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Clin. Exp. Obstet. Gynecol. 2022; 49(3): 058
http://doi.org/10.31083/j.ceog4903058
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
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Original Research
The effect of carvacrol on oxido-inflammatory ovarian injury and
infertility induced by ischemia-reperfusion in rats
Nurullah Sahin1, Ilhan Bahri Delibas2, Unal Isaoglu3, Bahadir Suleyman4, Gulce Naz Yazici5,
Taha Abdulkadir Coban6, Kemine Uzel7,*, Halis Suleyman4, Veysel Arslan8
1Department of Gynecology and Obstetrics, Private Kastamonu Anadolu Hospital, 37200 Kastamonu, Turkey
2Department of Gynecology and Obstetrics, Faculty of Medicine, Tokat Gaziosmanpasa University, 60250 Tokat, Turkey
3Department of Gynecology and Obstetrics, Private Adatıp İstanbul Hospital, 34912 İstanbul, Turkey
4Department of Pharmacology, Faculty of Medicine, Erzincan Binali Yildirim University, 24100 Erzincan, Turkey
5Department of Pathology, Faculty of Medicine, Erzincan Binali Yildirim University, 24100 Erzincan, Turkey
6Department of Biochemistry, Faculty of Medicine, Erzincan Binali Yildirim University, 24100 Erzincan, Turkey
7Department of Obstetrics and Gynecology, Erzincan Binali Yıldırım University Mengucek Gazi Training and Research Hospital, 24000 Erzincan,
Turkey
8Department of Bayburt Vocational High School of Health Services, Bayburt University, 69000 Bayburt, Turkey
*Correspondence: kemineuzel@hotmail.com (Kemine Uzel)
Academic Editor: Michael H. Dahan
Submitted: 17 May 2021 Revised: 28 July 2021 Accepted: 11 August 2021 Published: 4 March 2022
Abstract
Background: Increased oxidants and proinflammatory cytokines play a role in the pathogenesis of ovarian ischemia-reperfusion (OIR)
injury and related infertility. Carvacrol has antioxidant, antibacterial and anti-inflammatory properties. Methods: Protective effect
of carvacrol against ischemia-reperfusion (IR)-related ovarian damage and infertility was investigated. IR process were applied to the
ovaries of rats, which were divided into the following groups (n = 12): OIR, IR +50 mg/kg carvacrol (IRC-50), IR +100 mg/kg carvacrol
(IRC-100) and sham group (SG). After the reperfusion process, six rats from each group were killed and the removed ovaries were
examined biochemically and histopathologically. The remaining animals were kept two months with mature male rats to reproduce.
Results: At a dose of 50 mg/kg, carvacrol suppressed the oxidant parameter increase and antioxidant decrease caused by IR in ovarian
tissue. At a dose of 100 mg/kg, carvacrol antagonized both oxidant and proinflammatory cytokine increase and antioxidant decrease.
Histopathologically, severe degeneration of follicles in the ovaries of the OIR group, necrotic cell accumulations, hemorrhage in the
corpus luteum, edema in the interstitial tissue, polymorphous nuclear leukocyte (PNL) infiltration, and congestion and dilation of blood
vessels were detected. Inflammatory symptoms such as edema in the ovarian tissue, congested dilated blood vessels and PNL infiltration
were observed at a dose of 50 mg/kg of carvacrol, but these histopathological findings were not observed at a dose of 100 mg/kg.
Conclusion: A dose of 100 mg/kg carvacrol, which eliminated inflammatory damage, significantly prevented the development of IR-
induced infertility. Carvacrol may be beneficial in the treatment of IR-related ovarian damage and infertility.
Keywords: Carvacrol; Oxido-inflammatory; Infertility; Rat
1. Introduction
Ovarian ischemia is an emergency resulting from a
sprain (torsion) of the ovaries [1]. Ovarian torsion is more
common in patients with ovulation induction, pregnant
women, women of reproductive age, patients with a benign
or malignant ovarian mass greater than 5 cm, and women
who have experienced a previous ovarian torsion [2,3]. De-
lays in the diagnosis and treatment of ovarian torsion may
result in permanent tissue damage and organ loss [3,4].
