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