| BackgroundPremature ovarian failure(POF)refers to ovarian dysfunction caused by follicular dysfunction or follicle depletion before the age of 40,with manifestations such as amenorrhea,estrogen deficiency,and genital atrophy.In recent years,the incidence of POF has been increasing,which has seriously affected female fertility.Because of its unknown etiology,the main clinical methods are hormone replacement.There are currently no treatments that can effectively improve the patient's ovarian function and produce genetic offspring.Stem cell research has opened up a new approach for the treatment of POF.Studies have shown that mesenchymal stem cells(MSCs)can effectively improve ovarian function in POF animal models and some patients,but the specific mechanism of treatment is still unclear.Profound exploration of the relevant mechanism of MSCs in the treatment of POF will lay a theoretical foundation for its clinical treatment.Granular cells are important functional cells in ovulation and gonadotropin secretion.Granular cell proliferation and maturation play an important role in supporting and regulating follicle formation and development,and its apoptosis is also an important mechanism that affects ovarian reserve and function decline.Many studies have shown that the tissue and cell levels of the POF model have granulocyte apoptosis,and MSCs can promote granulocyte proliferation and reduce apoptosis after treatment.Therefore,granulosa cells may be targeted cells that MSCs improve POF ovarian activity and function.Paracrine is the main mechanism of MSCs involved in POF ovarian repair and functional improvement.As an important part of the paracrine mechanism,exosomes participate in cell-to-cell communication and signal transduction by carrying and transmitting important signal molecules.Studies have shown that exosomes are a nano-scale vesicular lipid bilayer membrane structure that can be secreted by a variety of cells,and that active substances such as proteins,liposomes,and RNAs participate in a variety of biological behaviors.Recent studies have shown that exosomes of MSCs can improve ovarian function by inhibiting apoptosis of granulosa cells by releasing microRNA(miRNA).For example,amniotic fluid MSCs exosomes can inhibit ovarian granulosa cell apoptosis,preventing follicular atresia,and restoring impaired ovarian function by down-regulate the expression of apoptotic proteins through miR10a.Therefore miRNAs carried by exosomes play a key role in MSCs-mediated tissue functional repair.Granular cell apoptosis is regulated by multiple signaling pathways.As a common tumor suppressor gene,p53 plays an important role in cell cycle arrest and cell aging.Previous studies have shown that p53 is also a key gene that regulates apoptosis of granulosa cells,and promotes the expression of apoptosis-related proteins by regulating pro-apoptotic and anti-apoptotic regulators.However,the role of p53 in MSCs in treating granular cell apoptosis has not been fully elucidated.In summary,MSCs exosomes are a new hope for POF therapy,and the related mechanisms that act on and regulate granulosa cells to improve ovarian function are still unclear.In this study,a mouse POF model was used to explore the molecular mechanism of MSCs exosomes for POF in order to provide new theoretical basis and ideas for the clinical treatment of POF with stem cells.ObjectiveIn this study,we aim to investigate the therapeutic effects of BMSCs-derived exosomes on POF mice,focusing on the regulation of miRNAs carried by exosomes on granulosa cell apoptosis and its related mechanism.Methods1)Isolate BMSCs from the femur and tibia of mouse by density gradient centrifugation.BMSCs were identified by cell differentiation potential and surface markers.2)BMSCs-derived exosomes were isolated by polymer precipitation and identified based on the morphological characteristics observed by TEM and the detection of exosomes surface markers by.WB.3)The control group(n=5)was intraperitoneally injected with normal saline.POF group(module group,n=5)was intraperitoneally injected with 5 mg/kg cisplatin,and 100 ?l PBS was injected into the tail vein on the 1st,5th,and 10th day after modeling.Serum E2 levels were detected by ELISA.HE staining was used to observe changes of ovarian tissue.4)The POF+BMSCs exosome group(treatment group,n=5)was intraperitoneally injected with 5 mg/kg cisplatin,and then exosomes(125 ?g dissolved in 100 ?l PBS)were injected into the tail vein on the 1st,5th,and 10th day after modeling.Fifteen days later,the mice were sacrificed and blood from eyeball and ovarian tissues were taken for the subsequent experiments.Histological changes of ovary were observed by HE staining.IHC was used to detect the expression of c-caspase 3.WB was used to detect p53 protein expression level;ELISA measures the concentration of E2 in serum.5)After isolation and culture of mouse primary