0.581
0.154± 0.0123
GroupⅤ(SZ+OLM-M)
2.24±0.086
16.21±0.653
0.138±.0054
NC
CC
SZ
OLM-M
SZ+OLM-M
0.00
0.05
0.10
0.15
0.20
0.25 +++
^^^ ^^ ^^^
groups
cololn weight /length ratio (g/cm)
Figure 2: The effects of SZ, OLM-M and their combination on colon weight / length in AA induced ulcerative colitis
in rats. Data are presented as means ± SEM (n =7).
+++ Significant difference as compared to normal control group at p<0.001.
^^^ Significant difference as compared to colitis group at p <0.001.
^^ Significant difference as compared to colitis control group at p <0.01.

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administration. On day 0 under light ether anesthesia, a
soft and flexible catheter lubricated with glycerine (2 mm
inner diameter) was inserted to the anus for 8 cm then
acetic acid was carefully injected. The rats were
maintained in a head-down position for 30 seconds in order
to prevent solution spreading out. After a 30-second period
of exposure, excess fluid was withdrawn and the colon was
then flushed with saline27. Before removing the
catheter, 2 mL of air was injected to spread the AA
completely in the colon28.
Figure 3: Macroscopic appearances of colons from acetic acid colitis in rats A: normal group (grade0) showing
normal mucosa with intact epithelial surface , B: Sulfasalazine-treated group, Olmesartan- treated group and
combination treated group (grade1) showing swelling or redness , C: Sulfasalazine-treated group (grade 2) showing
swelling and redness , E: acetic acid group (grade 4) showing more than two ulcers or one large ulcer , F: acetic acid
group (grade 5) showing mild necrosis and G: acetic acid group (grade 6) showing severe necrosis .
Table 3: Macroscopic score of different experimental groups.
Group Macroscopic
Score
GroupⅠ
GroupⅡ
GroupⅢ
GroupⅣ
GroupⅤ
0
-
-
-
-
7(100%)
1
7(100%)
5(71.42%)
5(71.42%)
-
2
-
2(28.57%)
2(28.57%)
1(14.28%)
-
3
-
-
-
-
-
4
-
-
-
1(14.28%)
-
5
-
-
-
2(28.57%)
-
6
-
-
-
3(42.85%)
-

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Tissue collection and preparation
24 hours following completion of the experiment, On the
8th day rats were sacrificed under deep ether anesthesia and
their distal colon was removed for the evaluation of body
weight, colon weight / length ratio, macroscopic damage,
and histological study. As well as by biochemical
measurement of reduced glutathione (GSH).
Measured parameters for assessment of colonic damage
Clinical finding
During the study, rats were checked daily for body weight,
behavioral changes, food intake, rectal bleeding and stool
consistency. The body weight of animals was measured at
regular time intervals from day 0 to 7. and change of body
weight (%) was calculated.
colon weight / length ratio
The abdomen was immediately opened. The entire colon
starting from caecum was excised, freed of adherent
adipose tissue, longitudinally split and washed with ice-
cold saline to remove fecal residues. The length(cm) and
weight (g) was measured. The colon was dried between
two filter papers then weigh. Each colon was gently
stretched and the distance from the colocecal junction to
anus was measured, from which weight/length (g/cm)
ratio, as indirect marker of inflammation was calculated.
Macroscopic scoring
The colonic samples were examined immediately by naked
eye and magnifying lens for gross inflammatory changes
according to the criteria as follows:
0=no inflammation; 1=swelling or redness; 2=swelling
and redness; 3=one or two ulcers; 4=more than two ulcers
or one large ulcer; 5=mild necrosis; 6=severe necrosis29.
Histopathological examination
Segments of colon were fixed in 15% formalin for 24 h.
