https://doi.org/10.52973/rcfcv-e34449
Received: 06/05/2024 Accepted: 07/08/2024 Published: 01/12/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34449
ABSTRACT
This study was conducted to investigate the protective effect of
Astragalus (AST) extract alone and in combination with vitamin E
(vitE) + selenium (Se) against the toxicity induced by cadmium (CdCl
2
)
in rat ovaries. Thirty–six female Wistar rats were divided into six
groups. AST was administered at a dose of 5 mg·kg
-1
, Cd at a dose of
2 mg·kg
-1
, and Vit E (60 mg·kg
-1
) + Se (1 mg·kg
-1
) orally for a duration of
15 days. The levels of MDA, GSH–Px, SOD, and CAT were analyzed in
the blood and tissue samples to assess oxidative stress. Additionally,
the levels of estrogen, FSH, LH, Inhibin B, and Antimullerian hormones
were measured in the serum samples. The ovarian tissues were
examined histopathologically and immunohistochemically for 8–OhDG,
Caspase3, and LC3B immunoreactivity. In the group exposed to CdCl
2
,
MDA levels signicantly increased, while antioxidant parameters
showed significant decreases (P<0.05). Although significant
improvements were observed in the groups treated with AST alone,
more signicant improvements were seen in the groups treated with
both AST and Vit E + Se (P<0.05). It was concluded that AST extracts
alone and in combination with Vit E + Se exhibited protective effects
against ovarian toxicity caused by Cd exposure and may be effective
against metal toxicity.
Key words: Cadmium; ovarium damage; Astragalus; oxidative
stress
RESUMEN
Este estudio se realizó para investigar el efecto protector del extracto
de astrágalo (AST) solo y en combinación con vitamina E (vit E) +
selenio (Se) contra la toxicidad inducida por cadmio (CdCl
2
) en ovarios
de rata. Treinta y seis ratas Wistar hembra se dividieron en seis
grupos. Se administró AST a una dosis de 5 mg·kg
-1
, Cd a una dosis
de 2 mg·kg
-1
y VitE (60 mg·kg
-1
) + Se (1 mg·kg
-1
) por vía oral durante
15 días. Se analizaron los niveles de MDA, GSH–Px, SOD y CAT en
muestras de sangre y tejido para evaluar el estrés oxidativo. Además,
en las muestras de suero se midieron los niveles de estrógeno, FSH,
LH, inhibina B y hormonas antimullerianas. Los tejidos ováricos
se examinaron histopatológica e inmunohistoquímicamente para
determinar la inmunorreactividad de 8–OhDG, caspasa 3 y LC3B.
En el grupo expuesto a CdCl
2
, los niveles de MDA aumentaron
significativamente, mientras que los parámetros antioxidantes
mostraron disminuciones significativas (P<0,05). Aunque se
observaron mejoras signicativas en los grupos tratados con AST sola,
se observaron mejoras más signicativas en los grupos tratados tanto
con AST como con Vit E + Se (P<0,05). Se concluyó que los extractos
de AST solos y en combinación con Vit E + Se exhibieron efectos
protectores contra la toxicidad ovárica causada por la exposición al
Cd y pueden ser ecaces contra la toxicidad de los metales.
Palabras clave: Cadmio, daño ovárico, astrágalo, estrés oxidativo
Effect of Astragalus microcephalus wild extract and Vitamin E–Selenium
combination on Cadmium–induced damage in rat ovaries
Efectos del extracto de Astragalus microcephalus y la combinación de vitaminaE
y Selenio sobre el daño tisular inducido por Cadmio en ovarios de rata
Begum Kurt
1
, Haki Kara
2
* , Mahmut Sahın
2
, Alper Serhat Kumru
2
, Mustafa Ozkaraca
3
1
Sivas Cumhuriyet University, Faculty of Medicine, Department of Obstetrics and Gynecology. Sivas,Türkiye.
2
Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology. Sivas,Türkiye.
