2. White adipose tissue is the major synthesis site of
ASP. Following its synthesis, ASP is released into
the blood, and its plasma concentration increases
during fasting. Besides peripheral target tissues,
ASP can also cross the blood-brain barrier and
have an effect on the central nervous system. ASP
has been reported to have systemic effects, and this
molecule is increasingly gaining popularity among
researchers.3 Meteorin-like protein (METRNL) is
a hormone released into the circulation mainly
after the skeletal muscle and adipose tissue are
exposed to cold or exercise.4 Increased circulating
METRNL results in increased energy expenditure
in the whole body and improves glucose tolerance
in people with obesity.5 In our study, changes in
skeletal muscle and serum levels of ASP and
METRNL were investigated using N-acetylcysteine
(NAC), a powerful antioxidant, in rats with experi
mentally induced IR injury.
Materials and Methods
This study received ethical approval from the local
Animal Research Ethics Committee (Decision No.
2020/01; Date: January 15, 2020) and was con
ducted in the Experimental Research Center.
The rats used in this study were maintained at
22–25°C room temperature with 12 hours of light
(7:00–19:00) and 12 hours of darkness (19:00–7:00).
They were fed in specially constructed cages the
bottoms of which were cleaned daily. Feed was
given in steel containers and drinking water (tap
water) was given in glass bottles. The same stan
dard rat feed was given to all rats, ad libitum water
and food intake was ensured, and the animals
were taken care of by cleaning their containers
daily. In total, 30 male Wistar Albino rats aged
8–10 weeks were divided into 5 groups as follows:
Group I (Control group) (n=6). No procedure was
applied to the rats in this group during the ex-
periment.
Group II (Sham group) (n=6). Laparotomy and ab-
dominal aortic dissection were performed on the
rats in this group. The amount of stress applied
and the duration of the surgical procedure were
the same as that in the other groups.
Group III (N-Acetylcysteine group) (n=6). A single
dose of 150 mg/kg NAC was administered intra
peritoneally to the rats in this group 120 minutes
before the end of the experiment.
Group IV (Ischemia-Reperfusion group) (n=6). The
infrarenal abdominal aorta was exposed by immo
bilizing the rats in the supine position and per
forming laparotomy in the midline of the abdo-
men. Later, the infrarenal abdominal aorta was
clipped using a nontraumatic microvascular clamp
for 120 minutes, followed by opening the clips
and terminating the experiment after 120 minutes
of reperfusion.
Group V (Ischemia-Reperfusion+N-Acetylcysteine
group) (n=6). The infrarenal abdominal aorta was
exposed by immobilizing the rats in the supine
position and performing laparotomy in the mid-
line of the abdomen. Later, the infrarenal abdom
inal aorta was clipped using a nontraumatic
microvascular clamp for 120 minutes, followed
by opening the clips, administering a single dose
of 150 mg/kg NAC intraperitoneally, and termi
nating the experiment after 120 minutes of reper-
fusion.
Anesthesia was achieved using intramuscular
administration of 30 mg/kg ketamine hydrochlo
ride (Ketalar; Pfizer, Groton, Connecticut, USA)
and 3 mg/kg xylazine hydrochloride 37 (Rompun;
Bayer, Leverkusen, Germany).
