The study investigated the protective effects of losartan, an angiotensin II type 1 receptor blocker, on intestinal ischemia-reperfusion injury in rats. Forty rats were divided into four groups: sham operation, ischemia, ischemia/reperfusion (I/R), and I/R + losartan treatment. Biochemical markers and histopathological analysis of the jejunum tissue were performed. Losartan treatment reduced oxidative stress markers, inflammation, and apoptosis compared to the I/R group. This suggests losartan may protect against intestinal damage caused by ischemia-reperfusion injury.

1. 269
OBJECTIVE: To investigate the protective effect of lo-
sartan, an angiotensin II type 1 receptor blocker with
antioxidative effect on intestinal ischemia-reperfusion
(I/R) injury in rats, against inflammation and apoptotic
development.
STUDY DESIGN: Forty male Wistar albino rats with
a mean weight of 200–250 g each were divided into
4 groups: (1) Sham operation (laparotomy only, sham
surgical preparation including isolation of the superior
mesenteric artery [SMA] without occlusion), (2) Ische­
mia model with SMA closure for 2 hours, (3) I/R group
(2 hours of ischemia followed by 3-hour reperfusion
(SMA occlusion for 120 minutes followed by 240 min­
utes reperfusion), and (4) Losartan group (2 hours of
ischemia, 40 mg/kg losartan was administered to the
animals; losartan was dissolved in 1 mL distilled water
and administered intraperitoneally after 2 hours of is-
chemia). Malondialdehyde (MDA), superoxide dismut­
ase (SOD), catalase (CAT), and glutathione (GSH) lev­
els were examined in jejunum tissue.
RESULTS: Losartan treatment reduced the I/R-induced
increase in MDA levels in the gut. Statistically, while
SOD, CAT, and GSH activities decreased significantly
in the I/R group, they increased in the I/R+Losartan
group. Villus loss and increase in inflammation after
ischemia persisted after reperfusion. Losartan treatment
played a role in the reduction of inflammation and apo­
ptosis and in the regulation of TNF-α and caspase-9
activity.
CONCLUSION: It has been thought that losartan in I/R
may reduce mucosal damage and cell apoptosis in the
direction of inflammation and may stabilize caspase-9
activity by inhibiting TNF-α stimulus. (Anal Quant
Cytopathol Histpathol 2021;43:269–277)
Keywords: caspase-9, ischemia, ischemia/reperfu­
sion, rat, reperfusion injury, TNF-α, tumor necrosis
factor-alpha.
In ischemia/reperfusion (I/R) damage of the small
intestine caused by the occlusion of the mesenteric
artery, the vascular structure in all layers of the
intestinal tissue is affected and the blood supply
decreases completely. Injury of the apical part of
the microcirculatory blood flow results in a de-
crease in intestinal mucosal blood perfusion, which
occurs simultaneously with the destruction of the
apical villi.1 Tissue damage is the initial ischemic
injury or changes in free oxygen radicals that
initiate reperfusion injury. Neutrophils, platelets,
endothelial factors, and cytokines are also among
the important pathogenic mechanisms of intestinal
I/R injury.2 It has been shown that free radical
formation triggers tissue damage by disrupting the
balance between oxidants and antioxidants, and
then inflammation increases.
Reperfusion in ischemic tissue also induces a
number of different events that cause ischemia-
related tissue damage and are milder than the
damage that occurs after reperfusion.3 However,
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4304-0269/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Prophylactic Effects of Losartan in Intestinal
Ischemia-Reperfusion Injury Model
Songül Beskisiz, M.D.
From the Department of Internal Medicine, Gazi Yaşargil Training and Research Hospital, Health Sciences University, Diyarbakır,
Turkey.
Songül Beskisiz is Physician.
Address correspondence to: Songül Beskisiz, M.D., Department of Internal Medicine, Gazi Yaşargil Training and Research Hospital,
Health Sciences University, Diyarbakır, Turkey (drsongul21@gmail.com).
