{"paper_id":"d728232c-9512-4467-8fd1-2683b709de36","body_text":"Despite the great benefits of chemotherapy in treating\ncancer patients, it has some side effects on ovaries ( 1 ).\nCytotoxic effects of chemotherapy damage the granulosa\ncells (GCs), so that folliculogenesis disruption may occur\n( 2 ). Unfortunately, this issue is disappointing for girls and\nyoung women who receive chemotherapy. Cyclophosphamide\nis one of the most administrated chemotherapy\ndrugs which directly affects ovaries ( 3 ). There are several\nmethods to treat ovarian damage, including hormone\ntherapy, freezing ovaries, stem cell therapy and applying\nantioxidants ( 4 ). Hormone therapy is not suitable for cancer\npatients, because it may increase the probability of\nthe cancer recurrence ( 5 ). As disadvantages of ovarian\ncryopreservation, it requires surgical procedures for tissue\nharvesting and transferring, while probability of returning\nits function is low ( 6 ). Recently, it has been observed that\ntransplantation of bone marrow stromal cells (BMSCs),\na type of mesenchymal stem cells, may treat ovarian\ndamage after chemotherapy ( 7 ,  8 ). BMSCs can produce\nsome growth factors, differentiate into other cell lines and\nreplace damaged cells ( 9 ,  10 ). On the other hand, it has\nbeen shown that some antioxidants such as L-carnitine\n(Lc) have beneficial effects on damaged ovaries ( 11 ). Lc\nis a flavonoid antioxidant that plays an essential role in\nfatty acid metabolism and is present in human serum and\ntissues ( 12 ,  13 ). However, the effect of Lc has not been\nassessed on damaged ovaries by chemotherapy.\nSeveral reports have shown that Lc has favorable effects\non mesenchymal stem cells, including suppression\nof apoptosis in BMSCs ( 14 ), modulating differentiation\nof adult mesenchymal stem cells ( 15 ) and improvement\nof the aged adipose tissue-derived human mesenchymal\nstem cells lifespan ( 16 ).\nAlthough the effects of individual BMSCs and Lc on \nthe repair of damaged ovaries have been investigated, \nthere is no report yet concerning the effect of simultaneous \nadministration of them on the recovery of damaged \novaries. So, in this study, due to the beneficial effects of \nLc on BMSCs, we evaluated for the first time the effect of \nco-administration of BMSC+Lc on ovarian function and \nstructure after creating a chemotherapy model with cyclophosphamide \nin rats.\n\nIn this experimental study, forty female wistar rats (180-\n200 g) were used. They had free access to food and water \nunder controlled temperature (25 ± 2.C). Vaginal smear \nwas daily obtained and only those showing at least two \nconsecutive normal vaginal estrus cycles were used in the \nexperiments. All procedures were approved by the Research \nCouncil of Semnan University of Medical Sciences \n(Semnan, Iran). The Ethical Code is IR.SEMUMS.REC.\nAfter sacrificing an adult rat, femurs and tibias were dissected \nout. Bone marrow was ejected with 10 ml of Dulbecco’s \nModified Eagle Medium (DMEM) and cultured \nin DMEM containing 10% fetal bovine serum (FBS) and \n1% penicillin/streptomycin (all from Gibco, Germany), \nincubated at 37.C, 95% humidity and 5% CO 2 . After 48 \nhours, non-adherent cells were removed by replacing the \nmedium. The cells were sub-cultured four times ( 17 ,  18 ).\nTo analyze expression of the stem cell surface markers, \nat least 100,000 cells were incubated with fluorescence-\nlabeled monoclonal antibodies against CD29, CD34, \nCD44, CD45 and CD90 (Sigma, China). Following a 10 \nminutes wash in phosphate-buffered saline (PBS, Sigma, \nUSA), the labeled cells were analyzed using a Becton \nDikinson FACS Calibur Flow Cytometer (BD, USA) ( 7 ).\nTo destroy the ovaries, a model of chemotherapy was \ncreated. Cyclophosphamide (Sigma, China) diluted in \nnormal saline was intraperitoneally (IP) injected at 50 mg/\nkg at the first day, followed by 13 days injection of 8 mg/\nkg daily cyclophosphamide ( 19 ).