https://doi.org/10.52973/rcfcv-e34315
Received: 19/09/2023 Accepted: 10/10/2023 Published: 02/01/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34315
ABSTRACT
Doxorubicin (Dox) is an anti–cancer agent used of ovarian, breast,
liver, lung cancers and solid tumors such as leukemia and lymphoma
treatment. However, since it is an antineoplastic agent with a high
toxic effect, it causes toxicity on many organs and tissues in the
organism. Spirulina spp. (SP) contains phytopigments such as β–
carotene, tocopherols and xanthophylls and is a natural source of
vitamin A as well as its anticarcinogenic effect. For these reasons,
this study was planned to experimentally reveal the antioxidant and
protective effects of SP on Dox–induced reproductive toxicity in
female rats. In the study, histopathological evaluation was performed
after Hematoxylin–Eosin staining in female rats with Dox toxicity.
The distribution of GSK–3β for cell proliferation, HIF–1α for oxidative
stress and VEGF for vascularization were evaluated. TUNEL staining
was performed to determine apoptosis. It was determined that SP
prevented tissue damage on the ovarian tissue of rats with Dox
toxicity, and this positive effect was achieved through factors such
as oxidative stress (HIF–1α), vascularization (VEGF) and inhibition of
cell death (Apoptosis). It has been observed that SP treatment to
be applied after chemotherapy is effective in protecting both the
developing follicles and the primordial follicle pool.
Key words: Spirulina spp.; apoptozis; ovary; infertility; doxorubicin
RESUMEN
La doxorrubicina (Dox) es un agente anticancerígeno que se utiliza
en el tratamiento de cánceres de ovario, mama, hígado, pulmón
y tumores sólidos como la leucemia y el linfoma. Sin embargo, al
ser un agente antineoplásico con un alto efecto tóxico, provoca
toxicidad en muchos órganos y tejidos del organismo. Spirulina
spp. (SP) contiene topigmentos como β–caroteno, tocoferoles y
xantolas y es una fuente natural de vitamina A, además de su efecto
anticancerígeno. Por estas razones, se planeó este estudio para
revelar experimentalmente los efectos antioxidantes y protectores del
SP sobre la toxicidad reproductiva inducida por Dox en ratas hembra.
En el estudio, la evaluación histopatológica se realizó después de la
tinción con hematoxilina–eosina en ratas hembra con toxicidad por
Dox. Se evaluó la distribución de GSK–3β para la proliferación celular,
HIF–1α para el estrés oxidativo y VEGF para la vascularización. Se
realizó tinción TUNEL para determinar la apoptosis. Se determinó que
SP previno el daño tisular en el tejido ovárico de ratas con toxicidad
por Dox, y este efecto positivo se logró a través de factores como el
estrés oxidativo (HIF–1α), la vascularización (VEGF) y la inhibición de
la muerte celular (Apoptosis). Se ha observado que el tratamiento con
SP aplicado después de la quimioterapia es ecaz para proteger, tanto
los folículos en desarrollo como el conjunto de folículos primordiales.
Palabras clave: Spirulina spp.; apoptosis; ovario; esterilidad;
doxorrubicina
Protective effect of Spirulina in the ovary of rats against Doxorubicin
toxicity
Efecto protector de la espirulina en el ovario de ratas contra la toxicidad de la Doxorrubicina
Şamil Öztürk
1
, Latife Ceyda İrkin
2
*
1
Çanakkale Onsekiz Mart University, Vocational School of Health Services. Çanakkale, Türkiye.
2
Çanakkale Onsekiz Mart University, School of Applied Sciences, Department of Fisheries Technology. Çanakkale, Türkiye.
*Corresponding author: latifeirkin@gmail.com
The role of Spirulina in female infertility / Öztürk and İrkin __________________________________________________________________________
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INTRODUCTION
Doxorubicin is classied as anthracycline group antibiotics. It is
commonly known by its trade name adriamycin and is the hydroxylated
analogue of Daunorubicin. Anthracyclines have varying effects
depending on the cell type. These drugs do not selectively enter
between base pairs close to each other and bind to the sugar–
phosphate structure of Deoxyribonucleic acid (DNA) and prevent its
synthesis [1]. Dox is used in the treatment of sarcomas, carcinomas
including breast and lung cancers, acute lymphocytic leukemias and
lymphomas [2]. The long–term effects of chemotherapy on ovarian
tissue are reduced premature ovarian failure and ovarian reserve,
which causes infertility. The number of follicles is an indicator
of ovarian reserve in the ovary. The ultrastructural indicators of
chemotherapy–induced ovarian damage are diffuse follicle loss and
ovarian brosis [3, 4]. Alkylating agents are the substances that cause
the highest follicle loss. In addition to follicular damage, they also
cause widespread granulosa cell damage characterized by decreased
steroid synthesis by forming DNA cross–links [3, 5].