3
Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Pathology. Sivas,Türkiye
*Corresponding author: hakikara@cumhuriyet.edu.tr
Effects of Astragalus on Cadmium induced ovarium damage / Kurt et al. __________________________________________________________
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INTRODUCTION
For the survival and growth of species, reproduction is seen as
essential [1]. The reproductive system inuences the organism’s
behavior and controls the morphological evolution and physiological
distinctions between males and females. Exposure to hazardous
compounds can cause teratogenic, carcinogenic, and mutagenic
consequences, infertility, tissue damage, oogenesis decits, and
other reproductive diseases [1, 2].
Due to increased industrial and agricultural activities, metal–
induced environmental pollution and contamination of heavy metals
in the food chain are rising. Toxic mechanisms can occur as ion–like
effects, disruption of cellular signaling pathways, oxidative stress,
disruption of gene structure, apoptosis, inammation, and impaired
endocrine metabolism [3]. Cadmium (Cd), which is found among
heavy metals, is a highly toxic element. The increase in its quantity
in soil and water due to intensive industrial activities, consumption
of foods grown in contaminated areas, consumption of contaminated
animal products, and consumption of tobacco products with high Cd
content can pose signicant risks to human health [4, 5]. Due to the
effects of Cd toxicity on the body’s major organs, it is considered one
of the most toxic compounds to human health [5].
Cadmium entering the body is associated with Metallothionein
(MT) protein. MT is known to be responsible for both the transport
and detoxification of cadmium in organ tissues. Due to its low
elimination rate, cadmium accumulation in the liver and kidneys
increases cadmium toxicity in long–term exposures. Cadmium
disrupts apoptotic mechanisms by causing the formation of reactive
oxygen species (ROS) in the kidney, liver, lungs, brain, bone tissue,
blood components, testes, and ovaries, leading to cellular and DNA
damage. As a result, it can lead to cancer [6]. In cadmium–induced
ovarian dysfunction, the increase in ROS, changes in gene expression,
DNA damage, apoptosis, and increased membrane lipid peroxidation
occur due to oxidative stress [7, 8].
Cadmium chloride (CdCl
2
) is a potential endocrine disruptor [9].
It has been reported that exposure to Cd can cause reproductive
and developmental disorders, especially in embryonic and young
animals and humans [10]. CdCl
2
can cause damage to both male and
female reproductive organs. It induces histopathological disorders,
disturbances in spermatogenesis, decreases testosterone levels and
has carcinogenic effects on the testes. At the same time, in female
reproductive organs, it causes histopathological disorders, delayed
pubertal disorders, prolonged estrus periods, and oxidative stress
[2]. It also increases enzyme activity in granulosa cells of the ovaries,
decreases gonadotropin binding, and decreases serum progesterone
and estradiol levels [8, 10, 11].
The formation of ROS and disruption of fundamental molecular
mechanisms through oxidative stress, which is associated with
mitochondrial damage, has been reported to play a signicant role
in Cd toxicity, involving both caspase–dependent and caspase–
independent apoptotic pathways [12, 13].
In addition to classical chelating agents, certain antioxidants such
as vitamin E, Selenium, and melatonin have been successful against
Cd toxicity [14]. Recently, extracts obtained from medicinal and
aromatic plants have started to be used in health. The benecial
effects of avonoids found in plant extracts, in particular, have
attracted the attention of an increasing number of researchers. Some
previous studies have indicated the protective effects of herbal and
natural substances such as quercetin, tualang honey, and Hibiscus
sabdariffa extract against ovarian toxicity [15, 16, 17].
This study examines the effectiveness of Astragalus microcephalus
wild extract, known for its potent antioxidant properties, against the
toxic effects of cadmium in the ovaries. Astragalus (AST) species are
plants that can be found in countries with temperate climates around
the world and have approximately 2,000 different species [18]. The
root parts are more commonly used [19]. It is stated that it has been
used in China for about 2,000 years [20]. Astragalus tea and capsules
are sold as over–the–counter dietary supplements in the US health
food market [21]. AST species have applications as food additives
and nutritional supplements in many countries worldwide.