The rats were anesthetized with additional
doses of the anesthetic (1/3 of the initial dose)
when necessary, while ensuring spontaneous ac
tivity of their respiratory muscles throughout the
procedure. To prevent possible hypothermia, the
operation was performed in the supine position
under a heating lamp. The skin was prepared asep
tically, and laparotomy was performed in the mid-
line of the abdomen. To maintain fluid balance,
10 mL warm saline was administered into the
physiological peritoneal cavity. The abdominal
aorta was accessed by pulling the intestines to
the left with wet gas. To achieve anticoagulation,
150 U/kg heparin (Nevparin; Mustafa Nevzat I
∙
laç
San, Istanbul, Turkey) was administered intra
venously through the tail vein 2 minutes before
placing the aortic clamp. A lower extremity ische
mia rat model was created by placing a non
traumatic micro
vascular clamp on the infrarenal
abdominal aorta. The absence of vascular flow
was confirmed using a HADECO brand ES-101 EX
model handheld doppler device on the distal side
of the clamp. The abdominal incision was closed
to minimize heat and fluid loss. The induced is-
chemia was observed for 120 minutes. After oc-
clusion, the abdomen was reopened and the mi-
68 Analytical and Quantitative Cytopathology and Histopathology®
Ozguler and Ustunel
3. crovascular clamp in the infrarenal abdominal
aorta was removed, followed by reperfusion for
120 minutes. To evaluate the ischemia and reper
fusion procedure with aortic clamping, handheld
doppler device was used to confirm the loss of
flow in the distal aorta during the clamping pro-
cedure. Similarly, to evaluate the reperfusion, a
handheld doppler device was used to confirm the
maintenance of flow in the distal aorta after clamp
removal.
Retrieval of Samples
At the end of the experiment, following intracar-
diac blood collection under anesthesia from the
rats of all groups, muscle tissues of the lower ex
tremity were removed rapidly. The blood samples
were centrifuged at 4,000 rpm for 5 minutes, and
the sera obtained were kept at −80°C until further
analysis.
The muscle tissue samples were fixed in 10%
formaldehyde solution for immunohistochemical
analysis. After fixing, the samples were washed
using tap water and were analyzed using rou
tine histological analysis. The tissues were embed
ded in paraffin blocks cut into 4–6 µm thick sec
tions that were then placed on polylysine-coated
slides.
Biochemical Analysis
Total Oxidant Status Measurement. Serum total oxi
dant status (TOS) levels were measured using a
Rel Assay Total Oxidant Status Test Kit (Mega Tip,
Gaziantep, Turkey).6
Serum METRNL and ASP Measurement. Serum lev
els of METRNL (Rat METRNL ELISA kit, SunRed
201-11-3875, China) and ASP (Rat Asprosin ELISA
kit, SunRed 201-11-5748, China) were measured
using ELISA.
Immunohistochemical Analysis
The 4–6 µm thick sections retrieved from the
paraffin blocks were placed on polylysine-coated
slides. Deparaffinized tissues were passed through
graded series of alcohol and boiled in a citrate buf
fer solution of pH 6 in a microwave oven (750 W)
for 7+5 minutes for antigen retrieval. After boil
ing, the tissues were kept at room temperature for
approximately 20 minutes to cool down, washed
3× using phosphate-buffered saline (PBS; P4417,
Sigma-Aldrich, USA) for 5 minutes each time, and
incubated for 5 minutes with hydrogen peroxide
block solution (Hydrogen Peroxide Block, TA-125-
HP, Lab Vision Corporation, USA) to prevent
endogenous peroxidase activity. The tissues were
again washed 3× using PBS for 5 minutes each
time. After applying Ultra V Block (TA-125-UB,
Lab Vision Corporation, USA) solution to the tis
sues for 5 minutes to prevent background stain-
ing, METRNL and ASP were diluted to 1/200 and
incubated with primary antibodies (METRNL Po-
lyclonal Antibody, PAH662Ra01, CLOUD-CLONE
Corp., Katy, Texas, USA) and with antiasprosin
antibody (FNab09797, Fine Test, China) for 60
minutes at room temperature in a humid envir-
onment. After this, the tissues were washed using
PBS as previously mentioned and incubated
with the secondary antibody (Biotinylated Goat
anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-
BN, Lab Vision Corporation, USA) for 30 min
utes at room temperature in a humid environ
ment. Then, the tissues were washed using PBS
as previously mentioned, incubated with strepta
vidin peroxidase (TS-125-HR, Lab Vision Corpo
ration, USA) for 30 minutes at room temperature
in a humid environment, and then kept in PBS.