The specimens were first dehydrated by immersion in
progressively increasing concentrations of ethanol, then
were cleared in xylene. Following this, the dehydrated
tissue was immersed in melted paraffin at 55-60 0C for 3
h. Sections 5 microns thick were cut by using microtome
(Leica RM2155). The sections were then deparaffinized by
treatment with xylene, ethanol and water. Tissues were
stained with haematoxylin and eosin (H&E) and were
evaluated microscopically by apathologist in blinded
fashion. All groups were histopathologically assessed by
using following score3.
0= normal
1=mild mixed infiltrates in the lamina propria.
2=focal superficial ulceration of mucosa only, moderate
cryptitis and crypt abscess
3=deep ulceration penetrating colonic wall through
mucosa till muscularis mucosa and severe inflammation
4=necrosis through large bowel wall
Reduced glutathione
Colonic tissue samples were frozen in liquid nitrogen and
stored at −80 °C until time of assay. Colon GSH levels
were determined as previously described by Lindenmaier,
Blenn, and Wang30,31 based on the reaction of 5, 5-
dithiobis-(2-nitrobenzoic acid) (DTNB) with the
glutathione present to form yellow product. The optical
density measured at 405 nm is directly proportional to
glutathione concentration in the sample by using
microplate reader (Elisys Uno Human Germany). The
glutathione content was expressed as μM/g tissue.
OD:optical density.
Statistical analysis
Data analyses were achieved using a software program
Graph Pad Prism version 5. Data were presented as means
± standard error (S.E.M(. For parametric data One way
analysis of variance (ANOVA) was used followed by
Tukey-Krammer multiple comparison test. Lesion score
and histological score (non-parametric values) analyzed
using the Kruskal–Wallis nonparametric analysis of
variance with Dunn's multiple comparison test. P values
less than 0.05 were considered Statistically significant.
RESULTS
Clinical findings, general observation and body weight
change.
After 24 h of administration of acetic acid, animals
developed hematochezia, diarrhea and progressively body
weight loss with weakness and decreased food intake. All
these symptoms began to be blunted in the Ⅲ,Ⅳ and Ⅴ
groups at day 4. At the end of the experiment, 9 rats died:
5 from groupⅡ, 3 from Ⅳ, and 1 from group Ⅲ, that was
due to bleeding or perforation of the colon. Figure(1)
shows that the intracolonic administration of acetic acid
has caused the body weight to decrease in groupⅡ .
Compared with that of the normal control group which
revealed increase in body weight (+6.18%), the body
weight of the AA control group at the end of the
experiment was significantly reduced by (-14.15%)
(p<0.001). The body weights of the groups treated with SZ,
OLM-M and combination between them were
significantly increased compared with colitis control group
at (p<0.001, p<0.05, p<0.001 respectively).( Table1,
Figure1).
colon weight / length ratio
Reduction in colon length, an increases in colon weight,
and a corresponding increase in the colon weight to length
ratio a reliable marker of colon inflammation were
Table 4: Histological assessment of inflammation in different experimental groups:
Group
histological score
GroupⅠ
GroupⅡ
GroupⅢ
GroupⅣ
GroupⅤ
0
7(100%)
-
-
-
-
1
-
-
5 (71.42%)
7(100%)
7(100%)
2
-
1(14.28%)
2(28.57%)
-
-
3
-
1(14.28%)
-
-
-
4
-
5(71.42%)
-
-
-

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Table 5: oxidative stress in rat colon was evaluated by
assessing GSH
Group
Glutathione (GSH)
Mµ / g tissue
Group Ⅰ(NC)
1.349±0.037
Group Ⅱ(CC)
0.734±0.040
Group Ⅲ(SZ)
1.059±0.060
Group Ⅳ(OLM-M)
1.164±0.016
Group Ⅴ(SZ+OLM-M)
1.341±0.019
observed in colitic animals relative to normal control. The
rats in the AA control group showed asignificant increase
in the colonic weight/length ratio (p<0.001)( Table 2)
compared with that of normal control rats. SZ treatment for
7 days exerted an intestinal anti-inflammatory effect that
included a decrease in the weight/length ratio compared
with that of AA control group (p<0.001) (Table2). As well
as OLM-M treated group rats showed asignificant decrease
(p<0.01) in the ratio of colon weight/length compared with
that of AA control rats. Combination of SZ and OLM-M
significantly improved colon weight / length ratio
compared with that of AA control rats (p<0.001).