Financial Disclosure: The author has no connection to any companies or products mentioned in this article.

2. malondialdehyde (MDA), glutathione (GSH), and
catalase (CAT) values were used to determine
tissue damage by measuring biochemical total an-
tioxidant and oxidant values in previous experi-
mental ischemia studies.4 I/R leads in part to intes­
tinal cell apoptosis as a result of reactive oxygen
species (ROS) and proinflammatory cytokine pro­
duction. Cas­
pases participate in processes such as
cell volume reduction (pyknosis), chromatin con­
densation, nuclear fragmentation (karyorexia), and
formation of plasma membrane blebs.5,6 Caspase-9
genes play a key role in the regulation of the apop­
totic cell and are used as markers in the reduc-
tion of cell degeneration and cell number.7 The cell
death receptor pathway involves members of the
TNF receptor superfamily.8 TNF-α is a multifunc-
tional cytokine with effects only in the proinflam­
matory response and apoptosis.9
Losartan, a blocker of angiotensin II type 1 re-
ceptor, inhibits oxidative damage in cells during
I/R damage. Losartan has several beneficial ef-
fects on the vasculatures, including decreases in
the total peripheral resistance and cardiac venous
return.10
The aim of this study was to investigate the
protective effect of losartan, an angiotensin II type
1 receptor blocker with antioxidative effect on in-
testinal I/R injury in rats, against inflammation
and apoptotic development.
Materials and Methods
All experimental protocols were conducted ac-
cording to the National Institutes of Health
Guidelines for the Care and Use of Laboratory
Animals. The study was approved by local ethics
committee. Forty male Wistar albino rats with a
mean weight of 200–250 g each were used. They
were housed in an air-conditioned room with
12-hour/12-hour light and dark cycles, where the
temperature (23±2°C) and relative humidity (65–
70%) were kept constant.
Group 1: Sham Operation Group. This group under­
went laparotomy only, sham surgical preparation
including isolation of the superior mesenteric ar-
tery (SMA) without occlusion.
Group 2: Ischemia Group. This group underwent
SMA closure for 2 hours.
Group 3: I/R Group. This group underwent a 2-
hour period of ischemia followed by 3 hours of
reperfusion (SMA occlusion for 120 minutes fol­
lowed by 240 minutes reperfusion).
Group 4: I/R+Losartan Group. This group under-
went a 2-hour period of ischemia treated with lo-
sartan. 40 mg/kg of losartan was administered to
the animals. Losartan was dissolved in 1 mL dis­
tilled water and administered intraperitoneally
after 2 hours of ischemia. After 2 hours of reper­
fusion, the jejunum tissues were removed for ex-
amination.
The jejunum tissues of all groups were removed
and divided into two equal pieces and stored
under suitable conditions for biochemical and his­
topathological investigations.
Surgical Procedure
All rats were fasted 12 hours before the experi-
ment. The rats were anesthetized with an intra­
muscular injection of ketamine (50 mg/kg; Keta­
lar, Parke Davis, Turkey) and xylazine (10 mg/
kg; Rompun, Bayer AG, Germany) under aseptic
conditions. The abdominal region was shaved and
a 2–3 cm abdominal midline incision was made.
In the intestinal I/R injury model, superior me-
senteric artery (SMA) was carefully applied and
blocked with a nontraumatic microvascular clamp
for 120 minutes. At the end of this period, the
clamp was removed and the mesenteric artery
was released. Then 240 minutes of reperfusion was
performed.
Biochemical Analysis
MDA, superoxide dismutase (SOD), CAT, and
GSH levels were examined in testicular tissue.
Tissue samples were homogenized with ice-cold
150 mMKC. MDA levels were assayed for prod­
ucts of lipid peroxidation, and the results were
expressed as nmol MDA/g tissue.11 The SOD
activity in the tissues was measured using the
RANSOD kit (Randox Laboratories, Crumlin, UK).
