This study aimed to evaluate the protective effects of zofenopril on intestinal ischemia-reperfusion injury using a rat model. Rats were divided into five groups: sham surgery, ischemia only, ischemia-reperfusion, ischemia-reperfusion with zofenopril pretreatment, and zofenopril only. Biochemical markers of oxidative stress and apoptosis were measured in intestinal tissue samples. Histopathological examination and immunohistochemical staining for caspase-3 was also performed. Results showed that ischemia-reperfusion caused intestinal damage including mucosal destruction and cell apoptosis. Zofenopril pretreatment reduced oxidative stress markers and inhibited apoptosis, protecting against ischemia-reperfusion injury on histological and biochemical evaluation. The

1. 95
OBJECTIVE: To provide information about the effects
of zofenopril on volvulus using an ischemia-reperfusion
model with histochemical and biochemical methods.
STUDY DESIGN: Experimental animals were divided
into 5 groups, under anesthesia. (1) The sham group
(n=7) received only laparotomy, and a 5 cm segment of
small intestine was removed approximately 2 cm from
the proximal part of the cecum. (2) The second group
(n=7) received laparotomy, and the intestine segment
was removed for examination. (3) The third group (n=
7) underwent 2 hours of ischemia followed by 2 hours
of reperfusion (2 hours of superior mesenteric artery
occlusion followed by 2 hours of reperfusion). (4) The
fourth group (n=7) received orally 15 mg/kg zofenopril
(before laparotomy) after 2 hours of ischemia. Then re-
perfusion was performed for 2 hours. (5) The fifth group
(n=7) received orally 15 mg/kg zofenopril without isch-
emia. Intestinal tissues were taken, fixed, and embedded
in paraffin blocks for histopathological examinations.
Blood samples were collected for biochemical analysis.
Total antioxidant status, total oxidative status (TOS),
and oxidative stress index (OSI) were measured in
jejunum tissue. Also, jejunum tissue histopathology was
evaluated under a light microscope.
RESULTS: Serum TOS levels were significantly dif-
ferent between all groups (p=0.759). OSI levels were
significantly different between all groups (p=0.180).
Histopathologically, ischemia reperfusion caused micro-
scopic intestinal damage such as mucosal destruction,
villus loss, and epithelial cell apoptosis, congestion, and
infiltration of inflammatory cells. Oral administration
of zofenopril following ischemia resulted in a marked
inhibition of apoptosis induction in the stroma and villi
epithelium.
CONCLUSION: Zofenopril improved the intestinal mu-
cosal damage by relieving oxidative stress and apopto-
sis after intestinal ischemia-reperfusion. (Anal Quant
Cytopathol Histpathol 2020;42:95–104)
Keywords: caspase-3 antibody, ischemia reperfu-
sion, jejunum, volvulus, zofenopril.
Volvulus is a condition of self-knotting with the
twisting of a redundant segment of intestine
around itself or another intestine segment. This
situation may lead to the malfunctioning of blood
flow in that zone. In the intrauterine period the
middle bowel takes its position in the abdomen
by rotating 270 degrees in the counterclockwise
direction around the superior mesenteric artery.
In the end, the cecum is placed in the right lower
quadrant. Defects of this rotation are characterized
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4203-0095/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Experimental Study on the Prophylactic Effects
of Zofenopril in an Ischemia-Reperfusion
Model with Intestinal Volvulus Injury
Hülya I
·
pek, M.D., and Gül Doğan, M.D.
From the Department of Pediatric Surgery, Erol Olçok Education and Research Hospital, and the Department of Pediatric Surgery, Med-
ical Faculty, Hitit University, Çorum, Turkey.
Hülya I
·
pek is Assistant Professor, Department of Pediatric Surgery, Erol Olçok Education and Research Hospital.
Gül Doğan is Assistant Professor, Department of Pediatric Surgery, Medical Faculty, Hitit University.