Therefore, it is recommended that patients receive reperfu-
sion of the torsioned ovaries with detorsion [5]. However,
detorsion of torsioned ovaries can cause more severe tissue
damage. This event is called ischemia-reperfusion (IR) in-
jury [6]. Studies show that oxidative stress is significant
in IR damage. As is known, nicotinamide adenine dinu-
cleotide is used in the metabolism of hypoxanthine (HX)
with xanthine dehydrogenase (XDH) in an anaerobic envi-
ronment. Therefore, reactive oxygen species (ROS) are not
produced as intermediates [7]. However, XDH produced
in an aerobic environment is converted to xanthine oxidase
(XO) in an anaerobic environment [8]. Since molecular
oxygen (O2) is used in the metabolism of HX with XO,
ROS are produced as an intermediate product. Therefore,
the metabolism of HX by the XO enzyme cannot occur un-
less there is reperfusion in ischemic tissue [9]. Providing
reoxygenation in reperfusion causes the metabolism of HX
and excessive ROS formation [10]. These ROS, known as
reperfusion mediators, cause the formation of toxic prod-
ucts such as aldehyde and malondialdehyde (MDA) from
lipids by oxidizing cell membrane lipids [11]. It is known
that reduced glutathione (GSH), known as an endogenous
antioxidant, decreases in ovarian tissue with high MDA
content [12].
Another event that is thought to contribute to the ex-
acerbation of IR injury is inflammation. Inflammation me-
diators induce an inflammatory response. Recently, proin-
flammatory interleukin 1 beta (IL-1β) and tumor necrosis
factor-alpha (TNF-α) have been shown to increase in tis-
sues exposed to IR [13]. In addition, ROS is known to
stimulate the expression of nuclear factor kappa B (NF-
κB), which controls the production of proinflammatory cy-
tokines. Fatma et al. [14] indicated that the rising in ox-
idative stress parameters in ovarian IR was associated with
a considerable rising the expression of NF-κB and TNF-
α. Unlubilgin et al. [15] reported in their study that the
increase of oxidant and proinflammatory cytokines in IR-
treated ovarian tissue was associated with infertility. This
information obtained from the literature reveals the impor-
tance of the early diagnosis and treatment of ovarian torsion
in preserving ovarian function and preventing future infer-
tility [1]. As mentioned above, detorsion of the torsioned
ovaries causes more severe damage to the ovarian tissue.
Therefore, a traditional operation option with detorsion is
mainly suggested [16].
Carvacrol is a monoterpene phenol produced from
Origanum vulgare and Thymus vulgaris [17]. Research
has been conducted on the clinical use of carvacrol. In
vitro and in vivo researches have indicated that carvacrol
has antioxidant, antibacterial, antifungal, anticancer, anti-
inflammatory and spasmolytic effects [18]. It has also been
reported that carvacrol has an inhibitory effect on proin-
flammatory cytokine and MDA production and a stimulant
effect on total glutathione (tGSH) production [19]. This in-
formation suggests that carvacrol may protect ovarian tis-
sue from the oxidative and inflammatory damage of IR. The
goal of our research is to analyze the impact of carvacrol
against IR-induced ovarian damage and reproductive dys-
function in female rats.
2. Materials and methods
2.1 Animals
A total of 48 female Albino Wistar rats, 8 months old
and weighing 248–256 grams were used in our study. The
animals were obtained from Atatürk University Medical
Experimental Application and Research Center. To allow
the animals to adapt to the environment, they were fed with
animal feed and tap water for seven days in the laboratory
(22 ◦C) in a 12 hours light/12 hours dark conditions. To
make sure that the female rats to be used in the experiment
were not pregnant, they were kept in cages without male
rats for one month. The protocols and procedures were ap-
proved by the local Animal Experimentation Ethics Com-
mittee (Date: 23.03.2021, meeting no: 236643897-000).
2.2 Chemicals
The carvacrol used in the assay was purchased from
Sigma (Sigma Chemical Co., Saint Louis, MO, USA), and
ketamine was purchased from Pfizer (İstanbul, Turkey).
2.3 Experimental groups
Two hours of ischemia and two hours of reperfusion
(IR) were applied to the ovaries of Albino Wistar female
rats, and they were split into the following groups (with
each group containing 12 rats): OIR, IR +50 mg/kg car-
vacrol (IRC-50), IR +100 mg/kg carvacrol (IRC-100), and
a control group that underwent a sham operation (SG).