GSH was determined by the spectrophotometric
method based on the use of Ellman’s reagent, and
the results were expressed as μmol glutathione/g
tissue.12 CAT activity was determined by the spec­
trophotometric method based on the ability of hy-
drogen peroxide to form a stable stained complex
with molybdenum salts.13
Histopathological Analysis
For microscopic evaluation, samples were taken
270 Analytical and Quantitative Cytopathology and Histopathology®
Beskisiz

3. from the sections of rat jejunum tissue with the
highest macroscopic damage. The colon of each
rat was put separately into a formaldehyde me-
dia. Tissues were detected in the 10% buffered
formalin, after which a routine tissue follow-up
was performed and they were placed in paraffin
blocks. Slides of 5 μm thickness were cut from the
paraffin blocks using a microtome and deparaf­
finized. The samples were dyed with hematoxylin-
eosin stain and examined under a light micro-
scope (Nikon Eclipse Ni) for edema, vascular con­
gestion, hemorrhage, inflammatory cell infiltration,
and mucosal damage.
A semiquantitative histological evaluation scor­
ing system was used to determine histopathologi­
cal changes. The criteria that were used to evalu-
ate for mucosal injury were damage/decomposition
in the surface epithelium (basal membrane pre­
served), vascular congestion, hemorrhage, and in-
filtration by inflammatory cells. The degree of in-
jury was evaluated using a modified Chiu’s score
method according to changes of the surface epi­
thelium and crypts of the intestinal mucosa.
Immunohistochemical Technique
Formaldehyde-fixed tissue was embedded in par-
affin wax for further immunohistochemical exam­
ination. Sections were deparaffinized in absolute
alcohol. Antigen retrieval process was performed
twice in citrate buffer solution (pH 6.0), first for
7 minutes, and second for 5 minutes, boiled in a
microwave oven at 700 W. They were allowed to
cool to room temperature for 30 minutes and
washed twice in distilled water for 5 minutes.
Endogenous peroxidase activity was blocked in
0.1% hydrogen peroxide for 20 minutes. Ultra V
block (catalog number 85-9043, Invitrogen, Carls­
bad, California, USA) was applied for 10 minutes
prior to the application of primary antibody TNF-α
(catalog number ab1793, Abcam, Cambridge, UK,
dilution rate: 1/100), capase-9 (catalog number
ab32539, Abcam, dilution rate: 1/100), secondary
antibody (catalog number 85-9043, Invitrogen) was
applied for 20 minutes. Slides were then exposed
to streptavidin-peroxidase for 20 minutes. Chro­
mogen diaminobenzidine (DAB) (Invitrogen, cata­
log number 34002) was used. Control slides were
prepared as mentioned above but omitting the
primary antibodies. After counterstaining with he-
matoxylin and washing in tap water for 8 minutes
and in distilled water for 10 minutes, the slides
were mounted with Entellan.14
Results
Statistical analysis of biochemical parameters and
immunohistochemical expressions are shown in
Table I. MDA and GSH were highest and SOD
and CAT were lowest in the I/R group as com­
pared to the Control, Ischemia, and I/R+Losartan
groups. These results were statistically significant.
Caspase-9 and TNF-α expressions were higher
than Control and I/R+Losartan group. These re-
sults were statistically significant. A graphical illus­
tration of Table I is shown in Figures 1 and 2.
In the histopathological examination of the
Sham group, the regular epithelial cells of the
villi structure were cylindrical and lay in the form
of a small goblet between them. In the lamina
propria, the connective tissue cells with regular
lumen of blood vessels were solitarily distributed,
and the muscle layer was observed in a circularly
arranged fusiform cell structure. In the ischemia
group, loss of most of the villi, intense conges-
tion and dilation in the vessels in the lamina
propria, significant leukocyte inflammation in the
connective tissue area, and hypertrophy in the
muscle cells were observed. Degeneration and
apoptotic changes were observed in the epithe-
lial cells at the bottom and cells in the intestinal
glands. In the ischemia/reperfusion group, while
the loss of villi continued, an increase in inflam-
matory cells in the connective tissue was observed.
