Document Type : Original Article
Authors
1 Nervous System Stem Cells Research Center, Semnan University of Medical Sciences, Semnan, Iran
2 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
Abstract
Keywords
Despite the great benefits of chemotherapy in treating
cancer patients, it has some side effects on ovaries (
Several reports have shown that Lc has favorable effects
on mesenchymal stem cells, including suppression
of apoptosis in BMSCs (
Although the effects of individual BMSCs and Lc on the repair of damaged ovaries have been investigated, there is no report yet concerning the effect of simultaneous administration of them on the recovery of damaged ovaries. So, in this study, due to the beneficial effects of Lc on BMSCs, we evaluated for the first time the effect of co-administration of BMSC+Lc on ovarian function and structure after creating a chemotherapy model with cyclophosphamide in rats.
In this experimental study, forty female wistar rats (180- 200 g) were used. They had free access to food and water under controlled temperature (25 ± 2.C). Vaginal smear was daily obtained and only those showing at least two consecutive normal vaginal estrus cycles were used in the experiments. All procedures were approved by the Research Council of Semnan University of Medical Sciences (Semnan, Iran). The Ethical Code is IR.SEMUMS.REC.
After sacrificing an adult rat, femurs and tibias were dissected
out. Bone marrow was ejected with 10 ml of Dulbecco’s
Modified Eagle Medium (DMEM) and cultured
in DMEM containing 10% fetal bovine serum (FBS) and
1% penicillin/streptomycin (all from Gibco, Germany),
incubated at 37.C, 95% humidity and 5% CO2. After 48
hours, non-adherent cells were removed by replacing the
medium. The cells were sub-cultured four times (
To analyze expression of the stem cell surface markers,
at least 100,000 cells were incubated with fluorescence-
labeled monoclonal antibodies against CD29, CD34,
CD44, CD45 and CD90 (Sigma, China). Following a 10
minutes wash in phosphate-buffered saline (PBS, Sigma,
USA), the labeled cells were analyzed using a Becton
Dikinson FACS Calibur Flow Cytometer (BD, USA) (
To destroy the ovaries, a model of chemotherapy was
created. Cyclophosphamide (Sigma, China) diluted in
normal saline was intraperitoneally (IP) injected at 50 mg/
kg at the first day, followed by 13 days injection of 8 mg/
kg daily cyclophosphamide (
After creating the chemotherapy model, the rats were
randomly divided into four groups (n=10 in each group):
i. Control group, 25 .l of culture medium was directly
injected into the bilateral ovaries, ii. BMSC group, 2×106
BMSCs suspended in 25 .l culture medium were directly
injected into the bilateral ovaries (
To track the transplanted BMSCs after four weeks in the
ovaries, the cells were labeled with DiI (1,1’-dioctadecyl-
Four weeks after the end of chemotherapy, serum estradiol
(E2) and follicle-stimulating hormone (FSH) levels
of these groups were measured by enzyme-linked immunosorbent
assay (ELISA) kits (East Bio-Pharm, China)
for rat, according to the manufacturer’s instruction (
Four weeks after the end of chemotherapy, the ovaries
were collected and fixed in 4% paraformaldehyde, dehydrated,
paraffin-embedded and serially sectioned at 5 .m
thickness. Five representative sections from each ovary
were randomly chosen and routine hematoxylin and eosin
(H&E) staining was performed for histological examination
with light microscopy. the number of primordial, primary,
secondary and antral follicles were measured (
Five ovaries in each group were lysed using RIPA buffer
(Cell Signaling Technology, Netherlands) supplemented
with protease inhibitor (Roche, Switzerland) on ice for
30 minutes. Then, the mixture was centrifuged at 13000
rpm for 20 minutes at 4°C. Equal value of proteins (80
.g) were loaded on sodium dodecyl sulfate (SDS, Sigma,
Japan) polyacrylamide gel (Merck, Germany) and separated
in a size manner by electrophoresis. The proteins
were transferred to nitrocellulose membranes (Amersham
Biosciences, USA). The membranes were blocked with
5% skim milk in tris buffered saline (TBS, pH=7.4). The
membranes were incubated with primary antibodies for
Bcl-2 (1:1000), Bax (1:1000) and .-Actin (1:1000, Abcam,
USA) overnight at 4°C. After washing, the membranes
were incubated with goat anti-rabbit secondary
antibody conjugated with horseradish peroxidase (HRP).