2.4 Surgical procedures
The 50 and 100 mg/kg carvacrol doses were injected
intraperitoneally (i.p.) to the rats in the IRC-50 and IRC-
100 groups, respectively, which had a unilateral ovariec-
tomy two weeks previously. Unilateral ovariectomy was
also performed in the OIR and SG groups. As a solvent,
25% dimethyl sulfoxide in normal saline was applicated to
the OIR and SG groups in the same way. One hour af-
ter administration of carvacrol and solvent, ketamine was
administered i.p. at a dose of 60 mg/kg to all of the rats
to provide anesthesia. In the previous study, antioxidants
were applied before IR treatment [20]. However, admin-
istration of antioxidations one hour before or after I/R has
been shown to provide beneficial treatment [21,22]. The
period when animals are immobilized in the supine position
is considered an appropriate period for surgical operation
[23]. During this time, the lower abdomens of all the rats
were opened 2–2.5 cm vertically to reach the ovaries. Vas-
cular clips were applied to the lower part of the right ovary
in the OIR, IRC-50, and IRC-100 groups, and two hours of
ischemia and two hours of reperfusion were performed [24].
The reason for our application of ischemia for two hours and
reperfusion for two hours is that the parameters that cause
oxidative and inflammatory damage of the ovary increase
significantly during this period [15]. The ovaries of the SG
group were closed without any procedure. At the end of the
reperfusion process, six rats from each group were killed
with high-dose anesthesia (ketamine 120 mg/kg). The right
ovaries of the killed rats were removed, and biochemical
and histopathological examinations were performed on the
ovarian tissue. The results obtained from the IRC-50, IRC-
100 and SG groups were compared with the results of the
OIR group. The remaining animals (six in each group) were
kept in the laboratory environment with mature male rats
for reproduction. The rats that became pregnant during this
period were taken to divided cages and kept alone in an ap-
propriated condition. Pregnancy times of animals SG: 25,
OIR: 32, IRC-50: 30, IRC-100: 28 days, respectively. The
rats that did not give birth within two months were consid-
ered sterile.
3. Biochemical analysis
3.1 Preparation of samples
At this stage, for biochemical examination, 0.2 g from
each removed tissue was weighed. Tissues were homoge-
nized with a high-speed homogenizer in an ice-cold phos-
2
phate buffers (50 mM, pH 7.4), which was appropriate for
the variable to be measured. The tissue homogenates were
centrifuged at 5000 rpm for 20 min at 4 ◦C, and the su-
pernatants were extracted to analyse for total glutathione
(tGSH), total oxidant status (TOS), total antioxidant sta-
tus (TAS), nuclear factor kappa B (NF- κB) IL-1β, and to-
tal protein. All tissue results were expressed as mg/g total
protein. All spectrophotometric measurements were per-
formed using a microplate reader (Bio-Tek, Winooski, VT,
USA).
3.2 Tissue MDA and tGSH determinations
MDA measurement is based on the method used by
Ohkawa et al. [25], which includes spectrophotomet-
ric measurement of the absorbance of thiobarbituric acid
(TBA) and the pink-colored complex formed by MDA. The
absorbance of the supernatant was measured at 532 nm. The
tGSH was measured using the method defined by Sedlak
and Lindsay RH [26].
3.3 Total Oxidant Status (TOS) and Total Antioxidant
Status (TAS) determinations
TOS and TAS levels of tissue homogenates were de-
termined using a novel automated measurement method
and commercially available kits (Rel Assay Diagnostics,
(İstanbul, Turkey), both developed by Erel [27,28]. The
TAS method was based on the bleaching of character-
istic colour of a more stable ABTS [2,2′-azino-bis (3-
ethylbenzothiazoline-6-sulfonic acid)] radical cation by an-
tioxidants and, measurements were performed at 660 nm.
The results were expressed as nmol hydrogen peroxide
(H2O2) equivalent/L. In TOS method, the oxidants pre-
sented in the sample oxidized the ferrous ion-o-dianisidine
complex to ferric ion. The oxidation reaction was enhanced
by glycerol molecules, which had been abundantly present
in the reaction medium. The ferric ion produced a coloured
complex with xylenol orange in an acidic medium. The
colour intensity, which could be measured at 530 nm spec-
trophotometrically, was related to the total amount of oxi-
dant molecules presented in the sample. The results were
expressed as µmol Trolox equivalent/L.