The active ingredients found in Astragalus species include saponins,
avonoids, polysaccharides, and trace elements such as selenium,
copper, zinc, iron, and volatile fatty acids. It has also been reported
to have a high selenium retention capacity [21, 22].
The main pharmacological effects of Astragalus polysaccharides
include anticancer, antiaging, antiviral, antibacterial, immune system
regulatory, blood sugar level–regulating, lipid–lowering, radiation–
protective effects, and antioxidant properties with very low toxicity
[17, 22, 23, 24, 25]. It is stated that AST exhibits a strong antioxidant
effect, reduces lipid peroxidation, increases superoxide dismutase
activity, decreases malondialdehyde (MDA) production, and exhibits
protective and anti–aging properties [26].
AST species have been reported to increase the proliferation
of T and B lymphocytes, increase cytokine production, activate
macrophages and B cells, increase the expression of IL2, IL3, IL4,
IFNy, IgM, and IgG, and decrease IgE levels [23, 24, 25, 27].
This study aims to determine the protective effect of AST extract,
which has a substantial antioxidant property, against the potential
damage caused by cadmium in the ovaries. Additionally, the study
aims to determine the feasibility of using AST alone or combined with
Selenium and vitamin E in treatment by performing applications with
AST, Selenium, and vitamin E combinations.
MATERIALS AND METHODS
The chemicals used in the study were of analytical purity, cadmium
chloride (CdCl
2
),vitamin E (α–tocopherol), and selenium (sodium
selenite) were purchased. (Sigma, St.Louis, MO, USA).
Experimental animals
This study used 36 Wistar female rats (Rattus norvegicus) (average
live weight 220–250 g). Animals were obtained from Sivas Cumhuriyet
University Experimental Animals Unit, and the study was carried out
in the same place. The rats were housed in 12 hours of light and 12
hours of darkness during the trial, and all guidelines for animal care
were observed. The rats received unlimited amounts of food and
water. Sivas Cumhuriyet University Animal Experiments Local Ethics
Committee granted clearance for this investigation with its letter
dated April 20, 2021, and number 538.
While forming the groups, a total of 36 female rats were used in 6
groups, with six animals in each group. The application period was
determined as 15 days in all groups.
Application groups were created as follows:
1. Control group (saline i.p.)
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2. Astragalus (AST) (5 mg·kg
-1
oral gavage)
3. Cadmium chloride (CdCl
2
) (2 mg·kg
-1
·day
-1
i.p.)
4. CdCl
2
+ AST (2 mg·kg
-1
·day
-1
i.p. + 5 mg·kg
-1
oral gavage)
5.
CdCl
2
+ Vitamin E + Selenium (2 mg·kg
-1
·day
-1
i.p. + Vit E
60mg·kg
-1
·day
-1
+ Se 1 mg·kg
-1
·day
-1
oral gavage)
6.
CdCl
2
+Vitamin E+Selenium+AST (2 mg·kg
-1
·day
-1
i.p.+ VitE 60
mg·kg
-1
·day
-1
+ Se 1 mg·kg
-1
·day
-1
oral gavage + 5 mg·kg
-1
oral gavage)
At the end of the application, the rats were euthaniased under
ketamine/xylazine (90/10 mg·kg
-1
, ip.) anesthesia. The blood taken from
the animals was centrifuged (Nuve, NF800, Turkey) and separated for
hormone analysis and other biochemical analyzes and kept at -18 C°
(Arçelik, 2350E, Turkey). The ovaries of the rats were removed, and one
was separated for biochemical analysis, while the other was preserved
in a 10% formaldehyde solution until histopathological examination.