\nAfter creating the chemotherapy model, the rats were \nrandomly divided into four groups (n=10 in each group): \ni. Control group, 25 .l of culture medium was directly \ninjected into the bilateral ovaries, ii. BMSC group, 2×10 6 \nBMSCs suspended in 25 .l culture medium were directly \ninjected into the bilateral ovaries ( 20 ), iii. Lc group, 200 \nmg/kg of Lc was injected IP, one day before beginning \nchemotherapy, until 7 days after chemotherapy ( 11 ), and \niv. BMSC+Lc co-administrated group, combined BMSCs \nand Lc was injected.\nTo track the transplanted BMSCs after four weeks in the \novaries, the cells were labeled with DiI (1,1’-dioctadecyl-\n 3 , 3 ,3’,3’-tetramethyl indocarbocyanine perchlorate) (Sigma, \nChina). Briefly, BMSCs were suspended in DMEM \nand 5 .l/ml DiI was added. After incubation for 20 minutes, \nthe cells were centrifuged and washed with PBS, \nand then suspended again for transplantation. Four weeks \nafter transplantation, prepared paraffin sections and the \nlabeled cells were detected by fluorescence microscope \n(Motic, Spain) ( 21 ).\nFour weeks after the end of chemotherapy, serum estradiol \n(E2) and follicle-stimulating hormone (FSH) levels \nof these groups were measured by enzyme-linked immunosorbent \nassay (ELISA) kits (East Bio-Pharm, China) \nfor rat, according to the manufacturer’s instruction ( 22 ).\nFour weeks after the end of chemotherapy, the ovaries \nwere collected and fixed in 4% paraformaldehyde, dehydrated, \nparaffin-embedded and serially sectioned at 5 .m \nthickness. Five representative sections from each ovary \nwere randomly chosen and routine hematoxylin and eosin \n(H&E) staining was performed for histological examination \nwith light microscopy. the number of primordial, primary, \nsecondary and antral follicles were measured ( 1 ).\nFive ovaries in each group were lysed using RIPA buffer \n(Cell Signaling Technology, Netherlands) supplemented \nwith protease inhibitor (Roche, Switzerland) on ice for \n30 minutes. Then, the mixture was centrifuged at 13000 \nrpm for 20 minutes at 4°C. Equal value of proteins (80 \n.g) were loaded on sodium dodecyl sulfate (SDS, Sigma, \nJapan) polyacrylamide gel (Merck, Germany) and separated \nin a size manner by electrophoresis. The proteins \nwere transferred to nitrocellulose membranes (Amersham \nBiosciences, USA). The membranes were blocked with \n5% skim milk in tris buffered saline (TBS, pH=7.4). The \nmembranes were incubated with primary antibodies for \nBcl-2 (1:1000), Bax (1:1000) and .-Actin (1:1000, Abcam, \nUSA) overnight at 4°C. After washing, the membranes \nwere incubated with goat anti-rabbit secondary \nantibody conjugated with horseradish peroxidase (HRP). \nAll antibodies were diluted according to manufacturer’s \ninstructions. Immunoreactive bands were visualized using \nan enhanced chemiluminescence detection system (Amersham \nBiosciences, USA). X-ray films were scanned, \nand then the relative protein levels were semi-quantified \nby densitometric analysis using image j software. .-actin \nwas tested as the internal control ( 23 ).\nAfter verifying the normality of variance assumptions, \ndata were analyzed by one-way analysis of variance\n(ANOVA) followed by the Tukey Test. Obtained data are \npresented as the mean ± SE, and a level of P<0.05 was \nconsidered statistically significant.\n\nBMSCs were cultured in the T25 flasks. After a few \ndays, the cells appeared to be spindle-shaped. By repeating \npassages, the cells became morphologically homogeneous. \nMost of the cells expressed the mesenchymal stromal \ncell markers (CD29, CD44 and CD90) and did not \nexpress the hematopoietic cell markers: CD34 and CD45 \n( Fig .1 ).\nIsolation and identification of bone marrow stromal cells (BMSCs). \n A.  Cultured BMSCs at passages 4 and  B.  The results of flow cytometry \nshow that BMSCs are positive for CD29, CD44 and CD90, while it is negative \nfor CD34 and CD45 (scale bar: 50 .m).