Address correspondence to: Hülya I
·
pek, M.D., Department of Pediatric Surgery, Erol Olçok Education and Research Hospital, 19040,
Çorum, Turkey (drhulyai@hotmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. by acute midgut volvulus, mobile cecum, Ladd
bands, internal hernia, and chronic midgut vol­
vulus. It is seen at a frequency of about 1 in 500
births. Acute middle-bowel volvulus is seen in
75% of patients within the first month and in
90% of the patients within the first year. At least
half of the patients who underwent surgery due
to rotation anomaly have middle bowel volvulus
at different grades. Bilious vomiting, abdominal
distension, bloody stool, change in abdominal skin
color, and, at later stages, shock and sepsis can
be seen in patients.1 Diagnosis and treatment are
truly urgent because if left untreated it can cause
gangrene in the intestine within the first few hours,
and this may result in extensive bowel resection.
There are no laboratory, radiological, ultrasono-
graphic, and Doppler findings specific to the in­
testinal volvulus. These tests are only helpful for
diagnosis, and the same findings can be seen in
cases of intestinal obstruction such as invagina-
tion, band ileus, and duodenal atresia-stenosis. For
this reason, it can delay the diagnosis and surgical
intervention may result in large bowel resection.2
Intestinal ischemia-reperfusion (I/R) injury re­
sults in morbidity and sometimes even mortality.
Intestinal I/R injury is a well-established model of
systemic inflammatory response syndrome where-
by ischemia is artificially induced in the small in-
testine followed by reperfusion of the blood supply.
Intestinal ischemia caused by complete vascular
occlusion of the superior mesenteric artery is an
easy and commonly used model of I/R in large
animals and rodents.3,4
The I/R injury of the small intestine caused by
the occlusion of the fine mesenteric artery affects
the vascular structures that exist in all layers of
the intestinal tissue. The injury of the apical part
of the microcirculation bloodstream is associated
with a decrease in intestinal mucosal blood perfu-
sion that simultaneously occurs with apical villous
destruction.5 Tissue damage largely occurs due to
I/R injury rather than due to the initial ischemic
insult or the oxygen free radicals, which initiate
reperfusion injury. Neutrophils, platelets, endothe-
lial factors, and cytokines are also believed to be
important pathogenic mechanisms of intestinal I/R
injury.6 Different biochemical methods are used
to determine tissue injury; total antioxidant status
(TAS) and total oxidative status (TOS) are among
the known methods. These markers were compre-
hensively evaluated in the reports of experimental
peripheral ischemia studies.7
Zofenopril is a derivative of the amino acid
proline and an inhibitor of angiotensin-converting
enzyme (ACE) and angiotensin II. Zofenopril 15
mg/kg given orally (before laparotomy) after 2
hours of ischemia ameliorates experimental cardi-
ac I/R injury or doxorubicin-induced cardiac in-
jury in animal models.8-10 Zofenopril is an antioxi­
dant agent used to regulate blood pressure as a
cardioprotective agent.11 The effect of zofenopril on
the sulfhydryl group is reported to be significant,
and the oxidation of the protein sulfhydryl group
is mentioned to have an important role in the path­
ophysiology of myocardial damage associated with
ischemia and reperfusion.12,13 It has been shown
that zofenopril reduces ischemia-reperfusion injury
in experimental ischemia-reperfusion models such
as renal, cardiac, cerebral, and testis tissues.14-16
Programmed cell death apoptosis defines damaged
cells. In normal cells, apoptosis is caused by devel-
opmental marks and cellular stress or damage.17
Caspases are known to modulate inflammation
and cell death, so they are involved in apoptosis.
Depending on their role, caspases differ from each
other, and caspase-3 is an executioner caspase.18
In this study, intestinal I/R-induced apoptosis oc-
curred by triggering signaling pathways in the
mitochondria.19
The aim of this study was to determine the pre-
ventive and apoptosis-reducing effect of zofeno-
pril in the treatment of intestinal I/R injuries by
biochemical histopathological and immunohisto-
chemical methods.