Plant extraction
The wild plant Astragalus microcephalus used in this study was
obtained from the rural area of Sivas/Türkiye. The identication
of the collected plants was made by Prof. Dr. H. Aşkın AKPULAT, a
faculty member of the Biology Department of the Faculty of Science,
Sivas Cumhuriyet University. The root parts of the collected events
were dried and ground. Then, 1 g of plant was extracted with 10 mL
of ethanol (10:1 mL extraction solvent·g
-1
herb) for 24 hours. After
the extraction, it was centrifuged (Nuve, NF800, Turkey) at 3075 G
for 10 min, passed through the evaporator (IKA, CMF300, People’s
Republic of China).
Biochemical assay
Rats were euthanasied, blood and the ovarian tissues collected.
Ovarium tissues were immediately removed under ice–cold conditions,
blotted free of blood and tissue uids, weighed, and kept at -80 C°.
(Antech, Eco Toch, Turkey).
Determination of MDA level
Lipid peroxidation was measured in terms of malondialdehyde
(MDA) as specied by Ohkawa et al. [28]. Thiobarbituric acid reactive
substances (TBARS), which are produced when MDA and thiobarbituric
acid react, were used to measure the MDA level in ovarian tissue
homogenate. Thiobarbituric acid and MDA reacted aerobically to
produce the MDA–(TBA) 2 complex, which was detected at 532 nm using
a spectrophotometer (Perkin Elmer, Lambda365, USA). For serum, MDA
concentration (TBARS) was estimated as “nmol·ml
-1
,” and for ovarium
tissues, “nmol·mg
-1
protein.” Standard solutions of 1, 1, 3, and 3–tetra
ethoxy–propane were used to compare absorbance readings (TEP).
Measurement of SOD activity
A commercially available standard enzymatic kit (Cayman Chemical
Company, Ann Arbor, MI) that uses a tetrazolium salt for the detection
of superoxide radicals produced by xanthine oxidase and hypoxanthine
was used to measure superoxide dismutase (SOD). All three forms of
SOD are measured by the SOD assay. The quantity of enzyme required
to demonstrate a 50% dismutation of the superoxide radical is referred
to as one unit of SOD activity. Using a plate reader, the absorbance
was read between 440 and 460 nm (Thermo, Multiskan GOMicroplate,
Massachusetts, USA). “U·mL
-1
” was used to express the SOD activity.
Measurement of GSH–Px activity
Glutathione peroxidase enzyme levels in blood and ovarian tissues
were measured with a commercial assay kit (Cayman Chemical
Company, Ann Arbor, Michigan,USA). Glutathione peroxidase is an
important antioxidant enzyme that helps protect cells from damage
caused by reactive oxygen species (ROS).
Measurement of CAT activity
Catalase enzyme activities (CAT) in blood and ovarian tissues were
determined with Catalase Colorimetrik Acitivity Kit (ThermoFischer
Scientic, cat no EIACATC, USA).
Histopathological Examination
Rats were necropsied, and the ovarian tissues were preserved in
a buffered formalin solution at a 10% concentration. The samples
were subsequently xed in paran blocks and subjected to standard
follow–up procedures. Hematoxylin–Eosin was used to analyze the
5 µm slices from the blocks to the slides under a light microscope
for histopathological results. Semiquantitative ratings of the
histopathological ndings included absent (0), mild (1), moderate
(2), and severe (3).
Immunohistochemical Examination
After washing with phosphate buffer solution (PBS) and xylol and
alcohol series on 5 µm slices on polyzed slides, endogenous peroxidase
inactivation is made sure of by soaking them in 3 percent H
2
O
2
for 10
min. Tissues were exposed to antigen retrieval solution for 2×5 min
at 500 watts in order to reveal the antigen within them. After protein
blocking, tissues were rinsed with PBS before being treated with 8–
OhDG, Caspase 3 (Biorbyte, Cat. No. Orb382909, 1/100 dilution ratio),
and LC3B (Santa Cruz, Cat. No. sc–271625, 1/300 dilution ratio) before
being incubated with primary antibodies overnight at +4 C
o
. In addition,
the manufacturer’s advice was followed when using the Large Volume
Detection System: anti–Polyvalent, HRP (Thermo Fisher, Catalog no:
TP–125–HL). As the chromogen, DAB (3,3–Diaminobenzidine) was
employed. It was coated with Stellan, counterstained with Mayer’s
Hematoxylin, and then viewed under a light microscope (Olympus,
IX70, Tokyo, Japan). The analysis was semiquantitative and classied
the immunopositivity in the ovarian tissues as absence (0), mild (1),
moderate (2), severe (3), and very severe (4).