Studies on microalgae species at the national and international
level continue intensively, and the number of commercial enterprises
serving in this eld is gradually increasing [6]. Spirulina spp. (SP) İs
the only type of blue–green algae grown and traded for use as a food
supplement among algae [7]. The properties of SP have attracted
the attention of researchers due to its ability to break the cell wall
barrier and easy access to its components. SP is consumed as a
food substance that has proven its safety in many toxicological
studies [8]. Recently, algae has emerged as a new food source with
the potential for multi–purpose use in Medicine Human. In addition,
algae are a rich source of natural bioactive compounds with various
biological activities. Apart from these, they carry many critical
compounds with their unique properties such as carotenoids, amino
acids and micronutrient accumulations, which have very important
roles for Human Health. Therefore, there is an increasing interest in
investigating the positive effect of algae on Human Health.
SP is a lamentous, spiral–shaped, multicellular and photosynthetic
cyanobacteria. This cyanobacterium is cultivated Worldwide and
is used as a primary human dietary supplement. It contains a wide
range of prophylactic and healing nutrients including vitamins,
minerals, proteins, γ–linolenic acid, β–carotene and undiscovered
bioactive compounds. The study was planned to consider the role
of chemical compounds in SP in preventing ovarian damage induced
by the chemotherapeutic agent Dox and improving damage repair
mechanisms. It is important to obtain a protective substance or
substances against the toxic effects of Dox on reproductive cells.
MATERIALS AND METHODS
In the study, 24 Wistar albino female rats (Rattus norvegicus), 2–3
months old, weighing 200–300 g, were preferred. The rats were
included in the study after being observed for two weeks to avoid
adaptation problems. Subjects included in the study were kept in
optimized conditions (12–hour day and night cycle, 22°C temperature,
humidity 30–70%). They were fed with standard feed and water.
Toxicity model and application of Spirulina
Dox was obtained as Adrimisin (50 mg/25 ml injectable solution)
from Saba Pharmaceuticals (Istanbul, Turkey). SP (Spirulina 99%
green powder) was purchased from (of Naturalebio, Italy). In order to
compose toxicity, Doxorubicin was administered as 1 dose (2 mg·kg
-1
/ i.p) every 3 days, a total of 12 mg·kg
-1
. The dose of doxorubicin was
selected minimal lethality [9]. After the administiration of Dox,
500mg·kg
-1
SP was given intragastrically (by gavage) once a day for 3
weeks. On the 21st day of the experiment, the animals were sacriced
under anesthesia and tissue samples were taken [10].
For experimental study, female rats were divided three groups
as follows:
Group–1 (n:6): Control group, no treatment was applied, only 100μL
volume of physiological saline (SF) was given intraperitoneally every
day during the experiment (n:6).
Group–2 (n:6): Doxorubicin group (DOX), Dox 2 mg·kg
-1
100 μL was
administered intraperitoneally every three days (cumulative doses
12 mg·kg
-1
) [9]. Since severe toxicity and lethal effects occurred with
the application of high amounts of Dox at a time, induction was done
in lower doses and at certain intervals.
Group–3 (n:6): DOX + SP group, Doxorubicin was administered at
a dose of 2 mg·kg
-1
in a volume of 100 μL intraperitoneally once in
three days (6 doses in total), while SP was administered once daily
as 500mg·kg
-1
intragastric (gavage).
Group–4: SP group, SP was dissolved in distilled water to achieve a
nal concentration of 200 mg·ml
-1
. 500 mg·kg
-1
of SP was administered
intragastric (gavage) once a day. All samples were taken on the 21st
day of the experiment.
Follicle count
After Dox and SP application, sections were taken from the right and
left ovarian tissue, stained with Hematoxylin–Eosin (H–E) staining and
examined under a light microscope. Ovarian reserve was determined by
counting primordial follicles. Primordial, primary, preantral, secondary
and tertiary follicles in the ovarian cortex, which can clearly distinguish
both nuclei and nucleoli, were counted separately in each section.