Materials and Methods
All experimental protocols conducted on animals
were consistent with the National Institutes of
Health Guidelines for the Care and Use of Labora-
tory Animals and approved by the Health Sciences
University, Ankara Education and Research Hos-
pital Ethics Committee of Animal Care and Usage.
In this study, 35 3-month-old male Wistar albino
rats weighing 230–280 g each were used. Rats were
randomly allocated into one of five groups (n=7
in each group). The jejunum tissue removed dur-
ing the operation was divided into two pieces of
equal size and stored under suitable conditions for
biochemical and histopathological investigations.
Group 1: sham operation (laparotomy only,
sham surgical preparation including isolation of
the superior mesenteric artery without occlusion).
Group 2: 2-hour period of ischemia (superior
mesenteric artery occlusion for 120 minutes).
96 Analytical and Quantitative Cytopathology and Histopathology®
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·
pek and Doğan

3. Group 3: 2-hour period of ischemia followed by
a 2-hour reperfusion (superior mesenteric artery
occlusion for 2 hours followed by 2 hours reperfu-
sion).
Group 4: a 2-hour period of ischemia in which
rats were treated with zofenopril 15 mg/kg oral
dose (before laparatomy), after 2 hours of ische­
mia. After 2 hours of reperfusion, the jejunum was
removed for examination.
Group 5: Only 15 mg/kg zofenopril was orally
administered without ischemia.
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 con­
ditions. The abdominal region was shaved, and a
2–3 cm abdominal midline incision was made. In
the intestinal I/R injury model, the superior mes-
enteric artery 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.
120 minutes of reperfusion was performed.
Biochemical Analysis
Intestinal tissue investigations were performed after
the thawing of the samples from −80°C to room
temperature. The tissue samples were weighed
with a scale of 0.001 gram sensitivity. The work-
ing solution was added in an amount 9 times the
sample quantity, after which the samples were
homogenized by using a mechanical homogenizer.
The samples were then centrifuged at 3,000 rpm
for 5 minutes, after which the supernatant was
discarded and calorimetrically analyzed by using
the Rel Assay E auto analyzer, as detailed below.
Rel Assay kits (Rel Assay Diagnostics, Gaziantep,
Turkey) were used in the analysis. The body’s to-
tal antioxidant status (TAS) against strong free
radicals was measured by using a fully automatic
method developed by Erel et al,20 and the total
oxidant status (TOS) was also evaluated by using
a fully automatic method developed. The oxida-
tive stress index (OSI) was calculated by dividing
the TOS into TAS.
Measurement of Total Oxidant Status (TOS). The
TOS was analyzed by using a fully automated cal-
orimetric method, in which the oxidants present
in the sample oxidize ferrous ion-o-dianisidine
complexes to ferric ion. The glycerol present in
the medium accelerates this reaction, increasing its
rate by almost three times. The ferric ions form a
colored complex with xylenol orange in an acidic
medium, and the intensity of the color, denoting
the amount of oxidants present in the medium,
can be measured spectro-photometrically. The re­
sults of the measurement are reported in a micro-
molar hydrogen peroxide equivalent per liter unit
(μmol H2O2 Equiv/L).
Measurement of Total Antioxidant Status (TAS). The
total antioxidant level is measured using the auto-
mated method developed by Erel et al,20 which is
based on the whitening of the characteristic blue-
green color of a stable radical cation 2,2′-azinobis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by
antioxidants. The reduced ABTS molecule is oxi-
dized by H2O2 to ABTS+ in an acidic medium.
