HiMSC-Exosome Inhibited Ovarian Granulosa Cell Apoptosis Induced By Cyclophosphamide

Premature ovarian failure(POF)is the end-stage of primary ovarian insufficiency(POI),which could cause amenorrhea,hypergonadotropism and infertility in patients no more than 40 years old.Previous studies demonstrated that iatrogenic factors such as radiotherapy and chemotherapy could cause POI in young women.Therefore,there is a clinical concern about reducing the injury to the ovaries caused by radiation and chemotherapy.Clinical research have proven that the side effects of the chemotherapy drug-cyclophosphamide(CTX)promoted ovarian granulosa cell apoptosis and follicular atresia,and its active metabolite 4hydroperoxycyclophosphamide(4-OHCP)could inhibit granulosa cell proliferation and promote cell apoptosis(including oocyte death).However,hormone replacement therapy is commonly used in clinical strategy,but its efficacy is not ideal and it has some side effects.Regenerative medicine research has found that the exosomes of stem cell have the potential to protect ovarian function.Stem cell-derived exosomes could both inhibit apoptosis and reduce intracellular reactive oxygen species(ROS)levels.We wonder if exosomes derived from human induced pluripotent stem cell-mesenchymal stem cells(hiMSCs)could alleviate the phenotype of POI-like mouse model induced by CTX,and if they could inhibit granulosa cell apoptosis and their possible mechanisms.Therefore,we conducted the following study.Based on a POI-like mouse model induced by CTX,the protective effect of in situ injection of exosomes derived from hiMSC on ovarian function,particularly on granulosa cell anti-apoptosis was validated.Meanwhile,KGN cell injury model was established by 4-OHCP,to investigate the mechanism of hiMSC-Exosome in repairing cell injury model.The results are as follows:(1)hiMSCs cultured in vitro exhibited characteristics of mesenchymal stem cells.Surface markers CD73,CD90 and CD 105 are expressed positively,while non-stem cell markers CD34,CD45,CD11b,CD 14 and HLA-DR are expressed negatively.Additionally,hiMSCs were able to differentiate into either adipocytes or osteocytes in response to the differentiation ability.Based on this,exosomes derived from hiMSCs are spherical with a lipid bilayer structure,and the average diameter of hiMSC-exosomes are 124.4 nm.Additionally,hiMSC-exosomes are enriched with positive markers such as Flotillin-1,TSG101,CD9 and CD63,while negative markers such as Calnexin and GAPDH expressed negatively.These results confirmed that the hiMSC-exosomes exhibited characteristics of extracellular vesicles and could be used for subsequent experiments.(2)The POI-like mouse model was successfully established by CTX.The body weight and ovarian weight of mice in the CTX group were significantly reduced.Serum levels of estradiol(E2)and anti-Müllerian hormone(AMH)were significantly decreased,while follicle-stimulating hormone(FSH)levels were significantly increased.The number of follicles at different stages in the ovaries was significantly reduced.The estrous cycle of the mice was disrupted,and they stayed in the estrus phase.The fertility rate was significantly decreased.Based on these results,after in situ injection of hiMSC-exosomes into mice ovarian bursa for 10 days.The results showed that hiMSC-exosomes increased the ovarian weight to body weight ratio of the mice.The levels of E2 and AMH in the sera of the mice were elevated,while the level of FSH was decreased,if compared with the mice in the CTX group.The number of primordial and total follicles lost were significantly reduced.Further investigation revealed that exosomes increased expression levels of proliferation markers Ki67 and PCNA in granulosa cells,increased expression of anti-apoptotic protein Bcl-2,and decreased expression of pro-apoptotic proteins Bax and cleaved Caspase-3.Additionally,nuclear factor erythroid 2-related factor 2(NRF2)and its downstream antioxidant stress proteins SOD1 and GCLC were upregulated.These findings suggested that hiMSCexosomes could improve mice ovarian function in CTX group,possibly by regulating NRF2 to inhibit ovarian granulosa cell apoptosis and promote cell proliferation.(3)4-OHCP was selected to establish a cell injury model in co-culture with KGN cells.The results showed that 4-OHCP inhibited KGN cell proliferation,promoted cell apoptosis,and increased ROS levels.Based on this cell injury model,hiMSC-exosomes were accepted when co-cultured with KGN cells for 24 hours.Additionally,hiMSC-exosomes significantly increased the protein expression of NRF2 protein and reduced oxidative stress levels.hiMSC-exosomes reduced the number of apoptotic cells and promoted cell proliferation.These results indicated that hiMSC-exosomes could restore KGN cell apoptosis induced by 4-OHCP.Conclusion:In situ injection of hiMSC-exosomes into the ovarian bursa of mice could restore ovarian injury induced by cyclophosphamide(CTX),reduce granulosa cell apoptosis and promote proliferation.This effect may be attributed to certain components of the exosomes that promoted the expression of NRF2 protein in cells,increased the expression of antioxidant enzymes,and downregulated cellular oxidative stress levels.This study lays the foundation for a deeper understanding of the pathological mechanisms of POI in women,and provides potential targets for the treatment of POI using exosomes derived from MSCs.