Evaluation of follicle quality is based on basement membrane integrity,
cell density, and oocyte integrity. Unhealthy follicles were differentiated
from healthy follicles by loss of granulosa cells and pycnotic nuclei.
Performing histochemistry, immunohistochemistry, TUNEL assay
At the end of the experiment, ovarian tissue samples were taken
from female rats and xed in 10% formalin solution for 72 hours. Then,
histochemical and immunohistochemical stainings were performed.
Since the severity of the staining in the tissue is important in this
evaluation, it was accepted as weak (+), moderate (++) and severe
(+++). The amount of stained cells was calculated by the H–score
method and the data were evaluated statistically [11]. Glycogen
synthase kinase (Gsk–3β), hypoxia inhibition factor (HIF–1 alpha) and
vascular endothelial growth factor (VEGF) staining was performed on
serially sectioned tissues using the indirect immunohistochemical
method. GSK–3β is a protein that is a member of protein kinases
and redox–sensitive multifunctional serine/threonine protein
kinase and is expressed in all cell types. Many studies have shown
that GSK–3 inhibition plays a protective role against oxidative and
apoptotic damage caused by chemotherapy [12]. The transcription
factor HIF– 1α is thought to be a critical regulatory factor during
the development of physiological systems and a key regulator of
body tissue homeostasis involved in the regulation of cell survival/
adaptation, anaerobic metabolism, immune response, cytokine
secretion, and angiogenesis [13]. New blood vessels are formed in the
FIGURE 1. Body weights of the subjects before and after the experiment
FIGURE 2. Number of ovarian follicles of the experimental groups; primary follicle:
Control–Dox (P<0.001), Dox–Dox+SP (P<0.01) and Dox–SP (P<0.001), secondary and
Graaan follicle: Control– Dox (P<0.001), Dox–Dox+SP (P<0.02) and Dox–SP (P<0.001)
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34315
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ovary to facilitate the distribution of oxygen, nutrients and hormone
substrates, as well as the transfer of hormones to target cells. The
ovarian follicle and corpus luteum produce a variety of angiogenic
factors, but vascular endothelial growth factor (VEGF) is thought to
play a crucial role in regulating normal and abnormal angiogenesis in
the ovary. Inhibition of VEGF expression prevents the development
of mature antral follicles by reducing follicle angiogenesis [14].
Terminal Transferase dUTP Nick End Labeling (TUNEL) staining
method was used to determine apoptotic cell death. TUNEL staining
method is used to detect DNA fragmentation resulting from apoptosis.
Follicles showing apoptotic activation were evaluated using the TUNEL
method. One of two serial sections taken from 5 different levels by
random sampling method from the right (n=6) and left (n=6) ovarian
tissue blocks was stained with the TUNEL method. The number and
percentage of follicles showing apoptotic activation were counted
and calculated separately. Apoptotic activation of ovarian follicles
was scored semiquantitatively between 0 and 2.
Score 0: Presence of at most 3–5 TUNEL positive follicle cells in
the granulosa cell layer, no staining in the primary oocyte
Score 1: Less than 50% TUNEL positive staining in the granulosa
cell layer, no staining in the primary oocyte
Score 2: Intense TUNEL positive staining covering more than
half of the granulosa cell layer and TUNEL positive staining of the
primary oocyte
Follicles with a TUNEL 2 score were considered as atretic follicles,
and the apoptotic follicle index was calculated using the formula given
below for follicles at different developmental stages.
%Apoptotic index = (number of positively stained apoptotic follicles
/ total number of follicles) × 100 [15].
Statistical analysis
The difference between the obtained ndings and the groups was
determined by one–way–ANOVA test and Tukey test was used for
multiple comparisons. P<0.05 was considered statistically signicant.
RESULTS AND DISCUSSION
Evaluation of body weights
The average weights of the rats were determined before starting
the experiment. Before the experiment was terminated after 21
days, each subject was weighed again and their average weight was
determined. Each group was evaluated statistically according to
its pre–experiment and post–experiment weights. No statistical
difference was detected in the subjects of the control group according
to their body weight (P<0.08). Post–experiment weight gain was
noted in the subjects in the SP group, and a statistical difference
was detected as P<0.02. A statistically signicant decrease was
determined in the post–experiment weights of the subjects in the
Dox group compared to the pre–experiment (P<0.001) (FIG. 1).
Evaluation of ovarian follicle counts
The number of healthy follicles is important in ovarian toxicity.