Hormone analysis
Anti–Mullerian Hormone (AMH), Estradiol 2 (E2), Folicul Stymulan
Hormone (FSH), Inhibin B (INH B), and Luteinizing (LH) hormone
levels were determined using the ELISA method following the
manufacturer’s manual (BT Lab, China). The manufacturer’s catalog
numbers are given. Rat Anti–Mullerian Hormone (AMH Elısa kit Cat.
No: EA0083Ra), Rat Estradiol (E2 ELISA Kit Cat.No: EA0011Ra), Rat
Follicle–stimulating Hormone (FSH ELISA Kit Cat.No: EA0015Ra), Rat
Inhibin B (INHB ELISA Kit Cat.No: EA0059Ra).
Statistical analysis
The SPSS version 20.00 application was used to analyze the data
that was obtained. The Kruskal Wallis test, one of the non–parametric
tests, and the Mann–Whitney U test for the group that caused the
difference were used to determine the difference between the groups.
(P<0.05). P–value less than 0.05 was considered statistically signicant.
FIGURE 1. Ovarium stromal degenerasyon and edeme levels. A– Control group and B– Astragalus group. Normal histological appearance, C– Cd group. Severe level,
D– Cd +AST group. Intermediate level, E– Cd +Vit E–Se group. At intermediate level, F– Cd +Vit E–Se group+AST group. Mild stromal degeneration (□) and edema (arrow),
Hematoxylin–Eosin staining
TABLE I
Immunohistochemical staining and histopatological (HE) ndings
Groups 8–OhDG Caspase 3 LC3B Stromal degeneration (HE) Edema (HE)
Control 1.00 ± 0.40
a
1.00 ± 0.00
a
1.16 ± 0.40
a
0.16 ± 0.40
a
0.33 ± 0.51
a
AST 1.00 ± 0.00
a
1.16 ± 0.40
a
1.16 ± 0.40
a
0.16 ± 0.40
a
0.16 ± 0.40
a
Cd 3.66 ± 0.51
b
3.83 ± 0.40
b
3.66 ± 0.51
b
2.83 ± 0.40
b
2.83 ± 0.40
b
Cd +AST 2.66 ± 0.51
c
2.83 ± 0.40
c
2.66 ± 0.51
c
2.00 ± 0.00
c
2.16 ± 0.40
c
Cd +Vit E–Se 2.83 ± 0.40
c
2.66 ± 0.51
c
2.83 ± 0.40
c
2.16 ± 0.40
c
1.83 ± 0.40
c
Cd +Vit E–Se+AST 1.83± 0.40
d
1.83± 0.40
d
2.00 ± 0.00
d
1.16 ± 0.40
d
1.00 ± 0.00
d
a,b,c,d
: Dierent letters in the same column represent dierences between groups (P<0.05)
Effects of Astragalus on Cadmium induced ovarium damage / Kurt et al. __________________________________________________________
4 of 11
RESULT AND DISCUSSION
Hematoxylin–Eosin staining ndings
Between the groups, a statistically signicant difference was
found (TABLE I, P<0,05). Rats in the control and AST groups’ ovarian
tissues displayed a typical histological appearance. Degeneration
and edema in the stromal tissue of the interstitial zones were the
identied histological ndings. While these histological results were
moderate in the Cd+AST and Cd+Vit E–Se groups and mild in the
Cd+Vit E–Se+AST group, they were severe in the Cd group (FIG. 1).