3.4 NF-κB, TNF-αand IL-1βanalysis
Tissue homogenate NF-κB and TNF-αconcentrations
were measured using sandwich enzyme-linked immunosor-
bent assays (Rat NF- NF-κBBELISA immunoassay kits,
cat. No. 201-11-0288; SunRed and Rat TNF-αand Rat IL-
1βELISA kits, cat no. YHB1098Ra, Shanghai LZ, Shang-
hai, China). Analyses were performed according to the
manufacturers’ instructions.
3.5 Histopathological examination
The tissue specimens were immersed in formaldehyde
solution (10%) for 72 hours. After fixation, the tissues
were placed in a cassette and washed in running water for
24 hours, and then were successively passed through in-
creasing strengths of alcohol (70%, 80%, 90%, and 100%).
Ovarian tissues, which were made transparent in xylol,
were embedded in paraffin blocks, and 4- to 5-micron-
thick sections were cut. The sections were stained with
hematoxylin-eosin dual staining and evaluated and pho-
tographed using the Olympus DP2-SAL firmware program
(Olympus® Inc. Tokyo, Japan). In the serial sections taken,
one center and five peripheral areas were selected at ×100
magnification for a total of six sections for each experimen-
tal group, and a score was assigned to each section for the
amount of degeneration, dilatation/congestion in the ves-
sels, interstitial edema, hemorrhage, and polymorphonu-
clear cell infiltration. For histopathological criteria, a score
was assigned between 0–3 points: 0 = not found, 1 = mild,
2 = moderate, and 3 = severe. Since there was a signifi-
cant difference in the number of follicles developing in the
ovarian tissue among the experimental groups, the develop-
ing follicles were classified and counted at 100×magnifica-
tion in serial sections taken from each experimental group.
Blind histopathological evaluation of the groups was con-
ducted by a histologist.
4. Statistical analysis
The experimental results were expressed as mean
value ±standard deviation (¯x±SD). The significance of
the differences between groups was determined using the
one-way ANOVA test followed by post-hoc Tukey tests.
Histopathological grading and the number of offspring are
presented as the median (min–max) value. While compar-
ing fertility between groups Chi Square was used. While
comparing the number of offspring in groups, Kruskal Wal-
lis test and then Dunn post hoc test were used and adjusted
pvalues were presented. All statistical processes were per-
formed using SPSS for Windows version 18.0 (IBM Corp.,
Chicago, IL, USA), and p<0.05 value was considered sig-
nificant. While performing histopathological scoring, the
median of the values was calculated.
5. Biochemical findings
5.1 MDA and tGSH analysis results
Fig. 1shows that the amount of MDA in the ovarian
tissue of the OIR group, to which only the IR procedure
was applied, was found to be significantly higher than the
amount of MDA in the ovarian tissue of the SG group (p<
0.0001). The amount of MDA in the IRC-50 and IRC-100
groups was found to be significantly lower than the OIR
group (p<0.0001). The same figure (Fig. 1) shows that
the amount of tGSH in the ovarian tissue of the OIR group
was lower than in the SG group (p<0.0001).
5.2 The results of TOS and TAS analysis
Our experimental results revealed that the TOS level
was significantly higher and the TAS level significantly
3
Fig. 1. MDA and tGSH levels in the ovarian tissue. IR, Ischemia-reperfusion; MDA, Malondialdehyde; tGSH, Total glutathione; OIR,
Ovary ischemia-reperfusion; IRC-50, IR +50 mg/kg carvacrol; IRC-100, IR +100 mg/kg carvacrol; SG, Sham operation group.
lower in the ovarian tissue of the OIR group compared to
the SG (p<0.0001). The TOS and TAS levels in the IRC-
50 and IRC-100 groups were significantly different from
those of the OIR group (Fig. 2) (p<0.0001).
5.3 The results of NF-κB, TNF-αand IL-1βanalysis
As presented in Fig. 3, the levels of NF-κB, TNF-α
and IL-1βin the ovarian tissue of the IR group increased
significantly compared to the SG group (p<0.0001).