ABTS+ can remain stable in an acetate buffer
(30 mmol/L, pH 3.6) for long periods. When the
ABTS+ molecule is diluted with an acetate buffer
with a higher pH and concentration (0.4 mmol/L,
pH 5.8), its color slowly and spontaneously whit-
ens, with the increasing rate of whitening accord-
ing to the antioxidant concentration present in the
sample. This reaction can be spectrophotometri-
cally monitored, and the rate of color whitening
is in reverse proportion to the total antioxidant
capacity of the sample. TAS levels were measured
by using commercially available kits (Relassay,
Turkey), which is a method that has less than
3% error margin. The reaction was calibrated with
Trolox, which is used to standardize the measure-
ments of total antioxidant levels. The results of
the measurements are reported as mmol Trolox
equivalent/L.
Measurement of Oxidative Stress Index (OSI). The
OSI value was calculated based on a formula,
being taken as the percentage of the TAS to TOS
ratio. TAS values were converted to μmol/L in
advance:
TOS (μmol H2O2 Equiv/L)
OSI (arbitrary unit) = __________________________ ×100
TAS (μmol Trolox Equiv/L)
Histopathological Examination
For microscopic evaluation, samples were taken
from the sections of rat intestine tissue with the
Volume 42, Number 3/June 2020 97
Zofenopril in Ischemia-Reperfusion Model

4. highest macroscopic damage. The colon of each
rat was separately put into a formaldehyde me-
dia. Tissues were detected in the 10% buffered for-
malin, a routine tissue follow-up was performed,
and they were embedded in paraffin blocks. Slides
of 5 μm thickness were cut from the parafin blocks
using a microtome and deparaffinized. The sam-
ples were dyed with hematoxylin-eosin stain and
examined under a light microscope (Nikon Eclipse
Ni) for the evaluation of parameters such as ede-
ma, vascular congestion, hemorrhage, inflammato-
ry cell infiltration, and mucosal damage.
A semiquantitative histological evaluation scor-
ing system was used to determine histopatholog-
ical changes. The mucosal damage was evaluated
with the modification of Schweizer et al21 and Chiu
semiquantitative scoring systems. The criteria used
to evaluate mucosal injury were damage/decom-
position in the surface epithelium (basal membrane
preserved), vascular congestion, hemorrhage, and
infiltration by inflammatory cells. Each specimen
was scored by using a scale ranging from 0 to 3
(0=none, normal histological structure, 1=mild,
damage in the surface epithelium, 2=moderate,
damage in the surface epithelium and lamina pro-
pria, and 3=severe, full-layer mucosal damage) for
each criterion.
Immunohistochemical Methods
Samples taken from the I/R rat small intestine
tissue were placed into 10% neutral formaldehyde
solution. Following the routine paraffin protocol,
4–6 μm paraffin sections were cut with a micro-
tome (Leica, Germany). Antigen retrieval process
was performed in citrate buffer solution (pH 6.0)
twice (2×5 minutes) in a 700 W microwave oven.
The sections were left to cool at room tempera­
ture for 20 minutes and washed in distilled water
twice for 4 minutes. Endogenous peroxidase ac-
tivity was blocked in 3% hydrogen peroxide so-
lution for 7 minutes. Ultra V block (catalog no.
1754084A, Histostain-Plus Kit, Novex Life Tech­
nologies, Frederick, Maryland, USA) was applied
for 8 minutes prior to the application of prima-
ry antibody caspase-3 (catalog no. sc-7272, Santa
Cruz Biotechnology, USA) for overnight. Second-
ary antibody (catalog no. 1754084A, Histostain-
Plus Kit, Novex Life Technologies) was applied
for 20 minutes. The sections were then exposed to
streptavidin-peroxidase for 20 minutes. Diamino-
benzidine (catalog no. 1636518A, DAB-Plus Sub-
strate Kit, Novex Life Technologies) was used as
a chromogen. After being counterstained with he-
matoxylin and washed in tap water for 3 minutes
and in distilled water for 2×3 min, the slides were
mounted. Sections were examined under light mi-
croscope (Carl Zeiss Imager A2, Germany).