Pyknosis was observed in the nuclei with degen­
erative changes in villi bases and intestinal gland
cells. In the cross-section of the I/R+Losartan
group there was a significant decrease in the loss
of villi, a significant decrease in the degenera­
tive appearance of epithelial and glandular cells,
the blood vessels in the connective tissue area
were slightly dilated and there was a significant
decrease in inflammatory cells, and no significant
pathological changes were observed in the muscle
tissue.
In the sham group, caspase-9 immunoactivity
was negative in many cells in the connective tis-
sue along the villus epithelium and intestinal gland
cells. In the ischemia group, caspase-9 expression
was positively observed in the jejunum lumen with
numerous pyknotic nuclei, intestinal gland cells,
and many connective tissue cells. In the I/R group,
it was observed that the expression of caspase-9
was increased in most of the degenerative cells
of villus epithelium cells and most of the connec-
tive tissue cells. Positive caspase-9 expression was
observed in a small number of degenerative cells
Volume 43, Number 4/August 2021 271
Losartan in Intestinal Ischemia-Reperfusion Injury

4. 272 Analytical and Quantitative Cytopathology and Histopathology®
Beskisiz
Table I Biochemical (MDA, SOD, GSH) and Immunohistochemical Scores (Caspase-9 and TNF-α Expression) of Control, Ischemia, I/R,
and I/R+Losartan Groups
Kruskal-Wallis
Mann-Whitney
H test U test
Parameter Group N Mean±SD
Mean rank p Value (p<0.05)
MDA (1) Control 10 6.28 19.58 33.255 (2)(3)
(2) Ischemia 10 9.22 43.19 p=0.001 (1)(4)
(3) I/R 10 13.43 48.23 (1)(4)
(4) I/R+Losartan 10 6.72 21.43 (2)(3)
SOD (1) Control 10 3.96 15.09 20.407 (2)(3)
(2) Ischemia 10 2.10 12.65 p=0.001 (1)(4)
(3) I/R 10 1.52 8.98 (1)(4)
(4) I/R+Losartan 10 3.43 15.09 (2)(3)
GSH (1) Control 10 8.76 34.32 21.294 (2)(3)
(2) Ischemia 10 12.60 45.84 p=0.001 (1)(4)
(3) I/R 10 15.44 48.32 (1)(4)
(4) I/R+Losartan 10 9.56 35.10 (2)(3)
CAT (1) Control 10 0.032 9.34 13.652 (2)(3)
(2) Ischemia 10 0.022 8.24 p=0.001 (1)(4)
(3) I/R 10 0.016 8.01 (1)(4)
(4) I/R+Losartan 10 0.037 10.76 (2)(3)
Caspase-9 (1) Control 10 1.20 10.29 20.027 (2)(3)
expression (2) Ischemia 10 3.70 12.45 p=0.001 (1)(4)
(3) I/R 10 3.90 14.34 (1)(4)
(4) I/R+Losartan 10 2.10 13.09 (2)(3)
TNF-α (1) Control 10 1.20 10.29 22.378 (2)(3)
expression (2) Ischemia 10 3.20 14.82 p=0.001 (1)(4)
(3) I/R 10 3.70 15.84 (1)(4)
(4) I/R+Losartan 10 1.60 11.30 (2)(3)
I/R = ischemia/reperfusion.
Figure 1
Graphical illustration of
MDA, SOD, GSH values,
and caspase-9 and TNF-α
expression.

5. and some cells in the intestinal glands due to
reorganization in the villi in the group treated
with I/R+Losartan. It was observed that there
was a slowdown in apoptotic change signals
due to decreased inflammation with the effect of
losartan.