The levels of NF-κB, TNF-αand IL-1βin the IRC-100
group significantly decreased compared to OIR group (p
<0.0001). However, there was no significant difference
in NF-κB and TNF-αlevels between the OIR and IRC-50
groups (p>0.05). The levels of IL-1βare statistically dif-
ferent between the OIR and IRC-50 groups (p<0.05).
5.4 Reproduction test results
As seen in Table 1, reproductive test results differ be-
tween groups (p= 0.009). One of the six rats in the SG
group taken for breeding was considered infertile. On the
other hand, in the IR group, none of the six rats taken for
breeding gave birth. In the IRC-50 group, one of the six
rats gave birth, and the remaining five rats were recorded
as infertile. In the IRC-100 group, four out of six rats gave
birth, and two remained infertile. When the median num-
ber of offspring born in the SG and OIR groups was com-
pared, a statistically significant difference was found (p=
0.007). When the median number of offspring in the IRC-
100 group was compared with the SG, the difference was
statistically insignificant (p= 0.932), while a significant dif-
ference was found between the number of offspring in the
SG and IRC-50 groups (p= 0.027). Atretic follicles were
significantly reduced in the OIR-100 group compared to the
OIR group. The numbers of primordial and developing fol-
licle of SG, OIR-50, and OIR-100 groups were found sig-
nificantly higher than the OIR group (Table 2).
5.5 Histopathological findings
In histopathological examination, the ovary tissue of
the SG group was evaluated as grade 0. The appearance of
the cortex and medulla in this group was normal; the cortex,
follicles, interstitial connective tissue, and blood vessels at
different stages of development were normal (Fig. 4A). De-
generation in the follicles and follicle cells, necrotic cell ac-
cumulations around the follicle, hemorrhage in the tissue
and corpus luteum, edema in the interstitial tissue, and con-
gestion and dilatation in the blood vessels were found to be
grade 3 in the follicles that developed in the ovary sections
of the IR group (Fig. 4B). Dilatation of blood vessels, con-
gestion, and PNL infiltration in surrounding connective tis-
sue were detected as grade 3 in large magnification images
(Fig. 4C). In the IRC-50 group treated with low-dose car-
vacrol, degeneration was observed in some of the follicles
at grade 2 severity, while necrotic cell debris was observed
in the lumen of grade 1 degenerated follicles and around the
follicle (Fig. 4D). Moderate PNL infiltration, dilatation and
congestion in the blood vessels were observed in the IRC-
50 group (Fig. 4E). Ovarian tissue samples belonging to the
IRC-100 group treated with high-dose carvacrol revealed
that the developing follicles generally had a normal struc-
ture and morphology (grade 0), the interstitial connective
tissue was partially edematous (grade 1), and mild conges-
tion in the blood vessels (grade 1) was present (Table 3). In
addition, the rarely seen PNL infiltration in the tissues be-
longing to the IRC-100 group and the presence of a small
amount of necrotic cell debris in some follicle lumen were
calculated as grade 0 (Fig. 4F).
4
Fig. 2. TOS and TAS levels in the ovarian tissue. IR, Ischemia-reperfusion; TOS, Total Oxidant Status; TAS, Total Antioxidant Status;
OIR, Ovary ischemia-reperfusion; IRC-50, IR +50 mg/kg carvacrol; IRC-100, IR +100 mg/kg carvacrol; SG, Sham operation group.
Fig. 3. NF-κB, TNF-α, and IL-1βlevels in the ovarian tissue. IR, Ischemia-reperfusion; IL-1β, Interleukin 1 beta; TNF-α, Tumor
necrosis factor-alpha; NF-κB, Nuclear factor kappa B; OIR, Ovary ischemia-reperfusion; IRC-50, IR +50 mg/kg carvacrol; IRC-100, IR
+100 mg/kg carvacrol; SG, Sham operation group.