All antibodies were diluted according to manufacturer’s
instructions. Immunoreactive bands were visualized using
an enhanced chemiluminescence detection system (Amersham
Biosciences, USA). X-ray films were scanned,
and then the relative protein levels were semi-quantified
by densitometric analysis using image j software. .-actin
was tested as the internal control (
After verifying the normality of variance assumptions, data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey Test. Obtained data are presented as the mean ± SE, and a level of P<0.05 was considered statistically significant.
BMSCs were cultured in the T25 flasks. After a few
days, the cells appeared to be spindle-shaped. By repeating
passages, the cells became morphologically homogeneous.
Most of the cells expressed the mesenchymal stromal
cell markers (CD29, CD44 and CD90) and did not
express the hematopoietic cell markers: CD34 and CD45
(
Isolation and identification of bone marrow stromal cells (BMSCs).
The transplanted BMSCs were labeled with dii, as red
spots in the sections of ovaries (
DiI labeled bone marrow stromal cells (BMSCs) in a section of ovary.
Hormonal examination was performed, by determining
levels of serum E2 and FSH, four weeks after treatment.
The results showed that levels of serum E2 in the
BMSC+Lc co-administrated group (P<0.001), BMSC
group (P<0.001) and Lc group (P<0.01) were significantly
higher than the control group. The results of BMSC+Lc
group were significantly higher than BMSC group
(P<0.05) and Lc group (P<0.001). The results of BMSC
group were significantly higher than Lc group (P<0.001,
The levels of serum FSH in the BMSC+Lc co-administrated
group (P<0.001), BMSC group (P<0.001) and
Lc group (P<0.01) were significantly lower than the control
group. The results of BMSC+Lc group were significantly
lower than BMSC group (P<0.05) and Lc group
(P<0.001). The results of BMSC group were significantly
lower than Lc group (P<0.01,
The levels of serum estradiol (E2) and follicle-stimulating hormone (FSH) in the experimental
groups four weeks after treatment.
H&E staining demonstrated that the number of all
follicles in different stages was significantly higher in
BMSC+Lc group compared to BMSC (P<0.01), Lc
(P<0.001) and control groups (P<0.001). Findings showed
that the number of all follicles in BMSC group was significantly
more than Lc group (P<0.05,
The number of follicles four weeks after treatment. H&E staining of ovaries in
Results of the hormonal, histological and expression of ovarian Bcl-2 and Bax proteins four weeks after treatment
Groups | Control | BMSCs | L-carnitine | BMSC+L-carnitine |
---|---|---|---|---|
E2 (pg/ml) | 25.18 ± 1.769 | 40.74 ± 0.63*** | 31.48 ± 0.533** | 45.2 ± 0.728*** |
FSH (mIU/ml) | 14.34 ± 0.682 | 6.9 ± 0.24*** | 10.5 ± 0.791** | 4.62 ± 0.338*** |
The number of ovarian follicles in different stages | ||||
Primordial | 17 ± 1.581 | 28.2 ± 0.86*** | 23.6 ± 0.51** | 33.2 ± 0.583*** |
Primary | 14.4 ± 0.748 | 25.4 ± 0.678*** | 20 ± 0.707** | 30 ± 1.03*** |
Secondary | 13 ± 0.316 | 20 ± 0.707*** | 17.4 ± 0.748** | 25.2 ± 0.583*** |
Antral | 5.6 ± 0.51 | 13.6 ± 0.51*** | 9.4 ± 0.61** | 18.2 ± 0.583*** |
Expression of ovarian Bcl-2 protein | 1.0 ± 0.0 | 1.473 ± 0.017*** | 1.89 ± 0.054* | 2.347 ±0.167*** |
Expression of ovarian Bax protein | 1.0 ± 0.0 | 0.806 ± 0.011*** | 0.392 ± 0.051** | 0.179 ± 0.027*** |
Bcl-2/Bax ratio | 0.723 ± 0.047 | 1.12 ± 0.026* | 1.143 ± 0.046* | 4.018 ± 0.127*** |
Data are presented as mean ± SE. E2; Estradiol, FSH; Follicle-stimulating hormone, *; P<0.05, **; P<0.01, and ***; P<0.001 versus control group.
