Abstract:

Objective: To investigate the ovarian damage and mechanism of human umbilical cord mesenchymal stem cells (hUC-MSCs) to improve the ovarian injury in mice with premature ovarian insufficiency (POI). Methods: A mouse model of POI was established by plantar injection of zona pellucida 3 peptide (pZP3), and the mice were divided into control group, adjuvant control group, pZP3 group and hUC-MSCs group, with 10 mice in each group. A vaginal smear was used to monitor the estrous cycle, enzyme-linked immunosorbent assay (ELISA) was used to detect serum follicle-stimulating hormone (FSH) and estradiol (E₂) levels, HE staining was used to observe the histology of the ovary, Western blotting was used to detect the expression levels of FOXO3a, p-FOXO3a, p53, Caspase-3 and Bax, and qRT-PCR was used to detect the markers of POI such as bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH), WNT, and follicle-stimulating hormone receptor (FSHR). qRT-PCR detected the expression levels of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6), and IL-1β, and RNA-seq detected the expression level of growth differentiation factor-15(GDF-15). The in vitro model was established by cyclophosphamide(Cy). Results: ①After 6 weeks of administration, compared to the control group, the pZP3 group and hUC-MSCs group showed disturbances in the estrous cycle, and the serum FSH level was increased and the E₂ level was decreased, indicating that the model construction was successful.②Compared with the control group, atretic follicles increased and primordial follicle number decreased in the pZP3 group, and the proportion of primordial follicles was significantly increased and the number of primordial follicles was increased after hUC-MSCs intervention(P<0.05). ③Compared with the control group, the expression levels of apoptotic proteins p53 and Bax were up-regulated in the ovaries of mice in the pZP3 group, and the expression of apoptotic proteins p53 and Bax were down-regulated after the intervention of hUC-MSCs (both P<0.05); the changes of Caspase-3 in the mice in the pZP3 group were not significantly different from that of the control group (P>0.05). pZP3 group inflammatory factors IL-1β and TNF-α expression were up-regulated, and their expression was significantly down-regulated after hUC-MSCs intervention (both P<0.05). ④The number of Treg cells in mice in the pZP3 group was significantly lower (P<0.01) and significantly higher after hUC-MSCs intervention (P<0.000 1). mRNA expression levels of BMP15, AMH, WNT and FSHR in ovarian tissues of mice in the pZP3 group were lower than those in the control group. Their expression levels were elevated compared with those in the pZP3 group after the intervention of hUC-MSCs (all P<0.05). ⑤The phosphorylation level of FOXO3a was increased in the pZP3 group compared with the control group (P<0.000 1) and decreased after hUC-MSCs intervention (P<0.000 1). The sequencing results suggested that GDF-15 expression was up-regulated in POI mice and down-regulated in the hUC-MSCs group. Conclusions: The expression of GDF-15 and p-FOXO3a was up-regulated in POI mice, hUC-MSCs improved the morphology of ovarian tissues in POI mice and achieved the therapeutic effect on POI mice by down-regulating the expression of GDF-15 and reducing the phosphorylation level of FOXO3a.

Key words: Human umbilical cord mesenchymal stem cell, Primary ovarian insufficiency, Growth differentiation factor 15, FOXO3, Ovarian reserve