Especially the number of secondary and tertiary follicles is very
important in drug toxicity. However, in the developing follicles, the
number of unilaminar (seen of primary oocyte, zona pellucida and a
single layer of cuboidal follicle cells surrounding it) and multilaminar
(seen of primary oocyte, zona pellucida and a single layer of
cuboidal follicle cells surrounding it) primary follicles is less due to
the formation of an atretic structure. is taken into account. In our
ndings, although there was no statistically signicant difference
in the number of unilaminar and multilaminar primary follicles, a
statistically signicant decrease was observed in the number of
secondary and graaf follicles. When the groups were compared in
terms of primary follicle numbers, a signicant difference was found
between Control–Dox, Dox–Dox+SP and Dox–SP. When the groups
were compared in terms of secondary and graaf follicle numbers, a
signicant difference was found between Control–Dox, Dox–Dox+SP
and Dox–SP (FIG. 2) . There was a signicant increase in the number
of atretic follicles in Dox administered subjects. Statistical data of
atretic follicles belonging to the ovaries of the subjects are shown
in FIG. 3. Between Control–Dox, Dox–Dox+SP, Dox–SP.
FIGURE 3. The number of atretic follicles in the ovarian tissues of the experimental
groups; Control–Dox (P<0.001), Dox–Dox+SP (P<0.01), Dox–SP (P<0.001)
FIGURE 5. Immunoreactivity graph of the experimental groups
The role of Spirulina in female infertility / Öztürk and İrkin __________________________________________________________________________
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Microscopic ndings
H–E stained ovarian sections of all experimental groups were used
to determine the general morphological features and follicle numbers
of the follicles in the ovarian cortex (FIG. 4A1). It was observed that the
normal histological structure was disrupted in the ovarian sections
of the DOX–treated experimental groups, and follicle degeneration,
vascular congestion, hemorrhage, and polymorphonuclear leukocyte
inltration (PMNL) were quite common (FIG. 4B1). In the groups given
Dox + SP together, the histological structure was preserved compared
to the experimental groups given Dox, histophatological features
were reduced, and there were newly developing follicles (FIG. 4C1).
When the ovarian tissues taken from rats belonging to SP group, a
generally normal histological appearance was detected. A very rare
pathological picture was observed in the ovarian sections, stroma
and parenchyma of the cortex. However, vascular congestion areas
were observed in some sections (FIG. 4D1).
Immunohistochemical ndings
In the ovarian sections stained with GSK–3β primary antibody, a
statistically signicant decrease was observed in the Dox group (FIG.
4B2) compared to the control group (FIG. 4A2) (P<0.001). A statistically
signicant increase was observed in the SP (FIG. 4D2) compared to
the control (P<0.05) (FIG. 5). Although the immunoreactivity intensities
increased in the groups where Dox and SP were administered together
(FIG. 4C2) compared to the Dox group, there was a lesser increase
compared to the SP and control groups.
It was observed that HIF–1α immunoreactivity was prominently
manifested in the oocytes, granulosa cells and theca cells of
secondary and early tertiary follicles in the Dox group (FIG. 4B3).
While it was observed that the immunoreactivity was quite low in
the ovarian tissue of the control group (FIG. 4A3), it was observed
that the immunoreactivity was mild in the ovarian tissue of the SP
group only (FIG. 4D3) and in the ovarian tissue of the Dox+SP (FIG. 4C3)
group. A signicant difference was also observed in the statistical
comparison between Dox and other groups in terms of the number
of positive cells (FIG. 5).
VEGF expression in ovarian follicles is dependent on follicle size.
Inhibition of VEGF expression results in decreased follicle angiogenesis
and failure to develop mature antral follicles. While the group with
the lowest VEGF expression was Dox (FIG. 4B4), the increase in
immunoreactivity was noted in the control (FIG. 4A4) and SP groups
(FIG. 4D4). A signicant difference was found between the Dox and
the control and SP (P<0.001) in terms of VEGF immunoreactivity, and
between the Dox–Dox+SP (FIG. 4C4) group (P<0.01) (FIG. 5).
Tunel ndings
Apoptotic activation and the number of atretic follicles in the
ovarian cortex and medulla stroma and in the granulosa cells of the
developing follicles were statistically increased in the Dox (FIG. 6A)
group compared to the other groups (FIG. 7). After Dox application, it
was observed that apoptotic activation decreased in the cortex and
medulla of the ovarian tissues of the subjects given SP, and there was
a decrease in the number of atretic follicles with an increase in the
number of follicles (FIG. 6C). It was observed that the TUNEL activity
in the ovarian tissues of the control group (FIG. 6A) and only the SP
group (FIG. 6D) was mild and in the follicle granulosa cells.