3-amino-9-ethylcarbazole (AEC) Substrate+AEC
Chromo
gen (AEC Substrate, TA-015 and HAS,
AEC Chromogen, TA-002-HAC, Lab Vision Cor-
poration, USA) solution was dropped on the tis
sues, and after detecting the image signal under
the light microscope, the tissues were washed with
PBS. The tissues that were counterstained with
Mayer’s hematoxylin were passed through PBS
and distilled water and covered with an appropri
ate closure solution (Large Volume Vision Mount,
TA-125-UG, Lab Vision Corporation, USA). For
negative control, PBS was applied in the other
tissues instead of the primer in the same way. The
prepared tissues were then examined, evaluated,
and photographed under a Leica DM500 micro
scope (Leica DFC295).
A histoscore was defined based on the preva-
lence (0.1: <25%, 0.4: 26–50%, 0.6: 51–75%, 0.9:
76–100%) and severity (0: none, +0.5: very little,
+1: low, +2: moderate, +3: severe) of immunoreac-
tivity. Histoscore = prevalence×severity.
Statistical Analyses
SPSS version 22 (IBM Corp. Released 2013. IBM
SPSS Statistics for Windows, Version 22.0. IBM
Corp., Armonk, New York, USA) was used for
statistical analyses. Numerical measurements were
presented as median and minimum-maximum.
Volume 43, Number 2/April 2021 69
N-Acetylcysteine on Meteorin-like Protein and Asprosin
4. The Kruskal-Wallis test was used for general com
parisons between more than 2 groups. The Mann-
Whitney U test was used for comparison between
2 groups. The statistical significance level was set
at 0.05 in all tests.
Results
Biochemical Findings
Serum TOS Levels. In the biochemical analysis
performed for evaluating the serum TOS levels of
all groups as compared with the control group,
no statistically significant difference was observed
between the Sham (p=0.548) and NAC (p=0.167)
groups; however, a statistically significant increase
was observed in the TOS levels of the IR group
(p=0.002). In addition, when compared with the
IR group, a statistically significant decrease in the
TOS levels was observed in the IR+NAC group
(p=0.024) (Table I).
Serum METRNL Levels. In the biochemical analy
sis performed for evaluating the serum METRNL
levels of all groups as compared with the control
group, no statistically significant difference was
observed between the Sham (p=0.229) and NAC
(p=0.133) groups; however, a statistically signifi
cant decrease was observed in the METRNL lev
els of the IR group (p=0.010). In addition, when
compared with the IR group, a statistically signifi
cant increase in the METRNL levels was observed
in the IR+NAC group (p=0.009) (Table I).
Serum ASP Levels. In the biochemical analysis
performed for evaluating the serum ASP levels
of all groups as compared with the control group,
no statistically significant difference was observed
between the Sham (p=0.262) and NAC (p=1.00)
groups; however, a statistically significant decrease
was observed in the ASP levels of the IR group
(p=0.002). When compared with the IR group, a
statistically significant increase in the ASP levels
was observed in the IR+NAC group (p=0.010)
(Table I).
Immunohistochemical Findings
METRNL Immunoreactivity. The examination of
immunohistochemical staining under the light
microscope for METRNL immunoreactivity result
ed in the observation of METRNL immunoreac
tivity in muscle tissue myocytes (black arrow).
Compared to the control group (Figure 1a), there
was no statistically significant difference between
the Sham (Figure 1b) (p=0.394) and NAC (Figure
1c) (p=0.394) groups in terms of METRNL immu
noreactivity, but there was a statistically signifi-
cant decrease in METRNL immunoreactivity in
the IR group (Figure 1d) (p=0.002). When com-
pared with the IR group, a statistically significant
increase in METRNL immunoreactivity was ob
served in the IR+NAC group (Figure 1e) (p=
0.002), and METRNL immunoreactivity was not
observed in the negative control (Figure 1f) (Ta-
ble II).