Statistical Analysis
Statistical analyses were conducted with SPSS
software (Version 22.0., Released 2013; IBM SPSS
Statistics for Windows, IBM Corp., Armonk, New
York, USA). Descriptive statistics were presented
as median (min-max) and mean±standard devia-
tion values. Distributions of the groups were valu-
ated by Shapiro-Wilk tests. The significance of the
difference among more than two groups was eval-
uated by using the Kruskal-Wallis test since data
did not meet the assumptions of the parametric
test ANOVA. Post hoc tests with Bonferroni cor­
rection were used to determine which groups dif-
fered with pairwise comparison. A value of p<0.05
was considered as statistically significant.
Results
Kruskal-Wallis ANOVA test results showed sta-
tistically significant differences among the groups
(p<0.001). To determine the difference, Bonferroni
correction and post hoc two-way comparison tests
were applied. Based on those tests, there was a
significant difference between control groups and
groups of ischemia (2 h), ischemia (2 h)+reperfu-
sion, and ischemia (1.5 h)+reperfusion (0.5 h)+
drug groups (p=0.010, 0.010, and 0.010, respec-
tively). There was also a significant difference
between the drug group and groups of ischemia
(2 h), ischemia (2 h)+reperfusion, and ischemia
(1.5 h)+reperfusion (0.5 h)+drug (p=0.010, 0.010,
and 0.010, respectively). Details are shown in Ta-
ble I. There was no significant difference among
the other groups (p>0.05). The zofenopril group
was different from the other groups. In other
words, there were significant differences between
the ischemia, ischemia/reperfusion, and ischemia/
reperfusion+zofenopril groups (p=0.003, 0.003,
and 0.003, respectively). However, there was no
difference between the drug group and the other
groups (p>0.05). The results are given in Table II,
and score distributions of the groups are shown in
boxplot graph in Figure 1.
Kruskal-Wallis ANOVA test results showed a
statistically significant difference between the zo-
fenopril scores of the rat groups (p<0.001). To de­
termine the difference, post hoc two-way compari-
98 Analytical and Quantitative Cytopathology and Histopathology®
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pek and Doğan

5. son tests were applied. Based on those tests, there
was a significant difference between the control
group and the groups of ischemia (2 h), ischemia
(2 h)+reperfusion, and ischemia (1.5 h)+reperfu­
sion (0.5 h)+drug (p=0.014, 0.014, and 0.024, re­
spectively). There was also a significant difference
between the drug group and groups of ischemia
(2 h), ischemia (2 h)+reperfusion, and ischemia
(1.5 h)+reperfusion (0.5 h) and drug (p=0.014,
0.014, and 0.024, respectively). Details are shown
in Table I. There was no significant difference be-
tween the other groups (p>0.05). The distribution
of zofenopril scores of the groups is shown by box-
plot graph in Figure 2.
Volume 42, Number 3/June 2020 99
Zofenopril in Ischemia-Reperfusion Model
Table I Comparison of Chiu’s Score According to Rat Groups with Kruskal-Wallis ANOVA
Post-hoc
Group N Mean SD Median Min Max p Value p value
1. Control 6 0 0 0 0 0 <0.001** 1-2: 0.014**
1-3: 0.014**
1-4: 0.024*
2. Ischemia (2 h) 6 4.5 0.84 5 3 5 2-5: 0.014**
3. Ischemia (2 h)+reperfusion 6 4.5 0.84 5 3 5 3-5: 0.014**
4. Ischemia (1.5 h)+reperfusion (0.5 h)+drug 6 4.5 0.55 4.5 4 5 4-5: 0.024*
5. Drug 6 0 0 0 0 0
Statistically significant: p<0.01**, p<0.05*.
1 = control, 2 = ischemia (2 h), 3 = ischemia (2 h)+reperfusion, 4 = ischemia (1.5 h)+reperfusion (0.5 h)+drug, 5 = drug, SD = standard deviation.