\nThe transplanted BMSCs were labeled with dii, as red \nspots in the sections of ovaries ( Fig .2 ). The results confirmed \npresence of the transplanted cells in the ovaries \nfour weeks after transplantation.\nDiI labeled bone marrow stromal cells (BMSCs) in a section of ovary. \n A.  The labeled BMSCs are visible as red spots and  B.  In the same section, \nthe labeled BMSCs are not visible with green fluorescence (scale bars: 100 \n.m). Arrows show the labeled cells.\nHormonal examination was performed, by determining \nlevels of serum E2 and FSH, four weeks after treatment. \nThe results showed that levels of serum E2 in the \nBMSC+Lc co-administrated group (P<0.001), BMSC \ngroup (P<0.001) and Lc group (P<0.01) were significantly \nhigher than the control group. The results of BMSC+Lc \ngroup were significantly higher than BMSC group \n(P<0.05) and Lc group (P<0.001). The results of BMSC \ngroup were significantly higher than Lc group (P<0.001, \n Table 1 ,  Fig .3A ).\nThe levels of serum FSH in the BMSC+Lc co-administrated\ngroup (P<0.001), BMSC group (P<0.001) and\nLc group (P<0.01) were significantly lower than the control \ngroup. The results of BMSC+Lc group were significantly \nlower than BMSC group (P<0.05) and Lc group \n(P<0.001). The results of BMSC group were significantly \nlower than Lc group (P<0.01,  Table 1 ,  Fig .3B ).\nThe levels of serum estradiol (E2) and follicle-stimulating hormone (FSH) in the experimental\ngroups four weeks after treatment.  A.  The results of serum E2 level\nand  B.  The results of serum FSH level. **; P<0.01, ***;\nP<0.001 versus control group, and BMSC; Bone marrow stromal cells.\nH&E staining demonstrated that the number of all \nfollicles in different stages was significantly higher in \nBMSC+Lc group compared to BMSC (P<0.01), Lc \n(P<0.001) and control groups (P<0.001). Findings showed \nthat the number of all follicles in BMSC group was significantly \nmore than Lc group (P<0.05,  Table 1 ,  Fig .4 ).\nThe number of follicles four weeks after treatment. H&E staining of ovaries in\n A.  Control,  B.  BMSCs,  C.  L-carnitine,\n D.  Co-administration of BMSC+Lc groups, and E. The number of follicles\nat different stages (scale bars: 200 .m). **; P<0.01, ***; P<0.001\nversus control group, and BMSC; Bone marrow stromal cells.\nResults of the hormonal, histological and expression of ovarian Bcl-2 and Bax proteins four weeks after treatment\nData are presented as mean ± SE. E2; Estradiol, FSH; Follicle-stimulating hormone, *; P<0.05, **; P<0.01, and ***; P<0.001 versus control group.\nExpression of ovarian Bcl-2 and Bax proteins was \ndetermined by Western blot. The results showed that \nBcl-2 expression in the co-administration of BMSC+Lc \n(P<0.001), BMSC (P<0.001) and Lc groups (P<0.05) \nwere significantly higher than the control group; while \nit was significantly higher than BMSC (P<0.05) and Lc \ngroups (P<0.01) in BMSC+Lc. In addition, it was significantly \nhigher in the BMSC, compared to Lc group \n(P<0.05). Bax expression in the BMSC+Lc co-administered \ngroup (P<0.001), BMSC group (P<0.001) and Lc \ngroup (P<0.001) were significantly lower than the control. \nIt was significantly lower in the BMSC+Lc compared to \nBMSC (P<0.01) and Lc groups (P<0.001). Additionally, \nit was significantly lower than Lc group, in the BMSC \ngroup (P<0.05). The Bcl-2/Bax ratio was significantly \nincreased in BMSC+Lc co-administered group, in comparison \nwith the control group (P<0.001), BMSC group \n(P<0.001) and Lc group (P<0.001,  Table 1 ,  Fig .5 ).\nAnalysis of Bcl-2 and Bax protein expressions by western blot assay four weeks after treatment.\n A.  The expression of ovarian Bcl-2 protein,  B.  The\nexpression of ovarian Bax protein,  C.  Immunoblot of Bcl-2, Bax and\n.-Actin proteins, and  D.  Bcl-2/Bax ratio in all groups. *; P<0.05,\n**; P<0.01, ***; P<0.001 versus control group.