In the immunohistochemical examination, mild
TNF-α expression was observed in villi epithelial
cells, solitary dispersed connective tissue cells,
and endothelial cells in the Sham group sections.
In the Ischemia group, positive TNF-α expression
was observed in inflammatory cells spilled on the
jejunum surface and prominent in the connective
tissue.
In the I/R group, TNF-α expression was posi­
tive in the connective tissue area at the bottom of
the villus, around the intestinal glands and in the
blood vessel endothelial cells. In the section of the
I/R+Losartan group, mild TNF-α expression was
observed in cells in the villus structure, intestinal
gland cells, and endothelial cells, while TNF-α ex-
pression was moderate in a small number of in-
flammatory cells in the connective tissue area. It
was thought that the inflammation due to losartan
activity showed a feature in the direction of re-
duction.
Discussion
Ischemia due to cell death and organ failure is
the insufficient production of oxygen and other
metabolites by the circulation. As a result, the
elimination of waste products is not possible with
the circulation. MDA is an indicator of lipid per­
oxidation in tissues, and increased levels cause
oxidative damage. In physiopathological condi­
tions, it has been reported that the harmful effects
of reactive oxygen species are inhibited by anti­
oxidant enzymes such as SOD and glutathione
peroxidase (GPx).15 During reperfusion of the gut,
the oxidant/antioxidant balance may change.
In a study to evaluate the tissue antioxidant
system, SOD and GPx activities were found to
increase in the treatment group, suggesting that
intestinal I/R damage was reduced by detoxify-
ing free oxygen radicals.16 MDA levels were
found to be significantly higher in the I/R group
as compared to the sham and I/R+Losartan
groups. The SOD, CAT, and GSH levels were
found to be significantly lower in the I/R group
as compared to the Control and I/R+Losartan
groups.
Acute cell damage, interstitial edema, and cel­
lular dysfunction occur.17 It has been stated that
reperfusion of ischemic tissue allows one side to
regain some functions lost during ischemia, while
cell loss on one side causes more cell damage.
Intestinal I/R injuries are characterized by altered
microvascular and epithelial permeability and villi
Volume 43, Number 4/August 2021 273
Losartan in Intestinal Ischemia-Reperfusion Injury
Figure 2
Graphical illustration of CAT
values.

6. damage.18 In the ischemia reperfusion model with
volvulus injury, it has been reported to cause
damage to intestinal cells microscopically, such
as mucosal destruction, loss of villi and epithelial
cell apoptosis, obstruction, and inflammatory infil­
tration.19 Hang et al20 showed that after I/R, there
was evidence of intestinal mucosal damage, ero-
sion, necrosis, significant loss of villi, occlusion of
blood vessels in the lamina propria, edema, in-
flammation, and intestinal morphological changes
such as mucosal and submucosal. After ischemia,
loss of most of the villi, intense congestion and
dilation of the vessels in the lamina propria,
significant leukocyte inflammation in the connec­
tive tissue region, degeneration, and apoptotic
changes in the cells of the intestinal glands were
observed (Figure 3B). In the I/R group, the loss
of villi continued, while an increase in inflamma­
tory cells was observed in the connective tissue.
Nuclei pyknosis was observed with degenerative
changes in intestinal gland cells (Figure 3C). In the
I/R+Losartan group, there was a significant de-
crease in the loss of villus, a significant decrease
in the degenerative appearance of epithelial and
glandular cells, slightly dilated blood vessels in
the connective tissue region, and a significant de-
crease in inflammatory cells (Figure 3D).