The protective effects of SP against Doxorubicin–induced ovarian
toxicity were evaluated at the histochemical and immunohistochemical
levels. It has been shown that when SP is administered at a dose of
500 mg·kg
-1
in vivo, it can provide a protective effect against the
gonadotoxic effect of Doxorubicin. This effect was seen as preserving
the number of primordial and developing follicles and decreasing the
number of atretic follicles, excluding reduction in body weight. This
is the rst study in the literature to show the protective effect of SP
against Dox–induced gonadotoxicity.
Considering the results of studies investigating the gonadotoxic
effect of Doxorubicin; Ben–Aharon et al. [16] reported that Doxorubicin
administration causes an increase in the number of atretic follicles in
the ovaries and this effect causes a signicant decrease in ovulation
rate [16]. Although follicular atresia is a physiological function of the
ovaries, damage to the follicles at the level of oocyte and granulosa
FIGURE 4. Histochemical and immunohistochemical staining of control and experimental groups
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H–E GSK-3β HIF-1α VEGF
ControlDoxDox+SPSP
B1 B2 B3 B4
C1 C2 C3 C4
D1 D2 D3 D4
A4
A3A2A1
FIGURE 6. TUNEL staining of control and experimental groups
A B C D
cells can lead to excessive follicle atresia, which may induce ovarian
dysfunction. As a result of histological examination of ovarian
biopsies of women receiving chemotherapy, it was observed that
germ cell damage occurred due to lower estradiol production [17,
18]. Therefore, in chemotherapy–induced gonadotoxicity, apart from
the interactions between granulosa cells and oocyte, disruptions
in the steroidogenesis mechanism due to losses in granulosa and
theca cells cause follicle loss. It has been reported that Doxorubicin
causes deterioration in endothelial function by inducing free oxygen
radicals and causing apoptosis in endothelial cells [19, 20]. It has been
FIGURE 7. Apoptotic index data graph of experimental groups
The role of Spirulina in female infertility / Öztürk and İrkin __________________________________________________________________________
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reported that after Doxorubicin administration, ROS production in
granulosa cells increased in a dose–dependent manner, decreased
mitochondrial membrane potential, increased Bax, Bcl–2 and p–53
mRNA expression, which led to apoptosis [21]. In this study, it has
been shown that Doxorubicin administration causes follicle loss by
increasing apoptotic activation in ovarian follicles.
Studies have reported that TUNEL positive cell staining in atretic
follicles in the ovary is more common in granulosa and theca cells [22].
When Slot et al. compared atretic and healthy follicles, they observed
that the release of Fas ligand, Bax, TUNEL and caspase–3 was more
intense in atretic follicles [23]. Vaskivuo et al. [24] reported TUNEL
positive staining in granulosa cells in the human ovary at the level
of secondary and graaf follicles. In a different study, it was stated
that after TUNEL staining and caspase–3 immunohistochemistry in
human ovaries, atretic follicle numbers were rare in the early follicle
stage, and apoptosis could not be demonstrated by caspase–3 and
TUNEL method in follicles at early maturation stage [25]. In this
study, TUNEL positivity was obtained in developing and graaf follicles.
It can be interpreted that apoptotic activation in primordial and
unilaminar primary follicles results in direct loss of follicle due to
rapid death of few follicle cells and degeneration of primary oocyte.
This interpretation also supports the differences in the number of
primordial follicles between the groups.
In studies evaluating the ecacy of tiopronin or cannabinoids
for the treatment of nephrotoxicity caused by cisplatin, a broad–
spectrum platinum–derived antineoplastic agent that has a toxic
effect on the gastrointestinal system similar to Doxorubicin in the
literature, it has been reported that therapeutic agents are similarly
ineffective in preventing weight loss. They interpreted this result
as the lack of effectiveness of the agents used for treatment on the
gastrointestinal system. However, although anorexia and weight loss
due to anorexia developed in rats due to the toxic effect of Doxorubicin
on the gastrointestinal system in this study, it is possible that there
will be reductions in weight loss due to the therapeutic effect of SP
on the gastrointestinal system. Kaygusuzoglu et al. emphasized in
their study that more apoptotic positive cells were detected in the
cisplatin group compared to the control group in ovarian toxicity
induced by cisplatin [26]. It was demonstrated increased apoptosis in
the ovarian tissues of rats treated with cyclophosphamide in ovarian
damage with cyclophosphamide, compared to the control group, with
increased caspase–3 immunoreactivity [27]. They also reported that
this caspase–3 immunoreactivity mainly occurs in the granulosa,
stromal cells and corpus luteum.