Immunohistochemical Staining Findings
In immunohistochemical staining, statistically significant
differences were found between the groups in terms of 8–OhDG,
Caspase 3, and LC3B immunoreactivity (TABLE I, P<0.05).
Staining with 8–OhDG, Caspase 3, and LC3B showed mild
immunopositivity in the control and AST group. Among the other
groups, very severe positivity was found in Cd, severe positivity in
Cd+AST and Cd+Vit E–Se, and moderate positivity in the Cd+Vit E–
Se+AST group. Immunopositivity was in stromal cells and granulosa
cells in the follicular (FIGS. 2, 3 and 4).
FIGURE 2. 8–OhDG Immunohistochemistry levels. A– Control group and B– Ast group. Mildly, C– Cd group. At very severe level, D– Cd+AST group and E– Cd+Vit E–Se group.
At severe level, F– Cd+Vit E–Se group+AST group. Medium–level. 8–OhDG immunopositivity is shown in stromal cells (arrow) and granulosa cells of follicles (arrowhead)
FIGURE 3. Caspase–3 Immunohistochemistry levels. A– Control group and B– Ast group. Mildly, C– Cd group. At very severe level, D– Cd+AST group and E– Cd+Vit E–Se
group. At severe level, F– Cd+Vit E–Se group+AST group. Medium–level. Caspase–3 immunopositivity is shown in stromal cells (arrow) and granulosa cells of follicles
(arrowhead)
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FIGURE 4. LC3B Immunohistochemistry levels. A– Control group and B– Ast group. Mildly, C– Cd group. At very severe level, D– Cd+AST group and E– Cd+Vit E–Se group.
At severe level, F– Cd+Vit E–Se group+AST group. Medium–level. LC3B immunopositivity is shown in stromal cells (arrow) and granulosa cells of follicles (arrowhead)
TABLE II
Blood and ovarium antioxidant parameters of rats given cadmium chloride
Groups
MDA (nmol/protein) SOD U·mg
-1
protein CAT U·mg
-1
protein GSH–Px nmol·mg
-1
protein
Blood Ovarium Blood Ovarium Blood Ovarium Blood Ovarium
Control 5.2 ± 1.0
a
2.8 ± 0.3
a
3.5 ± 0.7
a
1.5 ± 0.3
a
6.5 ± 0.8 a 1.3 ± 0.2
a
85 ± 7.4
a
55 ± 4.5
a
Cd 11.6 ± 2.1
a
5.6 ± 1.2
a
1.6 ± 0.2
a
0.6 ± 0.1 a 2.4 ± 0.2
a
0.5 ± 0
a
41 ± 3.6
a
28 ± 3.5
a
AST 6.1 ± 1.8
b
3.2 ± 0.5
b
3.2 ± 0.6
b
0.9 ± 0.2
b
5.2 ± 0.6
b
1.2 ± 0.3
b
88 ± 6.4
a
45 ± 3.6
b
Cd+AST 7.1 ± 1.5
c
3.8 ± 0.6
c
2.5 ± 0.3
c
1.0 ± 0.2
b
4.3 ± 0.4
c
1.0 ± 0.2
c
62 ± 6.5
b
41 ± 3.8
c
Cd+Vit E–Se 6.5 ± 0.9
b
3.2 ± 0.4
c
2.9 ± 0.5
c
1.1 ± 0.2
c
5.2 ± 0.6
b
1.3 ± 0.3
d
72 ± 5.8
c
52 ± 4.0
d
Cd+AST+ Vit E–Se 5.8 ± 0.8
d
3.0 ± 0.2
d
3.2 ± 0.5
d
1.2 ± 0.4
d
5.9 ± 0.8
d
1.4 ± 0.3
d
79 ± 6.7
d
50 ± 4.3
d
a,b,c,d
: Dierent letters in the same column represent dierences between groups (P<0.05).