However, the difference between SZand OLM_M group
was not statistically significant. also the difference
between combination group and SZ alone group and OLM-
M alone group was not statistically significant(p>0.05).
Macroscopic scoring
There was no evidence of mucosal injury in any specimen
from
control groups. In contrast, rectal administration of acetic
acid as an inducing agent resulted in the production of
severe colonic mucosal lesions. The colonic mucosa
appeared macroscopically edematous, hemorrhagic,
ulcerated and necrotic compared to the normal control
group. Thus the morphological score in the AA group was
significantly increased (p<0.001) as compared to normal
control group (fig 3). Administration of SZ and OLM-M
therapeutically reduced the severity of the gross lesion but
this reduction in morphological score was not statistically
significant as compared to AA group (p> 0.05). However,
combination of SZ and OLM-M effectively decreased the
morphological changes as compared to AA group
(p<0.05), but this decrease in morphological score in
combination group did not achieve significance compared
to SZ and OLM-M alone treated groups (P>0.05).
Histopatholgical study
There was a good correlation between macroscopic and
histological scores in each study group. Colon sections
from control group revealed an intact architecture of colon
tissues and hundred percent of the animals in this group
showed normal morphology. On the other hand, colons of
AA group revealed significant tissue injury with high
scores of microscopic damage indicating erosion of the
lining mucosal epithelium, degenerative changes in the
crypt epithelium deep ulceration of muscularis mucosa,
severe inflammation and necrosis through large bowel
wall. Diffuse mixed inflammatory cells infiltration
(neutrophils, eosinophile and lymphocytes) were detected
in the mucosa including the lamina propria, in addition to
submucosa and muscularis. As well as submucosal edema,
NC
CC
SZ
OLM-M
SZ+OLM-M
0.0
0.5
1.0
1.5
+++
^^^ ^^^ ^^^
 
groups
GSH( µM \g tissue)
Figure 4: The effects of SZ, OLM-M and their combination on endogenous anti-oxidant (GSH) levels in AA induced
ulcerative colitis in rats. Data are presented as means ± SEM (n =7).
+++ Significant difference as compared to normal control group at p<0.001. ^^^ Significant difference as compared
to colitis control group at p <0.001.
 Significant difference as compared to combination group at p <0.001.
 Significant difference as compared to combination group at p<0.01.

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vasculitis, dilatation and congestion in the blood vessels,
crypt abscesses, proliferation of fibroblast and
hemosiderin precipitation that indicative to haemorrhage
were observed. Administration of SZ as therapy resulted in
reduce in the severity of the injury of the large intestine but
the values obtained with this dose of SZ (360mg/kg) were
not significantly different from the acetic acid control
group (p>0.05). Significant histologic improvement was
observed and reduced the histopathological scores in
OLM-M treated group compared to the group treated with
only AA (P <0.05). In group Ⅴ, microscopic injury
observed with SZ(360mg/kg) plus OLM-M (3mg/kg) was
significantly less than AA group (P <0.05) and the anti-
inflammatory effect produced by this combination was
more distinct but was not statistically significant(p>0.05)
than that obtained after treatment with SZ (360 mg/kg,po.)
alone and similar to OLM-M (5 mg/kg, p.o.) alone.
Reduced glutathione (GSH)
AA-induced oxidative stress in rat colon was evaluated by
assessing GSH as shown in table (5) and fig(4). AA
significantly reduced GSH level in the colon by 45.9% as
compared to the control group (p< 0.001). Administration
of sulfasalazine resulted in an elevation of tissue GSH
concentration by about 30.47% as compared to AA-colitis
group (p<0.001). GSH content was signifiantly increased
in the group treated with OLM-M (5mg/kg) by 37.06% as
compared to animals that received AA alone (p< 0.001).
