The Recovery Role And Mechanisms Of The Ovarian Function In Premature Ovanan Insufficiency Mice With Umbilical-Derived Mesenchymal Stem Cells Transplantation

1.The role and mechanisms of heme oxygenase 1 in restoring the ovarian function of premature ovarian insufficiency mice with umbilical cord-derived mesenchymal stem cells transplantationObjective:To investigate the protective properties and mechanisms of HO-1 in restoring the ovarian function of POI mice with UCMSCs transplantation.Methods:1)Human UCMSCs and mice ovarian granulosa cells(GCs)were separately isolated,extracted,and cultured in in vitro experiments,and the HO-1/shHO-1 plasmids were transfected into UCMSCs,with the transfection efficiency analyzed by detecting the HO-1 mRNA and protein levels in each group.The GCs were grouped as the GCs group,the HN2 group,the UCMSCs group,the NC-UCMSCs group,the HO-1-UCMSCs group,the shHO-1-UCMSCs group,the DMSO group,the JNK activation group(anisomycin,0-20μM),and the JNK-inhibition group(SP600125,0-20μM)with different treatments,and the proliferation rate and apoptosis rate of GCs in different groups of mice were detected at 12 h,24 h,48 h,and 72 h,respectively.In in vivo experiments,the chemotherapy-induced POI mice model was built up,and the mice were grouped as the control group,the POI group,the UCMSCs group,the NC group,the HO-1 group,the shHO-1 group,the DMSO group,the SP600125 group、the anisomycin group with different treatments.The mice’s ovarian function was assessed by detecting the serum levels of hormone and observing the ovarian morphological changes.To investigate the involved molecular mechanisms,the JNK/Bcl-2 signal pathway-and the autophagy-related cytokines in mice ovaries were performed.And the intracellular autophagosome accumulation was observed by TEM.Additionally,the spleen levels of CD8+CD28-T cells and serum levels of interleukin 10(IL-10)were tested to evaluate the immune mechanisms involved.Results:In in vitro experiments,the GCs’ proliferation rates in mice of the shHO-1-UCMSCs group were significantly lower and the apoptosis rates were significantly higher in a time-dependent manner comparing with the LV-NC group.In in vivo experiments,results showed that the mice ovarian function in the shHO-1-UCMSCs group still unrecovered,which presented as the increased extent of ovarian fibrosis with decreased number of functional follicles,increased number of atretic follicles,and the disordered hormone production.Additionally,the JNK/Bcl-2 signaling pathway was inhibited in the ovaries,the expression of autophagy-related factors was significantly suppressed,and intracellular chromatin agglutination with autophagic vesicle fragmentation was observed by TEM.Additionally,the down-regulated levels of the CD8+CD28-T cells and the decreased serum levels of IL-10 were detected.On the contrary,the increased levels of GCs’ viability and the decreased levels of the GCs’ apoptosis in the HO-1-UCMSCs group in in vitro experiments.In in vivo experiments,the degree of ovarian fibrosis can be reduced,with the increased number of the functional follicles and the decreased number of the atretic follicles,and the reordered serum hormone levels.The JNK/Bcl-2 signal pathway can be activated,the expression of autophagy-related factors was increased,the intracellular autophagic vesicles were morphologically intact,lamellar structures were accumulated,and a large number of autophagic vacuoles were formed.At last,the spleen levels of the CD8+CD28-T cells were increased,along with the increased serum levels of IL-10.The tendency changes in mice with the JNK inhibitor treatment were similar to the shHO-1-UCMSCs group,and in mice with the JNK activator administration behaved similarly to that in the HO-1-UCMSCs group.Conclusions:HO-1 played a critical role in restoring the ovarian function in POI mice with UCMSCs transplantation,which may mediated by the activation of JNK/Bcl-2 signal pathway-regulated autophagy and up-regulating the circulating of CD8+CD28-T cells.2.The role and mechanisms of microRNA 21 expressed in umbilical cord-derived mesenchymal stem cells transplantation in restoring the ovarian function of premature ovarian insufficiency miceObjective:To explore whether and how the miR-21 played its role in the ovarian function recovery of POI mice with UCMSCs transplantation.Methods:After isolation,culture,and identification of UCMSCs,the optimal transfection conditions corresponding to LV-hsa-miR-21-5p/LV-hsa-miR-21-5p-inhibition/LV-NC were determined by pre-experiment,and the above lentiviruses were transfected into UCMSCs respectively,and the transfection efficiency was detected by QRT-PCT.The autoimmune-induced POI mouse model was constructed by the zona pellucida peptide fragment,and the mice were grouped with different treatment as the control group,the POI group,the UCMSCs group,the LV-NC-UCMSCs group,the LV-hsa-miR-21-UCMSCs group,and the LV-hsa-miR-21-inhibition-UCMSCs group.To evaluate the role of the miR-21 in the therapeutic process,the expression of pri-miR-21 and miR-21 in mice ovaries was first detected.To evaluate the changes in ovarian function in different groups of mice,the expression levels of serum hormone and the AZPAb were analyzed,the ovarian morphological changes were observed and the folliculogenesis was observed,with the apoptosis of cells in the ovaries were analyzed to confirm the successful construction of the autoimmune POI model,and to evaluated the changes of ovarian function.To confirm the underlying mechanisms,the mRNA and protein levels of PDCD4 and PTEN/AKT/FOXO3a signal pathway was analyzed.And the immunohistochemistry observation was used to further localize the expression changes of the PTEN/AKT/FOXO3a signal pathway-related cytokines in mice ovaries.Results:Autoimmune-induced POI mice model was successfully constructed,and UCMSCs partially repair the ovarian function in POI mice.In mice with the LV-hsa-miR-21-5p-inhibition-UCMSCs transplantation,the expression levels of pri-miR21 and miR-21 were significantly down-regulated,and it showed less therapeutic efficiency than mice with LV-NC-UCMSCs transplantation,presented as the enhanced extent of the ovarian fibrosis with the decreased number of functional follicles and increased number of atretic follicles.It presented as the increased levels of AZPAb and the disordered hormone levels,showed as the down-regulated levels of E2 and AMH,and the up-regulated levels of FSH and LH.Additionally,the apoptosis of the ovarian cells was detected increased.For the aspect of molecular mechanisms,the inclined levels of PDCD4 were analyzed,with the activation of the PTEN/AKT/FOXO3a signal pathway.In the contrast,with the enhancement of the miR-21 in mice ovaries with the LV-hsa-miR-21-5p-UCMSCs transplantation,the pri-miR-21 and miR-21 were significantly up-regulated in mice ovaries,and the injured ovarian function can be reversed,which presented as the alleviated levels of the ovarian fibrosis with the increased number of functional follicles and decreased the number of atretic follicles.The serum levels of the AZPAb were decreased,and the hormone levels were reordered.And the apoptosis of the ovarian cells was decreased.Besides,the suppressed expression of the PDCD4 and the inhibited activation of the PTEN/AKT/FOXO3a signal pathway were detected.Conclusions:MiR-21 played a critical role in restoring the ovarian function of POI mice with UCMSCs transplantation,which may be closely associated with the miR-21 expressed in UCMSCs,and the mechanisms may through inhibiting the PDCD4 expression and suppressing the PTEN/AKT/FOXO3a signal pathway in mice ovaries.