\n\nChemotherapy may damage the ovaries of girls and \nwomen, however, there are some ways to prevent from \nhappening this. In this study, for the first time, we evaluated \nthe effect of co-administration of BMSC+Lc on damaged \novaries after creating a chemotherapy model with \ncyclophosphamide in rat. Overall, the results showed that \nlevels of serum E2 and FSH, number of follicles in different \nstages and expression of Bcl-2 and Bax proteins \nin BMSC+Lc co-administrated group were significantly \nmore favorable than the control, BMSC and Lc groups.\nSome studies have shown that BMSC and Lc may individually \nimprove damaged ovaries ( 7 ,  8 ,  11 ). However, \nthe effect of BMSC+Lc co-administration has never been \napplied for the same purpose. Comparing the effect of \nBMSC+Lc co-administration with either of them alone \nmay introduce a novel clinical approach to the recovery \nof damaged ovaries by chemotherapy.\nBMSCs, as a mesenchymal stem cell type, are a suitable \ncandidate for cell therapy in damaged ovaries. Liu et al. \n( 24 ) have reported that mesenchymal stem cells improve \ntissue repair chiefly via differentiation and paracrine effects. \nSeveral studies have shown that BMSCs produce \nsome growth factors preventing cell apoptosis and repair \nthe ovaries. Some of these growth factors include vascular \nendothelial growth factor (VEGF), insulin-like growth \nfactor 1 (IGF-1), hepatocyte growth factor (HGF) and basic \nfibroblast growth factor (bFGF) ( 7 ,  8 ). VEGF is an \nangiogenic factor promoting formation of new capillary \nnetworks which provides nutrition for GCS ( 7 ,  8 ,  25 ). \nIGF-1 stimulates GC proliferation by regulating DNA \nreplication of granulosa and theca cells. IGF-1 increases \nthe function of gonadotropin hormones. Moreover, IGF-1 \nregulates aromatase activity, promotes follicular antrum \nformation and suppresses apoptosis in ovaries ( 7 ,  8 ). HGF \npromotes follicular maturation and inhibits apoptosis in \novarian follicles and GCS ( 7 ). Finally, bFGF works as \na starter of folliculogenesis by inducing primordial follicle \ndevelopment ( 25 ). In this regard, Badawy et al. ( 26 ) \nshowed that BMSCs could repair mouse ovarian insufficiency \nfollowing cyclophosphamide induction, and Fu \net al. ( 27 ) showed that overexpression of miR-21 in mesenchymal \nstem cells improved ovarian structure and function \nin rats with chemotherapy-induced ovarian damage. \nThe results of our study are in agreement with these reports.\nOn the other hand, Lc as an antioxidant may also improve \ndamaged ovaries. Zhang et al. ( 11 ) showed that Lc \ninhibits follicle apoptosis and increases the function of \nfrozen-thawed ovaries in mice. However, the effect of Lc \nhas not been assessed on rat ovaries damaged by a chemotherapy \nagent, cyclophosphamide. Some studies have \nshown that Lc has protective effects on other organs. For \nexample, Aktoz et al. ( 28 ) showed that Lc has protective \neffects against testicular toxicity in rat, Mescka et al. ( 29 ) \nshowed that Lc prevents oxidative stress in the brain of \nrats and Tousson et al. ( 30 ) showed that Lc has protective \neffects on rat cardiac injury.\nLc plays an important role in fatty acid transport and \nlipid catabolism of mitochondria. Lc produces ATP by increasing \n.-oxidation of fatty acid. Hence, it can provide \nenergy for follicular growth. Lc may also suppress apoptosis \nby increasing .-oxidation of fatty acids and reduce \nfatty acid toxicity. Moreover, accumulation of reactive \noxygen species (ROS) in follicles leads to evacuation of \nthe ATP reservoir, which decreases follicle quality. Lc, \nas a ROS scavenger and an energy generation facilitator, \ncan be responsible for useful effects on follicular survival \nand ovarian function ( 11 ,  12 ,  31 ). In relation to this issue, \nGiorgi et al. ( 32 ) showed that Lc prevents miotic oocyte \ndamage induced by follicular fluid from infertile women \nwith mild endometriosis and Xu et al. ( 33 ) showed that Lc, \nduring in vitro maturation of buffalo oocytes, improves \noocyte quality. The results of our study are in agreement \nwith these reports.