6. Discussion
Our study investigated the protective effect of two
different doses of carvacrol against IR-induced ovarian
damage and reproductive dysfunction in female rats. Our
biochemical test results showed that carvacrol at 50 and
100 mg/kg doses equally prevented the increase of MDA
in ovarian tissue by IR. As is known, lipids are the
biomolecules most affected by oxidative stress [29]. ROSs,
by lipid peroxidation (LPO), lead to the formation of toxic
aldehydes that exacerbate a variety of oxidative damage
[30]. MDA is one of the toxic products of the LPO reac-
tion, which is most commonly used in determining oxida-
tive stress [31]. MDA creates a cytotoxic effect by causing
cross-linking and polymerization of cell membrane compo-
nents and inactivation of receptor and membrane-bound en-
zymes [32]. Meeran et al. [33] reported that carvacrol and
its isomer, thymol, inhibited LPO; they also found that the
inhibitory effect of thymol on LPO was stronger than that
of carvacrol. It has been suggested that this is due to the
greater steric hindrance of thymol. Moreover, the high po-
tential of phenolic compounds to scavenge radicals may be
explained by their ability to donate a hydrogen atom from
their phenolic hydroxyl groups [34]. From the literature,
it is understood that the protective effects of carvacrol and
thymol on ovarian tissue are due to their common antioxi-
dant and anti-inflammatory properties [35]. Another com-
mon feature of thymol, carvacrol and other plant-derived
terpenes in their biological activities is the inhibition of
voltage-dependent Na+ion channels [36]. Ingec et al. [37]
reported that the increase in the amount of MDA was associ-
ated with the severity of the ovarian tissue injury. Kurt et al.
[38], on the other hand, argued that suppressing the increase
in the amount of MDA is important in reducing IR-related
ovarian damage. Dursun et al. [39] showed that carvacrol
suppressed the increase of MDA in testicular tissue after
torsion detorsion in rats, they also stated that can prevent
5
Table 1. The effect of carvacrol on IR-related infertility in female rats.
Groups (n = 24) Number of births Number of infertile animals* The average andmedian number of offspring born p
SG (n = 6) 5 1 4/6(0–6)
0.009
OIR (n = 6) 0 6 0/0(0–0)
IRC-50 (n = 6) 1 5 3/3(0–3)
IRC-100 (n = 6) 4 2 3/3(0–6)
SG, Sham group; OIR, Over ischemia-reperfusion; IRC-50, Ischemia-reperfusion +50 mg/kg carvacrol; IRC-100, Ischemia-
reperfusion +100 mg/kg carvacrol.
Number of offspring borns were presented as median (min–max) value for groups.
*While comparing fertility between groups Chi Square was used.
Table 2. Follicle staging in fertile rat groups.
Groups (n = 24) Primordial follicle Developing follicle Atretic follicle Corpus luteum
SG 14 23 3.1 13
OIR 12 19 6.6 12
OIR-50 13 22 4.5 13.1
OIR-100 14 23 3.3 13.5
SG, Sham group; OIR, Over ischemia-reperfusion; IRC-50, Ischemia-reperfusion +50 mg/kg car-
vacrol; IRC-100, Ischemia-reperfusion +100 mg/kg carvacrol.
Table 3. Histopathological damage grading in rat ovary
tissue.
Findings Groups and median score
Sham OIR IRC-50 IRC-100
Degeneration 0 3 (2–3) 2(1–2) 0(0–1)
Necrosis 0 3 (2–3) 2(0–2) 1(0–1)
Vascular dilatation/congestion 0 3 (2–3) 2(1–2) 1(0–1)
Interstitial edema 0 3 (2–3) 2(1–2) 1(0–1)
Hemorrhage 0 3 (2–3) 0(0–1) 0(0–1)
PNL infiltration 0 3 (2–3) 2(0–2) 0(0–1)
0 = Normal, 1 = Mild damage, 2 = Moderate damage, 3 =
Severe damage.
PNL, Polymorphous nuclear leukocyte.
apoptotic development and signaling of spermatogenic cells
with strong mitotic activity in the basement membranes of
seminiferous tubules. Taxifolin, another antioxidant that
prevents the increase of oxidants and the decrease of antiox-
idants in testicular tissue applied torsion/detorsion, has been
shown to alleviate histopathological damage. Taxifolin has
been proven to prevent the reduction in germinal epithelial
thickness caused by the torsion/detorsion, detached germi-
nal cell lines, cell necrosis of the seminiferous tubule, and
the development of degenerated germinal giant cells [40].