Also asignificant increase in the mucosal glutathione
concentration was observed in SZ & OLM-M group by
45.5% as compared to acetic acid group (p< 0.001). this
combination treatments group was able to reach the control
value. The increase was substantially higher with
combination treatments group than sulfasalazine and
Olmesartan medoxomile used alone (p <0.001 and
P<0.01).
Table (5): The effects of SZ, OLM-M and their
combination on endogenous anti-oxidant (GSH) levels
(μM /g tissue) in AA induced ulcerative colitis in rats.
Parametric data were expressed as mean ±S.E.M (n =7).
AA; Acetic Acid, (NC); normal control, (CC); colitis
control, SZ; sulfasalazine, OLM-M; Olmesartan
Medoxomile.
DISCUSSION
Various experimental models of colitis mimicking the
active phase of the disease have been developed to test the
potential beneficial effects of various drugs. One of the
most commonly employed models is acetic acid induced
colitis in rats. The model of induced colitis through intra
rectal injection of acetic acid presents advantage over other
experimental models. Such advantages include easy
availability of aggressor reagent, low cost, reproducibility
and similarity to UC in humans32. The mechanism by
which AA induces colitis involves the entry of protonated
form of acid into the epithelium where it dissociates to
liberate protons causing intracellular acidification that
might account for the epithelial injury33. Transient local
ischemia might contribute to the acute injury, Mucosa and
submucosal inflammation followed initial injury was
associated with activation of arachidonic acid pathways34.
AA metabolism by colonic enzymes provides superoxide
anions and H2O2 which contribute to its colonic toxic
effects35. The results of the present study demonstrate that
acetic acid induced colitis model in rats is a reproducible
technique and produces a large inflammatory response as
evidenced by body weight loss, reduction in food intake,
colonic shortening, increase colonic weight / length ratio,
characteristic acute colonic lesions such as mucosal
oedema, haemorrhage and necrosis. Furthermore, the
lesions are associated with changes in biochemical
parameters which include depletion of GSH. The RAS
from an evolutionary point of view is a very old system
with pro-inflammatory effects on different tissues. In
addition to endocrine effects, it also has paracrine and
autocrine actions36. It is noteworthy that colitis is likely to
be multifactorial, and recent data indicate that ARBs
prevented some, but not all of the inflammatory stimuli in
this model. Hence the importance of Investigation on the
participation of this class of drugs with other treatments
used in inflammatory bowel disease.
The present study highlights the therapeutic effects of
OLM-M, an Ang Ⅱ AT-1 receptor antagonist alone and in
combination with sulfasalazine in acetic acid induced
colitis, an experimental model of human IBD. There was
significant reduction in the body weight in the AA group
compared to normal control which exhibit marked increase
in body weight. Administration of SZ, OLM-M and
combination between them as therapy lowered the
incidence of diarrhea, improved food intake and attenuated
body weight loss as compared AA control group. This
effect of Olmesartan medoxomile is attributed to that Ang
Ⅱ causes a marked anorexigenic effect and weight loss,
and high circulating levels of Ang Ⅱ may contribute to the
anorexia, wasting, and cachexia37. These observations are
in accord with previous studies12,20.
Colonic weight /length ratio can be considered a reliable
and sensitive indicator of the severity of UC. Weight of
colon is raised due to the inflammation and also because of
the increased activity of the fibroblasts leading to the
overgrowth of muscularis mucosa33. consequently, the
increased colonic weight / length ratio confirms
intensification of intestinal infiltrations and consequent
intestinal oedema. Treatment of rats with SZ and OLM-M
(5mg/kg) for 7days following induction of colitis
significantly reduced colonic weight / length ratio. These
findings are in agreement with the earlier study which
showed that aliskiren at higher dose (10mg/kg,i.p.)
significantly improved colon weight/length ratio in
dextran sulfate sodium (DSS) induced colitis in mice12.