ASP Immunoreactivity. The examination of immu
nohistochemical staining under the light micro
scope for ASP immunoreactivity resulted in the
observation of ASP immunoreactivity in muscle
tissue myocytes (black arrow).
When compared with the control group (Fig
ure 2a), there was no statistically significant dif-
ference in the ASP immunoreactivities of the
Sham (Figure 2b) (p=0.589) and NAC (Figure 2c)
(p=0.818) groups, but there was a statistically sig
nificant decrease in the ASP immunoreactivity of
the IR group (Figure 2d). When compared with
70 Analytical and Quantitative Cytopathology and Histopathology®
Ozguler and Ustunel
Table I Serum Total Oxidant Status, Meteorin-like Protein, and Asprosin Levels
Total oxidant status Meteorin-like protein
(µmol/L) (ng/mL) Asprosin (ng/mL)
Group Median (min-max) Median (min-max) Median (min-max)
Control 7.88 (7.23–8.22) 2.52 (2.30–2.86) 39.21 (30.69–48.49)
Sham 7.92 (6.89–8.01) 2.18 (1.49–2.47) 44.99 (36.71–49.93)
NAC 7.11 (7.23–8.22) 1.97 (1.88–2.06) 41.41 (34.36–48.47)
IR 11.75 (10.09–13.44)a 0.68 (0.25–1.24)a 22.19 (17.98–26.12)a
IR+NAC 8.75 (8.71–9.89)b 2.11 (1.24–2.47)b 44.39 (41.03–48.65)b
Values are presented as median (min-max).
aCompared with the control group.
bCompared with the IR group (p<0.05).
5. the IR group, a statistically significant increase
in ASP immunoreactivity was observed in the
IR+NAC group (Figure 2e) (p=0.017), and ASP
immunoreactivity was not observed in the nega-
tive control (Figure 2f) (Table II).
Discussion
IR injury is a pathology wherein complex inflam
matory processes are involved in the process of
reestablishing blood flow to areas with impaired
circulation. The regional and systemic effects of
free oxygen radicals cause major damage. NAC,
a glutathione precursor, is a molecule that has
been reported to be beneficial in IR injuries be-
cause it prevents oxidative damage at the molec
ular level.7 In the IR model created in our study,
the increase in TOS levels and the significant
decrease in TOS levels in the NAC-administered
group are in line with previously reported data.1,7
In addition, the lower levels of TOS in the control
and sham groups as compared with the IR group
suggest that IR injury significantly increases oxi
dant molecules.
METRNL is a recently discovered molecule
which is produced in the adipose tissue. METRNL
has been reported to reduce lipid-mediated inflam
matory response and insulin resistance through
activation of 5′ AMP-activated protein kinase
(AMPK) or PPARγ signaling pathways in the ske
letal muscles of mice.5
In the H9C2 cell line, METRNL has been shown
to reduce oxidative stress-induced myocardial
damage by activating the AMPK-PAK2 pathway,
which attenuates the effects of oxidative stress
on the endoplasmic reticulum, thereby reducing
cardiomyocyte apoptosis. Hence, METRNL has
been suggested for the treatment of myocardial
ischemia.8 In another study on the H9C2 cell line,
METRNL reduced the harmful effects of doxoru
bicin, which causes oxidative damage on cardiac
cells.9 In the same study it was shown that while
the protective effects of METRNL were evident,
the antitumor potential of doxorubicin was also
preserved. Hence, it can be assumed that the anti
apoptotic characteristic of METRNL is cell type–
specific.
