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Human umbilical cord mesenchymal stem cell-derived extracellular vesicles harboring IGF-1 improve ovarian function of mice with premature ovarian insufficiency through the Nrf2/HO-1 pathway

Journal of Ovarian Research volume 17, Article number: 224 (2024) Cite this article

Abstract

Objective

Premature ovarian insufficiency (POI) is a disease with medical, psychological and reproductive implications, but its common therapies have limited efficacy and a likelihood of complications. This study delves into the therapeutic role of human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUC-MSCs-EVs) in POI mice through the insulin-like growth factor 1 (IGF-1)/nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/autophagy pathway.

Methods

hUC-MSCs were transfected with lentiviral short hairpin RNA of IGF-1 before EV extraction. Cyclophosphamide (CTX)-induced POI mouse models were administrated with hUC-MSCs-EVs. Mouse ovarian granulosa cells (GCs) were induced with CTX, then treated with hUC-MSCs-EVs and ML385. Ovarian histopathological changes were observed, changes in follicle number at all levels were counted and serum sex hormones were evaluated, as well as LC3II/I and Beclin-1 expression. GCs were subject to detection of proliferation, deaths, oxidative stress, and Nrf2 nuclear translocation.

Results

After CTX exposure, mice showed thinner GCs layer in the ovary, reduced number of GCs and follicles at all levels, disturbed serum sex hormones, enhanced oxidative stress and autophagy, and downregulated ovarian IGF-1; whereas, hUC-MSCs-EVs upregulated IGF-1 to improve the ovarian function. hUC-MSCs-EVs carrying IGF-1 activated Nrf2/HO-1 signaling to inhibit CTX-induced excessive autophagy of GCs, but this ameliorative effect was partially weakened by inhibiting Nrf2/HO-1 signaling. hUC-MSCs-EVs inhibited excessive autophagy of GCs and improved ovarian function of CTX-induced mice through IGF-1/Nrf2/HO-1 pathway.

Conclusion

hUC-MSCs-EVs activate the Nrf2/HO-1 signaling by carrying IGF-1, which in turn inhibits excessive autophagy and damage of GCs, thus improving ovarian function in POI mice.

Introduction

Premature ovarian insufficiency (POI) is a medical state in which sex hormones secreted are deficient and ovarian follicles are depleted and halt to maintain their reproductive and endocrine functions in women below 40 years old, which speeds up the onset of menopause [1]. It leads to menstrual disorder with infertility and various health problems mainly resulting from estrogen deficiency, and in the long term, patients with POI face an increased risk of cardiovascular diseases, osteoporosis, cognitive impairment and even earlier deaths [2]. Regarding to treatment, the recognized therapies for POI include in vitro activation, regenerative medicine and hormone therapy; however, the efficacy is limited and complications may occur [3, 4]. Therefore, finding a high-efficiency and safe therapy for POI is urgent.

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic stem cells that have potentials for reversing ovarian failure due to its therapeutic properties, including migration and paracrine effect [5,6,7]. Among the MSCs, human umbilical cord MSCs (hUC-MSCs) are featured by cost-efficiency, convenience and low immunogenicity, thereby being the ideal type for POI treatment [8]. Extracellular vesicles (EVs) are the active component in MSC secretome and the important carriers of DNA, RNA and proteins for intercellular communication [9, 10]. Insulin-like growth factor 1 (IGF-1) in synergy with growth hormone directly controls primordial follicles, folliculogenesis, and oocyte maturation [11]. Di-n-butyl phthalate reduced follicle number and ovarian reserve by tampering with ovarian IGF-1 signaling [12]. MSC treatment can increase the levels of anti-mullerian-hormone (AMH) and estradiol (E2) and the number of follicles, concurrent with elevated IGF-1 expression [13]. Therefore, we assume that hUC-MSC-derived EVs (hUC-MSCs-EVs) carry IGF-1 to rescue ovarian reserve in POI.

Granulosa cells (GCs) are the cell population that directly interact with oocytes and enable oocyte development and maturation within ovarian follicles [14]. Recently, oxidative stress-induced GC autophagy has been reported to result in follicular atresia and development arrest, indicating the implication of GC autophagy in POI [15, 16]. Inhibiting excessive autophagy of GCs can improve ovarian function in POI [17,18,19]. More importantly, hUC-MSCs can block excessive autophagy of GCs in mice with POI through the vascular endothelial growth factor A (VEGFA)/PI3K/AKT/mTOR signaling [20], suggesting that excessive autophagy of GCs may be a potential target o for POI treatment. Nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling is strongly linked with oxidative stress and autophagy in the ovary [21, 22]. For instance, icariin activated the Nrf2/HO-1/Sirt1 pathway to rescue ovarian structure and function of mice with autoimmune POI [23]. Despite that IGF-1 could restrain the generation of reactive oxygen species (ROS) by activating the Nrf2/HO-1 signaling [24], there is limited understanding on IGF-1/Nrf-1/HO-1 signaling-mediated GC autophagy in POI. This study mainly probed the mechanisms of hUC-MSCs-EVs inhibiting excessive autophagy of GCs and improving ovarian function in POI through IGF/Nrf2/HO-1 pathway, providing a new idea for POI treatment.