Moreover, Wengrower et al.(2004) demonstrated that
captopril reduced colonic weight / length ratio in a model
of TNBS colitis38. furthermore, Nagib et al( 2013)
documented the beneficial effects demonstrated with pre
treatment of rats with10mg /kg OLM-M are similar to or
even greater than those obtained with sulfasalazine in DSS
colitis model2. In the present study combination therapy
with SZ plus OLM-M caused asignificant reduction in

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colon weight/length ratio and this effect was superior to
those of either agent alone.
The morphological changes which occurred in this study
were similar to the other study that showed redness,
oedema, ulcer and necrosis in AA group29. In our study,
acetic acid administration caused a substantial degree of
tissue injury associated with congestion, haemorrhages,
oedema, leukocytic infiltration, deep ulceration
penetrating colonic wall through mucosa till muscularis
mucosa, severe inflammation and degenerative changes
such as necrosis .These histological features are consistent
with the features described in previous reports27. Our
macroscopic and histological analysis demonstrated the
ability of SZ and OLM-M to ameliorate intestinal damage
and inflammation. It was found that the morphological
scores were reduced by SZ and OLM-M but neither agent
caused a significant inhibition of colonic lesions as
compared to acetic-acid controls. The effects of
combination SZ (360mg/kg) plus OLM-M(3mg/kg) on
macroscopic injury were superior to those of either agent
alone and these values achieve significance compared to
AA alone. Microscopic findings showing attenuation of
tissue damage, reduction in cell infiltration and mucosal
ulceration with OLM-M (5mg/kg). These findings are in
line with El-Medany et al (2011) that demonstrated that
rats received captopril or valsartan, therapeutically showed
significant amelioration in these parameters as compared
to acetic acid group36. Interestingly, Dereli et al (2014)
confirm that administration of OLM-M can effectively
reduce fibrosis and limit the damage to the esophageal
tissue in the animal model of caustic burn24. These
favorable anti inflammatory actions of OLM-M, were
linked with inhibiting the release of TNF- α, was
previously reported in various organ maladies such as
hypertensive patients with microinflammation39 , insulin
resistance40, atherosclerosis41 and bleomycin-induced
pulmonary fibrosis42. Combination therapy with SZ
(360mg/kg) plus OLM-M (3mg/kg) was associated with a
significantly reduced in microscopic colonic damage. Data
derived from this study indicate that dual therapy has a
synergistic effect in reducing inflammation and promoting
mucosal repair than SZ (360mg/kg) alone. Our study
appears to be the first of its kind as we were not able to
find references in the english literature.
The gastrointestinal (GI) tract is a key source of ROS. They
are generated from several sources including stimulated
PMNs, eosinophils, xanthine oxidase, colonic bacteria and
epithelial lipoxygenase43. The depletion of glutathione is
considered a crucial event of colonic damage occurring
both in human UC and in animal.In the present work,
acetic acid-induced colonic lesions are associated with
significant depletion in GSH levels which is in agreement
with the previous findings36,44. Data obtained from the
treatment of animals with SZ and OLM-M shows
significant increase in GSH level as compared to the AA
group. It is noticeable that OLM-M showed better
improvements but not significant than sulfasalazine. Our
results are in agreement with earlier studies that showed
the antioxidant effect of OLM-M on experimentally
induced liver fibrosis in rats45.
Co-administration of SZ(360mg/kg, p.o.) with OLM-
M(3mg/kg, p.o.)
produced substantial elevation of tissue GSH
concentration, and a more documented antioxidant
response in comparison with the SZ(360mg/kg, p.o.) and
OLM-M(5mg/kg, p.o.) used alone.
CONCLUSION
In conclusion, this study indicates the efficacy of OLM-M
in AA-induced UC. These effects, which are comparable
or even better than sulfasalazine, are possibly attributed to
its anti-inflammatory via modulation of the immune
system and antioxidant properties. Combination of OLM-
M and SZ has shown greater efficacy than single SZ
treatment.
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