\nIn addition, several studies have shown that Lc has favorable \neffects on mesenchymal stem cells. Fujisawa et \nal. showed that Lc suppresses apoptosis in BMSCs, due \nto restoration of mitochondrial activity and suppression of \nsenescence induction by blocking TGF-., suggesting that \nLc is involved in mitochondrial activation even in senescent \ncells ( 14 ). Lu et al. ( 15 ) showed that carnitine could \naffect differentiation rate of adult stem cells by regulating \nmitochondrial metabolism, and it may enhance tissue development. \nFarahzadi et al. ( 16 ) showed that Lc improves \nthe lifespan of aged adipose tissue-derived human mesenchymal \nstem cells by overexpressing telomerase and \nlengthening telomeres.\nConsidering the beneficial effects of Lc on mesenchymal \nstem cells, in the present study, the combined effects \nof Lc and BMSCs were evaluated on the recovery of ovaries \ndamaged by chemotherapy agent. We cultured BMSCs \nand transplanted them into the rat ovaries after creating \nthe chemotherapy model. BMSCs expressed CD29, \nCD44 and CD90, but not CD34 and CD45. That was in \nagreement with other study ( 7 ). We labeled BMSCs with \ndii and transplanted them into the ovaries. It was shown \nthat transplanted BMSCs could be present in the ovaries \nafter four weeks. These results are in agreement with \nother report ( 21 ). To evaluate the ovarian function, levels \nof serum E2 and FSH were assessed by ELISA kit. To \nevaluate the ovarian structure, number of follicles at different \nstages was counted by HandE staining. Moreover, \nto evaluate apoptosis in the ovaries, expression of Bcl-2 \nand Bax proteins was measured by western blot, since the \nprotein products of Bcl-2 and Bax genes are respectively \ndescribed as anti-apoptotic and pro-apoptotic factors ( 23 ). \nFindings obtained from these evaluations showed that the \nresults of BMSC and Lc groups were significantly more \nfavorable than the control group. These results are in \nagreement with the other studies ( 8 ,  11 ,  23 ).\nIndeed, the results of hormonal, histological and expression \nof Bcl-2 and Bax proteins were in the same direction \nand confirmed each other. So that, these results \nin BMSC+Lc co-administrated group were significantly \nmore favorable than BMSC, Lc and control groups. The \nreasons are probably due to the combination of useful \nproperties of BMSCs and Lc with different mechanisms \nof action in the restoration of ovaries after chemotherapy. \nIn addition, considering that Lc has favorable effects on \ndifferentiation, increasing lifespan and decreasing apoptosis \nin BMSCs, it may increase survival of the transplanted \nBMSCs in the ovaries.\nThe results of BMSC group were significantly more \nfavorable than Lc group. In the present study, considering \nthat BMSCs were injected into the ovaries, these cells \nmight produce some growth factors or might replace damaged \ncells in the ovaries ( 7 - 9 ). In this regard, Liu et al. \n( 24 ) compared local and systemic administration of mesenchymal \nstem cells and reported that local administration \nof stem cells is the most efficient route for cell homing \nand immediate generation. So, probably due to these \nreasons, the recovery of damaged ovaries after chemotherapy \nwith in situ transplantation of BMSCs were more \nfavorable than intraperitoneal injection of Lc.\nThis study has some limitations which should be considered. \nThe number of samples was small, so a larger \nsample size is required. Additionally, more research is \nnecessary to clarify the molecular mechanisms underlying \nthe function of BMSC and Lc to repair damaged \novary after chemotherapy.\n\nThe results of this study suggest that the effect of \nBMSC+Lc co-administration is probably more effective \nthan the effect of their administrations individually on \nthe recovery of ovaries damaged by cyclophosphamide \nchemotherapy agent in rat.","source_license":"CC-BY-4.0","license_restricted":false}