Expression of ovarian Bcl-2 and Bax proteins was
determined by Western blot. The results showed that
Bcl-2 expression in the co-administration of BMSC+Lc
(P<0.001), BMSC (P<0.001) and Lc groups (P<0.05)
were significantly higher than the control group; while
it was significantly higher than BMSC (P<0.05) and Lc
groups (P<0.01) in BMSC+Lc. In addition, it was significantly
higher in the BMSC, compared to Lc group
(P<0.05). Bax expression in the BMSC+Lc co-administered
group (P<0.001), BMSC group (P<0.001) and Lc
group (P<0.001) were significantly lower than the control.
It was significantly lower in the BMSC+Lc compared to
BMSC (P<0.01) and Lc groups (P<0.001). Additionally,
it was significantly lower than Lc group, in the BMSC
group (P<0.05). The Bcl-2/Bax ratio was significantly
increased in BMSC+Lc co-administered group, in comparison
with the control group (P<0.001), BMSC group
(P<0.001) and Lc group (P<0.001,
Analysis of Bcl-2 and Bax protein expressions by western blot assay four weeks after treatment.
Chemotherapy may damage the ovaries of girls and women, however, there are some ways to prevent from happening this. In this study, for the first time, we evaluated the effect of co-administration of BMSC+Lc on damaged ovaries after creating a chemotherapy model with cyclophosphamide in rat. Overall, the results showed that levels of serum E2 and FSH, number of follicles in different stages and expression of Bcl-2 and Bax proteins in BMSC+Lc co-administrated group were significantly more favorable than the control, BMSC and Lc groups.
Some studies have shown that BMSC and Lc may individually
improve damaged ovaries (
BMSCs, as a mesenchymal stem cell type, are a suitable
candidate for cell therapy in damaged ovaries. Liu et al.
(
On the other hand, Lc as an antioxidant may also improve
damaged ovaries. Zhang et al. (
Lc plays an important role in fatty acid transport and
lipid catabolism of mitochondria. Lc produces ATP by increasing
.-oxidation of fatty acid. Hence, it can provide
energy for follicular growth. Lc may also suppress apoptosis
by increasing .-oxidation of fatty acids and reduce
fatty acid toxicity. Moreover, accumulation of reactive
oxygen species (ROS) in follicles leads to evacuation of
the ATP reservoir, which decreases follicle quality. Lc,
as a ROS scavenger and an energy generation facilitator,
can be responsible for useful effects on follicular survival
and ovarian function (
In addition, several studies have shown that Lc has favorable
effects on mesenchymal stem cells. Fujisawa et
al. showed that Lc suppresses apoptosis in BMSCs, due
to restoration of mitochondrial activity and suppression of
senescence induction by blocking TGF-., suggesting that
Lc is involved in mitochondrial activation even in senescent
cells (
Considering the beneficial effects of Lc on mesenchymal
stem cells, in the present study, the combined effects
of Lc and BMSCs were evaluated on the recovery of ovaries
damaged by chemotherapy agent. We cultured BMSCs
and transplanted them into the rat ovaries after creating
the chemotherapy model. BMSCs expressed CD29,
CD44 and CD90, but not CD34 and CD45. That was in
agreement with other study (
Indeed, the results of hormonal, histological and expression of Bcl-2 and Bax proteins were in the same direction and confirmed each other. So that, these results in BMSC+Lc co-administrated group were significantly more favorable than BMSC, Lc and control groups. The reasons are probably due to the combination of useful properties of BMSCs and Lc with different mechanisms of action in the restoration of ovaries after chemotherapy. In addition, considering that Lc has favorable effects on differentiation, increasing lifespan and decreasing apoptosis in BMSCs, it may increase survival of the transplanted BMSCs in the ovaries.
The results of BMSC group were significantly more
favorable than Lc group. In the present study, considering
that BMSCs were injected into the ovaries, these cells
might produce some growth factors or might replace damaged
cells in the ovaries (
This study has some limitations which should be considered. The number of samples was small, so a larger sample size is required. Additionally, more research is necessary to clarify the molecular mechanisms underlying the function of BMSC and Lc to repair damaged ovary after chemotherapy.
The results of this study suggest that the effect of BMSC+Lc co-administration is probably more effective than the effect of their administrations individually on the recovery of ovaries damaged by cyclophosphamide chemotherapy agent in rat.