Similar to the results of these studies, in our
study METRNL decreased in response to increased
TOS in the tissue and serum in the IR model,
suggesting that exogenous supplementation of
METRNL in cell cultures can protect cardiomyo
cytes and skeletal muscle, thereby providing effec
Volume 43, Number 2/April 2021 71
N-Acetylcysteine on Meteorin-like Protein and Asprosin
Table II Meteorin-like Protein and Asprosin Immunoreactivity
Histoscore
Meteorin-like protein Asprosin
Group Median (min-max) Median (min-max)
Control 0.90 (0.80–2.70) 0.40 (0.30–0.60)
Sham 1.05 (0.60–1.80) 0.35 (0.20–0.45)
NAC 1.80 (0.80–2.70) 0.42 (0.10–0.60)
IR 0.42 (0.30–0.80)a 0.10 (0.10–0.20)a
IR+NAC 1.05 (0.80–1.80)b 0.30 (0.20–0.45)b
Values are presented as median (min-max).
aCompared with the control group.
bCompared with the IR group (p<0.05).
Figure 1
Meteorin-like protein
immunoreactivity of the study
groups.
6. tive protection against IR injury. It is also known
that a significant amount of METRNL is synthe
sized in the skeletal muscle during exercise.10 The
reduction in METRNL to its normal levels in the
period following NAC administration strengthens
the link between METRNL and oxidative stress.
In addition, changes in circulation indicate that
METRNL may also be effective in distal organ
pathologies of IR injury.
ASP, a hormone synthesized in the white adi-
pose tissue, regulates hepatic glucose release.2
In addition, ASP has been reported to cross the
blood-brain barrier, stimulate orexigenic neurons
through G protein–related pathways, and inhibit
anorexigenic neurons.11 ASP has been shown to be
present in various tissues, such as liver, pancreas,
kidney, testis, skeletal muscle, and heart.12,13 The
biological effect of ASP on the liver depends on
the G protein receptor, resulting in glucose pro
duction and release.2 It has been reported that
beta cells are also synthesized under hyperlipid-
emic conditions.14 In addition, it has been shown
that the expression of superoxide dismutase in-
creases via activation of the ERK1/2 signaling
pathway in mesenchymal stromal cells treated
with ASP.15 In another study, ASP was found to
inhibit the production of malondialdehyde and
oxidation products in mouse cardiomyocytes.16
With this effect, ASP inhibits cell death by de-
creasing the level of reactive oxygen radicals.
Contrary to the protective effects of ASP on the
heart, ASP increases free oxygen radicals and pro
inflammatory cytokines via toll-like receptor in
beta cells.14 Therefore, it may be thought to cause
the loss of ASP beta cells. It has been claimed that
METRNL and ASP can be biomarkers in differ
entiating between malignant mesothelioma and
benign diseases.13 Jung et al17 suggested that, con
trary to its effects on the heart, ASP has no effect
on the formation of free oxygen radicals in the
skeletal muscles. However, the receptor that ASP
binds to in the skeletal muscles has not yet been
elucidated.3 The data on the effects of ASP on
skeletal muscles and IR injury is preliminary and
conflicting. This is because ASP is a newly discov
ered protein. In our experiment, the reduction in
ASP levels in the oxidative environment of IR in-
jury and the elevation in ASP levels in NAC or
control groups will provide key insights for future
studies on this topic.
It has long been known that adipokines and
myokines are effective in inflammatory pro
cesses.3,8,18,19 We demonstrated changes in the
tissue and serum levels of METRNL and ASP,
which are newly identified adipokines, in lower
extremity IR injury. We proved their presence
in the skeletal muscle immunohistochemically. In
addition, the simultaneous changes in the levels
of METRNL and ASP in the serum and the region
of IR injury suggest that the IR injury may be
the source of the circulating levels of these mole
cules. Hence, we believe that ASP and METRNL
may be useful in the diagnosis or treatment of
lower extremity injuries, which are an important
clinical pathology as they cause both local and dis
tal organ damage.
72 Analytical and Quantitative Cytopathology and Histopathology®
Ozguler and Ustunel
Figure 2
Asprosin immunoreactivity of
the study groups.
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