Effects of Astragalus on Cadmium induced ovarium damage / Kurt et al. __________________________________________________________
6 of 11
Antioxidant parameter ndings
Ovarian tissue and blood MDA, SOD, CAT, and GSH–Px values
obtained in the study are given in TABLE II.
Hormon Levels Findings
Estradiol, Luteinizing hormone, Follicle stimulating hormone,
Antimullerian hormone, and inhibin B levels were measured in the sera
obtained from the rats used in the study. While there were signicant
decreases in hormone levels in all groups given cadmium (P<0.05),
hormone levels in the treatment groups increased significantly
(P<0.05) and approached control levels (FIG. 5).
With the advancement of technology and industrialization, exposure
to heavy metals, particularly cadmium, is becoming increasingly
common. Heavy metal toxicity can lead to damage in various organs
and tissues. It is well–known that exposure to cadmium can cause
significant damage to the reproductive systems and hormonal
changes in both males and females. Accumulation in the female
reproductive organs can lead to cytotoxicity, decreased FSH, LH,
and Estradiol hormone levels, and menstrual cycle delays [29]. This
study is the rst to look into the adverse effects of AST on cadmium
toxicity–related ovarian damage.
Ovaries play a crucial role in the production of female steroid
hormones. Teca and granulosa cells regulate the estrous cycle in
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FIGURE 5. A: Estradiol, B: Follicle stimulating hormone, C: Luteinizing hormone, D: Inhibin B and E: Antimullerian hormone levels were measured in the serum (*,# P<0.05)
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response to these steroids [29]. It is known that cadmium reduces
the number of follicles and leads to folliculogenesis dysfunction [8,
13, 30]. Follicular dysfunction due to ovarian toxicity affects fertility
and accelerates the menopausal process, increasing the risk of
osteoporosis and cardiovascular diseases. Gonadotropic hormones
(LH, FSH) need to bind to surface–specic receptors on granulosa
cells to stimulate the production of gonadal steroid hormones. It has
been shown that cadmium toxicity inhibits the binding of LH and FSH
hormones to these receptors [30]. The effects of cadmium on ovarian
follicles were shown to be connected with changes in gonadotropin
hormones and decreases in follicle–stimulating hormone (FSH)
and luteinizing hormone (LH) in a study by Ruslee et al. that was
comparable to this study [15]. In our study, observed a decrease
in all hormone levels in rats exposed to cadmium. When AST was
added to the treatment groups, the adverse effects were observed
to decrease. The best results were observed in the group where AST
was combined with vitamin E–Se, known for its antioxidant activity.
Histopathological examination of ovaries exposed to cadmium
revealed severe degeneration and edema in the stromal tissue.
Although the use of AST in the treatment groups reduced the level of
histopathological damage, the most effective protection was observed
in the groups where vitamin E–Se was used in combination with AST.
There is growing proof that one of the primary molecular processes
behind cadmium toxicity is oxidative stress, which results in the
production of ROS and mitochondrial damage [31]. For many cell
lines exposed to cadmium, apoptotic processes involving both
caspase–dependent and caspase–independent pathways have been
discovered [32]. In this study, immunohistochemical parameters
(8–OhDG, Caspase 3, and LC3B) were examined, and it was found
that intense staining occurred only in the ovarian tissues exposed
to cadmium toxicity. In contrast, the staining decreased when AST
was administered. Furthermore, the combination of vitamin E–Se
and AST resulted in the least staining.
It is commonly acknowledged that one of the main mechanisms of
cellular malfunction and death brought on by cadmium exposure is
oxidative stress (OS) [31]. According to numerous studies, cadmium
can cause granulosa cells to undergo apoptosis by producing ROS
that come from the mitochondria. Cellular homeostasis depends on
maintaining a balance between the generation of reactive oxygen
species and the ability of the antioxidant system. The equilibrium
of oxidation–antioxidant systems can be upset by excessive ROS
generation, which can result in illness and malfunction in cells [12].