Table II Comparison of Histological Evaluation Scores According to Rat Groups with Kruskal-Wallis ANOVA (n=6)
Ischemia
Ischemia (1.5 h)+
Ischemia (2 h)+ reperfusion Post-hoc
Control (2 h) reperfusion (0.5 h)+drug Drug p Value p value
Damage/decomposition in the 0 (0–0) 3 (2–3) 3 (2–3) 3 (2–3) 0 (0–0) <0.001* 1-2: 0.010*
surface epithelium (basal 0±0 2.83±0.41 2.83±0.41 2.83±0.41 0±0 1-3: 0.010*
membrane preserved) 1-4: 0.010*
Zofenopril 2-5: 0.010*
3-5: 0.010*
4-5: 0.010*
Vascular congestion 0 (0–1) 3 (2–3) 3 (2–3) 3 (2–3) 1 (0–2) <0.001* 1-2: 0.003**
Zofenopril 0.17±0.41 2.83±0.41 2.83±0.41 2.83±0.41 1.17±0.75 1-3: 0.003**
1-4: 0.003**
Hemorrhage 0 (0–0) 3 (3–3) 3 (3–3) 3 (3–3) 0 (0–0) <0.001* 1-2: 0.005**
0±0 3±0 3±0 3±0 0±0 1-3: 0.005**
1-4: 0.005**
2-5: 0.005**
3-5: 0.005**
4-5: 0.005**
Inflammation 0 (0–0) 2 (1–3) 2 (2–3) 2 (1–3) 0 (0–0) <0.001* 1-2: 0.016*
Zofenopril
0±0
2.17±0.75
2.17±0.41
2.17±0.75
0±0 1-3: 0.019*
1-4: 0.016*
2-5: 0.016*
3-5: 0.019*
4-5: 0.016*
Statistically significant: p<0.01**, p<0.05*.
1 = control, 2 = ischemia (2 h), 3 = ischemia (2 h)+reperfusion, 4 = ischemia (1.5 h)+reperfusion (0.5 h)+drug, 5 = drug.

6. Histopathologic Results
Group 1 (Control Group). Jejunum mucosa structure
was observed in normal histological morphology
(Figure 3A).
Group 2 (2 Hours of Ischemia). Microscopic exam-
ination of the small intestinal tissue of the 2-hour
ischemic group revealed an evident villus dam-
age, hemorrhage, and cellular inflammation in
lamina propria and necrosis of epithelial cells,
Chiu’s score 3 (Figure 3B).
Group 3 (2 Hours of Ischemia and 2 Hours of Reper-
fusion). Mucosal damage was evident in the jeju-
num mucosa. Lamina propria showed hemorrhage,
congestion, and cellular inflammation, Chiu’s score
2 (Figure 3C).
Group 4 (1.5 Hours of Ischemia+Zofenopril). Micro-
scopic examination revealed an ischemic damage
in the villi, congestion, and mild inflammation in
lamina. Reparative reactive changes were observed
in the surface epithelium, and glandular structures
were preserved, Chiu’s score 2 (Figure 3D).
Group 5 (Only Zofenopril). Jejunum mucosa struc-
ture was observed in normal histological morphol-
ogy, Chiu’s score 1 (Figure 3E).
Representative mucosal morphological changes
are presented in Figure 3. The mucosal injury
100 Analytical and Quantitative Cytopathology and Histopathology®
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pek and Doğan
Figure 2 The boxplot graph of zofenopril scores in all groups.
Figure 1
The boxplot graph of damage
to the epithelium, vascular
congestion, hemorrhage, and
inflammation in all
experimental groups.

7. was quantified as Chiu’s score in Figure 1. Chiu’s
scores in Group 2 (ischemia 2 h), Group 3 (is-
chemia 2 h+reperfusion), and Group 4 (ischemia
2 h+reperfusion 2 h+zofenopril 15 mg/kg) were
significantly higher than those in the control group
(p=0.005, 0.002, and 0.039, respectively).