As an activated marker of the caspase family
of caspase-9 signaling induction of mitochondrial
damage and progression and activation of apopto-
sis by initiating cytochrome c release causes an in-
crease.21 Inhibition or genetic excision of caspase-9
from endothelial cells has been reported to have
the property of restoring vascular integrity and
274 Analytical and Quantitative Cytopathology and Histopathology®
Beskisiz
Figure 3 (A) Sham group: regular epithelial cells of villi structure cylindrical, interspersed goblet cells (arrow), blood vessels are normal
in structure, connective tissue cells are solitarily distributed, and the muscle layer is fusiform (hematoxylin-eosin stain). (B) Ischemia
group: loss of most of the villi, intense occlusion (arrow), and dilation of the vessels in the lamina propria, significant leukocyte
inflammation in the connective tissue region (thin arrow), and hypertrophy of muscle cells, degeneration and apoptotic changes in the
cells of the intestinal glands (hematoxylin-eosin stain). (C) Ischemia/reperfusion group: loss of villi, increase in inflammatory cells in
connective tissue, degenerative (arrow) changes in intestinal gland cells, and nuclei pyknosis (hematoxylin-eosin stain). (D) I/R+Losartan
group: decrease in loss of villus (thin), decrease in degenerative appearance of epithelial and glandular cells, decrease in inflammatory
cells (hematoxylin-eosin stain).

7. restoring neuronal activity in an adult model of
retinal hypoxia/ischemia. It was emphasized that
targeting endothelial cell pathways could provide
effective treatments to alleviate ischemic neu­
ronal damage and identify a non-apoptotic role
for caspase-9 and caspase-7 in vascular barrier
dysfunction.22 In an intestinal study, ischemia-
reperfusion was reported to significantly increase
the expression of mucosal TNF-α at both mRNA
and protein levels. It activated caspase-9/caspase-
3, in which it induced mucosal injury and cell apo­
ptosis by activating the JNK signaling pathway.23
In our study, increased caspase-9 expression was
observed due to increased inflammation and en-
dothelial dysfunction after ischemia (Figure 4B).
Caspase-9 activity showed rapid progression in
apoptotic cells with reperfusion (Figure 4C). In
the group treated with Losartan, the decrease in
inflammation and degenerative cells caused mod­
erate and mild expression of caspase-9 activity
(Figure 4D).
TNF-α mediates injury of endothelial cells and
infiltration of neutrophils.24,25 It has been shown
that TNF-α mediates I/R-induced injury and ex-
erts a protective effect by inhibiting its function or
expression.26 TNF-α is thought to activate path-
ways that promote both cell survival and apo-
ptosis, depending on the cell type and biological
context. The effect of TNF-α, along with increased
inflammation, was thought to induce I/R-induced
intestinal cell apoptosis. Oxygen deficiency caused
by ischemia caused aggregate formation of inflam­
matory cells and initiation of apoptotic process
and increased TNF-α expression (Figure 5B). After
reperfusion, an increase in TNF-α expression was
observed (Figure 5D). Losartan administration pro­
moted the reduction of inflammation and had an
effect on fixing TNF-α signaling.
Volume 43, Number 4/August 2021 275
Losartan in Intestinal Ischemia-Reperfusion Injury
Figure 4 (A) Sham group: caspase-9 expression is negative in many cells in connective tissue along the villus epithelium and in
intestinal gland cells (arrow) (caspase-9 immunostaining). (B) Ischemia group: numerous pyknotic nuclei and intestinal gland cells
and caspase-9 expression positive in the jejunum lumen (arrow) (caspase-9 immunostaining). (C) I/R group: an increase of caspase-9
expression in degenerative cells of the villus epithelial cells (arrow) and most of the connective tissue cells (caspase-9 immunostaining).
(D) I/R+Losartan group: positive caspase-9 expression in a small number of degenerative cells (arrow) and some cells in the intestinal
glands.

8. Conclusion
It has been thought that losartan in I/R may
reduce mucosal damage and cell apoptosis in
the direction of inflammation and may stabilize
caspase-9 activity by inhibiting TNF-α stimulus.
Acknowledgement
I would like to thank Research Assistant Dr. Fırat
Asir (Department of Histology and Embryology,
Medical Faculty, Dicle University, Diyarbakır) for
his contribution while performing the experiments
and writing the manuscript.
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