Cd has a high anity for the sulfhydryl groups of proteins, which
can affect the activities of specic enzymes, particularly glutathione,
the major sulfhydryl reserve in granulosa cells [29]. Furthermore,
investigations have shown that in the group exposed to Cd, SOD activity
is signicantly inhibited, while CAT activity shows a signicant increase.
Another study has also found that cadmium reduces antioxidants in rats’
ovaries and increases MDA and hydrogen peroxide (H
2
O
2
) [33]. TABLE II
showed the tissue and blood antioxidant values. The Cd–exposed group
had signicantly higher MDA levels than the control group, whereas SOD,
CAT, and GSH–Px levels were signicantly lower. These indicators are
seen to vary signicantly across all treatment groups and get closer
to the levels of the control group (P<0.05).
In previous studies, it has been reported that 8–OHdG, a commonly
used biomarker for DNA damage, is formed in damaged tissues by
removing a hydrogen atom from nucleic acids by toxic oxygen radicals
such as hydroxyl radicals [34]. There is a relationship between the
Effects of Astragalus on Cadmium induced ovarium damage / Kurt et al. __________________________________________________________
8 of 11
formation of 8–OHdG and the production of ROS, indicating that ROS
can contribute to the formation of 8–OHdG. Studies demonstrate
signicantly increased levels of 8–OHdG in damaged ovarian tissue
caused by oxidative stress compared to healthy tissues [34].
Immunohistochemical staining using 8–OHdG as a DNA damage
marker revealed intense immunopositivity only in ovarian tissues
exposed to Cd toxicity. However, when AST was administered, the
immunopositivity started to decrease, and when AST was combined
with vit E–Se, the immunopositivity was found to be minimal. Similar
ndings were also observed in the staining performed with Caspase–3
and LC3B. In the group treated with Cd alone, advanced levels of
positivity were detected, which started to decrease with the use
of AST and Vit E–Se. The combination group, Cd + Vit E–Se + AST,
showed the lowest levels of positivity, indicating that this combination
reduced cell death. Caspase–3, one of the markers used in the study,
is an indicator of apoptosis, which is dened as a typical cellular death
process that maintains tissue homeostasis and is triggered by various
pathological–physiological stimuli such as oxidative damage [35, 36].
LC3B, another marker used in the study, is a marker that indicates
autophagic cell death. It has been noted that oxidative stress can
trigger autophagy, causing tissue and cellular damage. LC3B is a
structural protein that can bind to the membrane of autophagosomes
and plays a key role in autophagosome formation [37]. Therefore,
in this study, the minimal immunopositivity of both Caspase–3 and
LC3B in the Cd + Vit E–Se + AST group indicates that the combination
treatment reduced apoptotic and autophagic cell death induced by Cd.
CONCLUSION
In conclusion, when histopathological, immunohistochemical,
hormonal, and antioxidant parameters were examined, Cd–induced
ovarian toxicity was observed consistently. It has been concluded
that the phenolic and avonoid compounds found in AST extract may
play a role in these effects. While AST alone was relatively effective in
protecting against this toxicity, the combination of Vit E–Se and AST
showed the highest ecacy in mitigating the toxic effects.
ACKNOWLEDGEMENT
Funding
This study was supported by Sivas Cumhuriyet University Scientic
Research Projects (CUBAP) with project number V–2021–119.
Ethical Approval
All experimental procedures applied in this study were examined
by Sivas Cumhuriyet University Experimental Animal Research Ethics
Committee and approved on 20.04.2021 with the number 538.
Author Contributions
All authors contributed to the study conception and design. Material
preparation, data collection and analysis were performed by Begum
Kurt, Haki Kara, Mahmut Sahin, Alper Serhat Kumru and Mustafa
Ozkaraca. The rst draft of the manuscript was written by Begum Kurt
and all authors commented on previous versions of the manuscript.
All authors read and approved the nal manuscript.
Data availability statement
The data are available by the corresponding author upon.
Conicts of interest
The authors declare no conict of interest.
Sample availability
Samples of the compounds are available from the authors.
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