The present study shows that the IR procedure caused a de-
crease in tGSH in the ovarian tissue. GSH is known to
be a tripeptide antioxidant consisting of L-glutamate, L-
cysteine and glycine [41]. GSH has been proven to play a
critical role in protecting cells from oxidative damage and
the toxicity of xenobiotic electrophiles while maintaining
redox homeostasis [42]. Isaoglu et al. [43] demonstrated
that an IR event causes a decrease in the amount of tGSH
in the ovarian tissue. Another study found that drugs that
prevent the decrease of tGSH levels protect ovarian tissue
from IR damage [44]. Kadioglu et al. [12] emphasized the
importance of keeping MDA low as well as keeping tGSH
high in reducing IR-related damage. It has been reported
that carvacrol significantly prevents the depletion of cellu-
lar GSH content by hydrogen peroxide (H2O2) [45]. It has
also been reported that carvacrol is a good scavenger of per-
oxyl radicals and inhibits the peroxidation of phospholipid
liposomes in the presence of iron (III) and ascorbate [46].
In our study, TOS and TAS levels were measured to
gain an understanding of the way that IR in ovarian tis-
sue changes the oxidant-antioxidant balance in favor of
oxidants. MDA, tGSH and other oxidant-antioxidant pa-
rameters are known to be used to determine the oxidant-
antioxidant balance. TOS and TAS reflect the total effects
of all antioxidants and oxidants in tissues [25,26]. There-
fore, TOS levels are used for practical measurement of
ROS, and TAS levels are used to evaluate total antioxidant
status. In a study supporting our TOS and TAS findings, it
was observed that the IR procedure increased TOS levels in
ovarian tissue and decreased TAS levels [47].
Our study showed that proinflammatory cytokine lev-
els such as NF-κB, TNF-αand IL-1βwere increased in
the IR-applied ovarian tissue. As noted in the literature,
the production of NF-κB, TNF-α, IL-1βand oxidant is in-
creased in tissues exposed to IR [48]. Keskin et al. [49]
showed that histopathological damage developed in IR-
induced testicular tissue with high NF-κB, TNF-αand oxi-
dant levels and low antioxidants. NF-κB plays an important
6
Fig. 4. Histopathological examination of ovary tissue. (A) Ovarian tissue of SG group stained with hematoxylin-eosin; DF, developing
follicle; Int, interstitial area; CL, corpus luteum; ⋆, blood vessel, ×100. (B) Ovarian tissue of the IR group stained with hematoxylin-
eosin; dDF, degeneration in developing follicles; , accumulation of necrotized cells in the follicle and surrounding connective tissue;
Int, edema in the interstitial area; CL, hemorrhage in the corpus luteum; , hemorrhagic areas; , densely dilated and congested blood
vessel, ×100. (C) Ovary tissue of the OIR group stained with hematoxylin-eosin; at large magnification, , dense dilated and congested
blood vessel; , polymorphonuclear cell infiltration, ×200. (D) Ovarian tissue of the IRC-50 group stained with hematoxylin-eosin;
locally degenerated developing follicle (dDF), Int, edema in the interstitial area (Int); CL, corpus luteum; , necrotic cell debris in the
lumen of the follicles; ⋆, dilated and congested blood vessels, ×100. (E) Ovarian tissue of the IRC-50 group stained with hematoxylin-
eosin; at large magnification, , dilated and congested blood vessel and , PNL infiltration, ×200. (F) Ovarian tissue of the IRC-100
group stained with hematoxylin-eosin; normal structure DF, developing follicle; Int, edema in the interstitial area (Int); CL, corpus
luteum; , small amount of necrotic cell debris in the lumen of some follicles; , congested blood vessels in patches, ×100.
role in regulating the expression of other proinflammatory
cytokine genes [50]. In addition, it has been reported that
NF-κB activation is induced by ROS, and oxidative stress
triggers inflammation [51,52]. However, in our study, al-
though carvacrol inhibited the increase of IR-related oxi-
dants at 50 and 100 mg/kg doses to the same extent, it was
able to inhibit the increase of NF-κB, TNF-αand IL-1β
only at a dose of 100 mg/kg. No information was found
in the literature to support these findings. However, car-
vacrol is known to protect lung tissue from damage due
to oxidative stress and proinflammatory cytokine increase
[53], and carvacrol has been shown to have a stronger in-
hibitory effect on oxidant and proinflammatory cytokines
at high doses [53,54]. Li et al. [55] suggested that the anti-
inflammatory effect of carvacrol is due to inhibition of the
pro-inflammatory NF-κB pathway. This information indi-
cates that the IR process causes damage in different tissues
with a similar mechanism.