Biochemical Results
Serum TOS levels were significantly different be-
tween groups 1–5 (p=0.759). The levels of serum
TAS in the I/R group as compared with the zo-
fenopril and zofenopril+I/R groups were found to
be significantly lower (p=0.098). OSI levels were
significantly different between groups 1, 2, 3, 4,
and 5 (p=0.180). Serum OSI levels were high in the
I/R group, and they decreased in the zofenopril
and zofenopril+I/R groups (Table III).
Discussion
Ischemic damage is seen in the intestinal feeding
vessels due to a variety of reasons, including em-
boli, thrombosis, or atherosclerosis-related obstruc-
tions, and vascular reasons such as volvulus, invag-
ination, and congestion in the bloodstream.22,23
As a consequence of intestinal ischemia reper-
fusion, the induction of bacterial translocation is
important for the release of pro-inflammatory cyto-
kines and the loss of intestinal barrier integrity for
intestinal function. Ischemia caused by cell death
and organ failure leads to an inability of the cir-
culation to provide oxygen and other metabolites,
and the resulting residual products cannot return
to their original state by circulation again. Eventu-
ally, acute cellular enlargement, interstitial edema,
and cellular dysfunction occur. Reperfusion of isch-
emic tissue allows one side to recover some of the
functions which were lost during ischemia, while
cell loss on one side leads to further damage.
Intestinal I-R injuries are characterized by altered
microvascular and epithelial permeability and vil-
lus damage.24 In our study, ischemia reperfusion
model with intestinal volvulus injury caused mi-
croscopic intestinal damage such as mucosal de­
struction, villus loss, and epithelial cell apoptosis,
congestion, and infiltration of inflammatory cells.
Zofenopril calcium, a pro-drug of the active
compound zofenoprilat, is a highly lipophilic ACE
inhibitor that has been successfully and safely ap­
Volume 42, Number 3/June 2020 101
Zofenopril in Ischemia-Reperfusion Model
Figure 3 (A) Jejunum mucosa with normal villus crypts (H&E, ×100). (B) Abnormal structure of jejunum mucosa with common
mucosal damage, epithelial breakdown, hemorrhage, and congestion (H&E, ×100). (C) At the surface of the jejunum mucosa, extensive
mucosal damage, villus enlargement, surface epithelial damage, epithelial disintegration, hemorrhage, and congestion (H&E, ×100).
(D) Jejunum mucosa with damaged villi, extensive dissociation of the surface epithelium, hemorrhage, and congestion (H&E, ×100).
(E) Jejunum mucosa with normal villi’s crypt structure (H&E, ×100).
Table III Comparison of the Biochemical Results for Groups
Group 1, Group 2, Group 3, Group 4, Group 5,
mean±SD mean±SD mean±SD mean±SD mean±SD
p Value
TAS (mmol Trolox equivalent/L) 1.47±0.34 1.18±0.46 1.14±0.41 0.91±0.30 1.46±0.41 0.098
TOS (μmol H2O2 equivalent/L) 11.60±4.47 11.24±4.71 14.17±4.99 12.73±2.92 13.37±4.40 0.759
OSI (arbitrary unit) 0.80±0.33 1.06±0.51 1.52±0.99 1.54±0.66 0.98±0.37 0.180
Values are demonstrated as mean±standard deviation (SD).
OSI = oxidative stress index, TAS = total antioxidant status, TOS = total oxidant status.

8. plied in the treatment of essential hypertension.25
The successful use of this drug in cardiac patients
with previous I/R injury in the heart and kidneys,
without clinically significant side effects, also sug-
gests the use of the drug in testicular torsion as well
as in the intestinal ischemia model of the drug.
Uzar et al26 demonstrated that zofenopril de-
creased reactive oxygen radicals and apoptosis
with an experimental cerebral model of I/R. In a
model of testis torsion in rats, Altunoluk et al27
revealed that malondialdehyde and nictric oxide
levels significantly decreased and, on the con-
trary, superoxide dismutase and glutathione per­
oxidase levels significantly increased in the zo-
fenopril group. They also demonstrated the effect
of zofenopril histopathologically.