granulosa cells,they were divided into five groups according to different treatment methods:sequential,cisplatin treatment group,cisplatin+exosomes group,cisplatin+exosome+RNase group.And cisplatin+3T3-,exosome group.The cisplatin and exosome concentration groups were added 10 ?g/ml,after 48 hours of co-culture,the granulosa cell activity was detected by MTT assay,the granulosa cell viability was detected by flow cytometry,and the expression of the related protein was detected by WB assay.6)Using bioinformatics to predict the miRNA of p53,and to screen and verify the expression level,select the target miRNA.The substitution was verified by DLR assay.The target miRNA in the granulosa cells is overexpressed,and the inhibitory effect is detected by WB.7)Isolate exosomes from BMSCs after miR-664-5p knockout and observed its effect on granulosa cells apoptosis.Results1)Microscopic observation showed that BMSCs formed calcium nodules after osteogenic differentiation induction,and the radioactive center was orange-red after staining with alizarin red.After BMSCs were induced to differentiate into adipogenesis,fine lipid droplets appeared in the cells.Flow cytometry results showed that BMSCs were immune-positive for markers of mesenchymal stromal stem cells namely CD90,and immune-negative for hematopoietic markers namely CD34.2)By transmission electron microscopy,membranous vesicles of uniform size,round or oval shape,with clear margins and double lipid membranes surrounding them can be seen.WB results showed that the expression of exosome surface marker protein CD63 was significantly higher than BMSCs lysate.3)HE staining ovarian tissues experiment showed that the mice had large and abundant follicles,abundant follicular fluid and multiple corpus luteum in the control group.The follicles in the POF group were few and mostly primitive or initial follicles,and the atresia follicles formed by granule cell damage increased,and the interstitial increased.4)Compared with the POF group,the atresia follicles in the POF+exosome group decreased,while the corpus luteum increased.IHC detection of cleaved-caspase 3 was performed on the ovarian tissues.Compared with the control group,the expression of c-caspase3 was significantly increased in the POF group,while this increase of c-caspase3 was inhibited after exosome injection.WB showed that the expression level of P53 in the POF group increased compared with the control group,while the expression of P53 was markedly lower in the POF+exosome group than that in the POF group.It was found that the concentration of E2 in the POF group decreased compared with the control group,and the concentration of E2 in the POF+ exosome group was increased relative to the POF group.5)Compared with the control group,the proportion of early apoptotic cells was increased in cisplatin group,while this increase was reversed by BMSCs-derived exosomes.When BMSCs-derived exosomes were treated with RNase,the proportion of early apoptotic cells was increased compared with cisplatin+BMSCs-exosome group.The same therapeutic result was reached in the cell viability assay.WB showed that compared with the cisplatin group,the expression of P53 protein and c-caspase3 protein was down-regulated and the expression of Bcl2 was up-regulated in the cisplatin+BMSCs-exosome group.When BMSCs-derived exosomes were treated with RN ase,the expression of P53 protein and c-caspase3 protein were up-regulated and the expression of Bcl2 was down-regulated compared to cisplatin+BMSCs-exosome group.6)Two databases,Target scan and microT-CDS,were used to search for the miRNA intersection predicted to target p53.We chose the top 10 miRNAs from total miRNAs to verify their expression level by using qRT-PCR.The results showed that miR-664-5p was highly expressed in BMSC-derived exosomes.DLR assay showed that miR-664-5p bound to the 3'UTR of p53,which resulted in decreased relative luciferase activity in response to miR-664-5p mimic.When miR-664-5p was overexpressed in mouse ovarian granulosa cells,cisplatin damage to granulosa cells was inhibited,and the expression of p53 protein was down-regulated.7)Results showed that the proportion of early apoptotic granulosa cells induced by cisplatin was increased.Knockout of miR-664-5p in BMSC reversed the inhibition of p53 and the promotion of Bcl2 expression in granulosa cells by BMSC-derived exosomes.ConclusionThis study confirmed that BMSC-derived exosomes played a key role in restoring ovarian function in a cisplatin-induced POF mouse model,mainly by delivering miR-664-5p to granulosa cells to regulate p53 expression of cells and thereby inhibited ovarian granules apoptosis.Our current study clarified the potential molecular mechanisms of MSC-derived exosomes-mediated ovarian function recovery and provided new strategies and directions for the treatment of POF. |
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