Said et al. examined the uterus and ovaries of female rats exposed to
radiation in their study and showed that caspase–3 immunoreactivity
was more intense in the granulosa, theca interstitial cells and uterine
epithelial cells compared to the control group [28]. They also
emphasized in their study that uterine degeneration was prevented by
sodium selenite treatment. Similarly, in the present study, was found
that DOX induced apoptosis in both follicle cells and stromal cells in
the ovary and uterus. It was observed that the number of caspase–3
positive apoptotic cells increased in DOX–induced ovarian damage
compared to the control, and this number decreased signicantly in
the DOX+SP group. As in this study, Maltaris et al [29]. showed that
Doxorubicin has negative effects on ovarian reserve, with a decrease
in the number of ovarian follicles and a signicant increase in the
number of degenerated follicles in the mouse (Mus musculus) group
given Doxorubicin, and follicular apoptosis was determined by the
TUNEL method, as in this study.
The steroid content in green algae can contribute to balanced
hormonal regulation by forming hormones that play an important
role in female fertility and regulate the function of the reproductive
system in the best way. Therefore, algae can be used as an alternative
herbal treatment to overcome the toxic effects of free radicals that
cause female infertility as a result of disruption of the hormonal
regulation of the reproductive system [30]. However, although the
effect of SP, a microalgae species, on reproductive functions is not
well known, it has been reported that S. maxima extract inhibits body
and testicular weights, metabolic parameters, normal seminiferous
tubules degeneration, increases Leydig cell number, Testosterone
levels and steroidogenic enzymes in studies conducted in rats so
far [31]. SP is a powerful antioxidant molecule and is known for
its antioxidant and antiapoptotic properties. It consists primarily
of various components such as B–complex vitamins, β–carotene,
chlorophyll, vitamin E, superoxide dismutase, and numerous minerals.
It highlights that the antioxidants in SP, particularly C–phycocyanin,
SOD, B–complex vitamins, β–carotene, chlorophyll and vitamin E,
may act synergistically to restore the antioxidant status of the ovary
[32]. In addition to its nutritional advantages, SP has extra benecial
characters such as antibacterial, antifungal, antiviral, anticancer,
anti–inammatory and antioxidant activities. Also SP is used as a
feed supplement in the aquaculture and poultry industry. SP aqueous
extract has been reported to act against MSG–induced ovarian
dysfunction in mice (Mus musculus) by weakening the changed oocyte
quality, ovarian histopathology, sex hormone and antioxidant enzymes
as a result of mono sodium glutamate (MSG) supplementation [33].
CONCLUSION
Chemotherapy has extremely harmful effects on the female
reproductive system, especially on the ovary, which has a fast
cellular cycle. Although most women with cancer are treated with
chemotherapy or radiotherapy, they are faced with infertility and
gonadal insuciency due to the toxic effects of the agents used after
the treatment. It has been observed that SP treatment to be applied
after chemotherapy is effective in protecting both the developing
follicles and the primordial follicle pool. The apoptotic activation
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caused by Doxorubicin in the follicular granulosa cells and stromal
cells by causing calcium accumulation in the cell and causing DNA
damage together with the ROS accumulation, the inhibiting effect
of the calcium in the cell into the cell and the free oxygen radicals
minimizing the developing follicles in the normal ovarian tissue by
chemotherapeutic agents. It is thought to protect from toxic effects.
This study is the rst to show that SP has a protective effect on female
reproductive system toxicity after doxorubicin chemotherapy and will
guide approaches to preserve fertility as an alternative treatment
option. Further studies are needed to support this view.
Ethical approval
The research protocol was approved by Çanakkale Onsekiz Mart
University Animal Research Ethics Committee (2021–03/02).
Disclosure statement
The authors report there are no competing interests to declare.
Funding
This work was supported by the Çanakkale Onsekiz Mart University,
Scientic Research Project Unit (FBA 2021–3970).
Conicts of interest
The authors declare that they have no conicts of interest with
respect to the work presented in this report.
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