Donnarumma et al28 reported that zofenopril
treatment had effects on oxidative stress before
ischemic damage occurred. It has been stated that
reactive oxygen species (ROS) is a major factor in
cardiac tissue damage during hypoxia and I/R
conditions, and tissue antioxidant defense can be
improved following ischemic injury during reper-
fusion. Therefore, zofenopril shows its effect by
preventing ROS formation.
According to Gonzalez et al,3 ischemia and re-
perfusion have direct effects on intestinal epithe-
lial cells in vivo. As expressed in their study, cell
death has critical pressure for sustaining normal
barrier function and preserves epithelial integrity
throughout the lumen surface while releasing
dying cells. Cells gradually mature and migrate
along the crypt villus axis. When they approach
the luminal surface, they are exposed to epithelial
cell depletion.
In a previous study, TAS, TOS, and OSI mea­
surements were reported not only to show oxi-
dative and antioxidative status during diagnosis,
but also to play a role in the follow-up of the treat-
ment. The TOS-TAS ratio and OSI are indicatives
of the degree of oxidative stress, showing the
antioxidation and oxidation redox balance.29 TOS
measurement provides a sensitive lipid peroxi-
dation and oxidative stress index.30 Our findings
are consistent with the results of previous studies
measuring oxidative stress as we defined above—
that the levels of serum TAS in the I/R group as
compared to the zofenopril and zofenopril+I/R
groups were found to be significantly lower. How-
ever, OSI levels were high in the I/R group and
they decreased in the zofenopril and zofenopril+
I/R groups. As a consequence, we may suggest
that zofenopril decreased oxidative stress levels in
treated groups.
Apoptosis is a major form of cell death induced
by I/R and gives damage to the intestinal mucosa.
Disruption of intestinal epithelial homeostasis
triggers apoptosis.31,32 Mitochondria-mediated apo­
ptosis plays a central role in tissue homeostasis. It
has been reported that Bcl-2/Bax ratio reduction
activates the mitochondrial apoptotic pathway and
thus divided caspase-3, which is a sign of the
apoptotic delivery protein.33 Type I cell death and
caspase-8 activation directly induces caspase-3
activation by pro-caspase-8. Type II cell death was
reported to be mediated by cytochrome c release,
followed by activation of caspase-3, directly to
the death signal. Therefore, type I cell death path­
way has been reported to be triggered by I/R
induction of apoptosis in the lamina propria of the
small intestine.34 Uzar et al26 demonstrated that
zofenopril decreased reactive oxygen radicals and
apoptosis with an experimental cerebral model
of I/R. Lian et al35 stated that anti-inflammation,
102 Analytical and Quantitative Cytopathology and Histopathology®
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pek and Doğan
Figure 4 (A) Negative caspase-3 expression in jejunum villus epithelial cells, caspase-3 (caspase-3 immunostaining, ×100).
(B) Positive caspase-3 expression in multiple apoptotic cells, intense degeneration of epithelial cells and lamina propria layer (caspase-3
immunostaining, ×100). (C) Positive caspase-3 expression in dense apoptotic cells towards lumen (caspase-3 immunostaining, ×100).
(D) Positive caspase-3 expression, low number of degenerative cells (caspase-3 immunostaining, ×100).

9. anti-oxidation, restoration of barrier function, and
inhibition of apoptosis are indicators of intestinal
I/R damage improvement. In this study the ex-
pression of caspase-3 was increased in the ische­
mia and ischemia/reperfusion group, whereas
the expression of caspase-3 was decreased in the
zofenopril-treated group after I/R (Figure 4).
When ischemia-reperfusion is considered as a
model of cell damage, apoptosis-inducing signal-
ing molecules seem to alter the epithelial and
stromal cell structure, and the use of angiotensin
inhibitor zofenopril may reduce cellular damage.
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