Carvacrol at a dose of 100 mg/kg, which significantly
inhibited the overproduction of NF-κB, TNF-αand IL-1β,
reduced ovarian IR injury histopathologically better than
the 50 mg/kg dose. In addition, inflammatory symptoms
such as edema, congested and dilated blood vessels and
PNL infiltration were observed in the ovarian tissue of the
carvacrol group administered a dose of 50 mg/kg, while
these histopathological findings were not observed in the
rats that received a dose of 100 mg/kg. Previous stud-
ies have found that IR caused histopathologically dilated
and congested blood vessels, PNL infiltration, hemorrhage,
edema, follicular degeneration and necrosis in the ovarian
tissue [56,57]. Unlubilgin et al. [15] explained that this
IR-related histopathological damage leads to infertility. In
our study, proinflammatory cytokine production was sig-
nificantly suppressed, and the number of infertile animals
decreased significantly in the carvacrol group (at a dose of
100 mg/kg), in which, histopathologically, no inflamma-
tory symptoms were observed. Bhandari et al. [58] found
7
that the development of infertility was associated with an
increase in proinflammatory cytokines and a decrease in
anti-inflammatory cytokines. which supports our experi-
mental results. As a result, the IR event caused an increase
in oxidant and proinflammatory cytokines and a decrease
in antioxidants in ovarian tissue. Ovarian IR caused tissue
degeneration, necrosis, vascular damage and inflammatory
events histopathologically. At a dose of 100 mg/kg, car-
vacrol, which inhibits the overproduction of oxidant and
proinflammatory cytokines increased by IR in ovarian tis-
sue, prevented the development of inflammation in the tis-
sue. The 50 mg/kg dose of carvacrol, which inhibited the
IR-related oxidant release in ovarian tissue to the same ex-
tent, did not inhibit the increase of proinflammatory cy-
tokines and the development of inflammation. Carvacrol
significantly prevented IR-related infertility at a dose of 100
mg/kg but not at a dose of 50 mg/kg. This indicates that the
anti-inflammatory effect of carvacrol at a dose of 100 mg/kg
is not related to its antioxidant activity. It also shows that
inflammation is an important factor in the pathogenesis of
ovarian IR-related infertility. Induction of inflammation in
IR-related ovarian tissue suggests that it is not limited to
oxidants.
7. Conclusions
Our experimental results suggest that effective doses
of antioxidants and anti-inflammatory drugs may be bene-
ficial in the clinical treatment of ovarian IR injury due to
torsion and detorsion and in preventing the development of
infertility. In addition, the information obtained from our
study and literature indicates that it may be beneficial to
apply antioxidant and anti-inflammatory treatment before
and after reperfusion.
Abbreviations
IR, Ischemia-reperfusion; HX, Hypoxanthine; XDH,
Xanthine dehydrogenase; ROS, Reactive oxygen species;
XO, Xanthine oxidase; MDA, Malondialdehyde; tGSH, To-
tal glutathione; TOS, Total Oxidant Status; TAS, Total An-
tioxidant Status; IL-1β, Interleukin 1 beta; TNF-α, Tu-
mor necrosis factor-alpha; NF-κB, Nuclear factor kappa
B; PNL, Polymorphous nuclear leukocyte; OIR, Ovary
ischemia-reperfusion; IRC-50, IR +50 mg/kg carvacrol;
IRC-100, IR +100 mg/kg carvacrol; SG, Sham operation
group.
Author contributions
NS, IBD and UI designed the research study. BS per-
formed the research. HS provided help and advice on the
experiments. GNY and TAC, VA analyzed the data. BS,
HS and KU wrote the manuscript. All authors contributed
to editorial changes in the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
The study was conducted in accordance with the Dec-
laration of Helsinki, and the protocol was approved by the
Ethics Committee of Atatürk University Animal Experi-
ments Local Ethics Committee, Erzurum, Turkey (Date:
23.03.2021, meeting no: 236643897-000). All animals re-
ceived care in compliance with the institution’s guidelines,
as outlined in the Guide for the Care and Use of Laboratory
Animals, published by the National Institutes of Health.
Acknowledgment
Thanks to all the peer reviewers for their opinions and
suggestions.
Funding
This research received no external funding.
Conflict of interest
The authors declare no conflict of interest.
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