Melatonin enhances autologous adipose-derived stem cells to improve mouse ovarian function in relation to the SIRT6/NF-κB pathway

Abstract

Background: Premature ovarian insufficiency (POI) is the main cause of female infertility. Adipose-derived stem cells (ADSCs) are ideal candidates for the treatment of POI. However, some deficient biological characteristics of ADSCs limit their utility. This study investigated whether melatonin (MLT)-pretreated autologous ADSCs were superior to ADSCs alone in the treatment of the POI mouse model.

Methods: Autologous ADSCs were isolated and cultured in MLT-containing medium. Surface markers of ADSCs were detected by flow cytometry. To determine the effect of MLT on ADSCs, CCK-8 assay was used to detect ADSCs proliferation and enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokines. The POI model was established by intraperitoneal injection of cyclophosphamide and busulfan. Then, MLT-pretreated autologous ADSCs were transplanted into mice by intraovarian injection. After 7 days of treatment, ovarian morphology, follicle counts, and sex hormones levels were evaluated by hematoxylin and eosin (H&E) staining and ELISA, and the recovery of fertility was also observed. The expressions of SIRT6 and NF-κB were detected by immunohistochemical (IHC) staining and quantitative real-time polymerase chain reaction (qRT-PCR).

Results: Flow cytometry showed that autologous ADSCs expressed CD90 (99.7%) and CD29 (97.5%). MLT can not only promote the proliferation of ADSCs but also boost their secretory function, especially when ADSCs were pretreated with 5 µM MLT for 3 days, improving the interference effect. After transplantation of autologous ADSCs pretreated with 5 µM MLT, the serum hormone levels and reproductive function were significantly recovered, and the mean counts of primordial follicle increased. At the same time, the expression of SIRT6 was remarkably increased and the expression of NF-κB was significantly decreased in this group.

Conclusions: MLT enhances several effects of ADSCs in restoring hormone levels, mean primordial follicle counts, and reproductive capacity in POI mice. Meanwhile, our results suggest that the SIRT6/NF-κB signal pathway may be the potential therapeutic mechanism for ADSCs to treat POI.

Keywords: Autologous adipose-derived stem cells; Melatonin; Premature ovarian insufficiency; SIRT6/NF-κB pathway.

Fig. 1

The estrous cycle of mice. Regular estrous cycles include proestrus, oestrus, metoestrus, and dioestrus. Scale bar: 50 or 10 μm

Fig. 2

The experimental design. ADSCs, adipose-derived stem cells; POI, premature ovarian insufficiency; MLT, melatonin; E2, estradiol; FSH, follicle-stimulating hormone; AMH, anti-Mullerian hormone; H&E, hematoxylin and eosin; qRT-PCR, quantitative real-time polymerase chain reaction; and IHC, immunohistochemical

Fig. 3

The morphology and characterization of autologous ADSCs. a The fibroblastoid shape of ADSCs from passage 0 (P0) to P3 (scale bar: 100 μm). b Flow cytometry analysis of cells surface markers in ADSCs. CD 90: 99.7%; CD 29: 97.5%; CD 45: 0.68%; and CD 34: 0.091%

Fig. 4

The differentiation of autologous ADSCs. a Lipid droplets secreted by differentiated adipocytes were observed under bright-field microscope. The arrow indicates lipid droplets. b–d The adipogenic, osteogenic, and chondrogenic differentiation of ADSCs was assessed by Oil Red O staining, Alizarin Red staining, and Alcian blue staining, respectively. The arrows in b-d indicate lipid droplets, calcium nodules, and mucopolysaccharides in chondrocytes, respectively. Scale bar: 50 μm

Fig. 5

Assay of the activity of ADSCs treated with MLT. a Dose-dependent and time-course tests to detect the optimal concentration of MLT for promoting ADSCs proliferation. The cell proliferation levels were determined by cck-8 assay on days 1, 3, 5, and 7. (**p < 0.01, and ***p < 0.001 vs. control). b ADSCs morphology with or without 5 μM MLT. Scale bar: 50 μm

Fig. 6

The levels of BDNF, VEGF, and HGF were measured by ELISA assay in ADSCs and 5 μM MLT-ADSCs (ns = not significant, **p < 0.01 vs. ADSCs groups). a The levels of BDNF in ADSCs and 5 μM MLT-ADSCs. b The levels of VEGF in ADSCs and 5 μM MLT-ADSCs. c The levels of HGF in ADSCs and 5 μM MLT-ADSCs. BDNF, brain-derived neurotrophic factor; VEGF, vascular endothelial growth factor; and HGF, hepatocyte growth factor

Fig. 7

Effects of ADSCs and 5 μM MLT-ADSCs transplantation in mice with POI. a Changes in serum E2 levels. b Changes in serum FSH levels. c Ovarian histological changes were analyzed using H&E staining. Scale bar: 100 μm. Yellow arrows indicate PrFs located in the ovarian cortex. d The number of follicles at different stages was counted and compared in the control, PBS, ADSCs, and 5 μM MLT-ADSCs groups. PrF, primordial follicle; PF, primary follicle; SF, secondary follicle; AF, antral follicle; E2, estradiol; and FSH, follicle-stimulating hormone. (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001)

Fig. 8

Results of the fertility experiment (*p < 0.05 and ****p < 0.0001) a Reproductive outcomes. b The cubs counts of per group. c The pregnancy rate of per group

Fig. 9

In vivo tracking after cell transplantation. a Fluorescence microscopy analysis of ovarian tissue sections. Scale bar: 100 μm. b The number of positive cells was counted and compared in the ADSCs and 5 μM MLT-ADSCs groups (*p < 0.05)

Fig. 10

In vivo tracking after cell transplantation. Fluorescence microscopy analysis of heart, lungs, and kidney sections from ADSCs and 5 μM MLT-ADSCs groups. Scale bar: 100 μm

Fig. 11

The expression levels of SIRT6, NF-κB, and BDNF in the ovaries were detected using immunohistochemical staining. a–c A brown-yellow color indicates positive expression of the target protein. The white arrow represents SIRT6 expression, the red arrow represents NF-κB expression, and the green arrow represents BDNF expression. Scale bar: 200 μm. d–f The statistical charts of the three kinds of protein expression in the four groups. SIRT6, sirtuin 6; NF-κB, nuclear factor kappa B; and BDNF, brain-derived neurotrophic factor (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001)

Fig. 12

qRT-PCR results. a Relative expression of SIRT6 mRNA in four groups. b Relative expression of NF-κB mRNA in four groups. c Relative expression of BDNF mRNA in four groups. SIRT6, sirtuin 6; NF-κB, nuclear factor kappa B; and BDNF, brain-derived neurotrophic factor (ns = not significant, *p < 0.05, **p < 0.01, ****p < 0.001)