Materials and methods

Culture and identification of hUC-MSCs

hUC-MSCs were procured from Pricella (CP-CL11, Wuhan, Hubei, China). hUC-MSCs were isolated and purified using the tissue explant method, and kept in specialized complete medium. Once the density reached 80%, the cells were disassociated using 0.25% trypsin (Pricella) and cultured for passage. The third- to fifth-passage cells were collected for subsequent analysis.Thereafter, their morphology was visualized through an inverted microscope (Olympus). Surface markers of hUC-MSCs (CD29, CD44, CD90, CD43, CD14 and HLA-DR) were detected using flow cytometry. The following flow cytometry antibodies (BD Biosciences, San Jose, CA, USA) were added at a concentration of approximately 1 µg/1 × 106 cells (optimal concentration was determined by the recommended concentration in the products’ instructions and by antibody titration experiments): purified anti-human CD29 antibody (303001), purified anti-human CD44 antibody (397502), purified anti-human CD90 (Thy1) antibody (328101), purified anti-human CD43 antibody (343202), FITC anti-human CD14 (982502) and purified anti-human HLA-DR antibody (327002). Cell purity of hUC-MSCs was determined using a flow cytometer (BD Biosciences) and analyzed by FlowJo V10 (FlowJo, BD Biosciences).

hUC-MSC-EVs isolation and identification

Supernatant collected from hUC-MSCs were centrifuged for 30 min at 2000×g and added with total exosome isolation reagent (Thermo Fisher Scientific, Waltham, MA, USA). After that, the supernatant was concentrated and placed at 2–8ºC overnight before centrifugation at 10,000×g and 4ºC for 1 h. Resuspended in 100 µL phosphate buffer solution (PBS), the collected EVs were observed with a transmission electronic microscope (TEM) (Olympus, Tokyo, Japan) and their diameter distribution and concentration were analyzed through nanoparticle tracking (NTA). Surface markers (CD9, CD63, CD81 and Calnexin) were quantified through western blot The supernatant of hUC-MSCs treated for 2 h with 20 µM GW4869 (inhibitor of EV secretion, Sigma Aldrich, St. Louis, MO, USA) [25] was served as the negative control (GW group).

EVs used in this study were divided into the following groups: (1) EVs: directly extracted from hUC-MSCs; (2) EVs + PK: hUC-MSCs-EVs treated with Proteinase K (20 mg/mL, Beyotime, Shanghai, China); (3) EVs + PK + T: hUC-MSCs-EVs treated with 20 mg/mL Proteinase K and 1% Triton-X-100 (Sigma Aldrich) [26]; (4) EVs-negative control of short hairpin RNA (sh-NC): EVs extracted from hUC-MSCs that were infected with blank lentiviral vector (pLenti-III-Blank Vector) (sh-NC); (5) EVs-sh-IGF-1: EVs extracted from hUC-MSCs that were infected with IGF-1 lentiviral vector (human; cytomegalovirus, pLenti-GIII-CMV) (sh-IGF-1). sh-IGF-1 and sh-NC were from Applied Biological Materials Inc. (Richmond, BC, Canada), with a titer of 1.5 × 108 TU/mL.

Animals

Specific pathogen-free 8-week-old female C57BL/6 mice [Shanghai SLAC Laboratory Animals Co., Ltd. (China)] were reared in standard animal rooms at temperature of 22–24 °C and humidity of 40–70% with 12 h/12 h light and dark cycles and free access to feed and water. All experiments were carried out after the review and approval from the ethics committee of Changzhi Medical College and conformed to internationally recognized animal research guidelines and ethics norms [Approval No. (2021)043].

Animal groups and treatment

Experimental POI was induced in mice by intraperitoneal injection of 120 mg/kg cyclophosphamide (CTX) once a week for 2 consecutive weeks [27], and mice administrated with normal saline at the equal amount and frequency served as the controls (control group).

According to the random number table, the mice were separated into the control, POI, POI + PBS, POI + EVs, POI + EVs-sh-NC, and POI + EVs-sh-IGF-1 groups (N = 6). In addition to intraperitoneal injection of 120 mg/kg CTX at the aforementioned amount and frequency, mice in the POI + PBS and POI + EVs groups were given 0.2 mL PBS or suspension encompassing 1 × 108/mL hUC-MSCs-EVs through their tail veins once every two days for 6 consecutive weeks [28], while mice in the POI + EVs-sh-NC and POI + EVs-sh-IGF-1 groups were administrated with 0.2 mL suspension comprising 1 × 108/mL EVs-sh-NC or EVs-sh-IGF-1 once every two days for 6 consecutive weeks.

One week after completion of all interventions, the mice were anesthetized using 0.3% pentobarbital sodium (50 mg/kg) intraperitoneally, and 0.1 mL of blood was sampled from the orbital venous plexus, with the serum separated for enzyme-linked immunosorbent assay (ELISA). Subsequently, the mice were euthanized by overdosing 3% pentobarbital sodium (100 mg/kg) intraperitoneally, and ovarian tissues were immediately collected. Part of the ovary was made into tissue sections for hematoxylin and eosin (H&E) staining and immunohistochemistry, and the others were made into tissue homogenates for western blot and detection of oxidative stress-related indexes.

ELISA

Mouse serum E2 (E-OSEL-M0008), follicle-stimulating hormone (FSH, E-EL-M0511), luteinizing hormone (LH, E-EL-M3053) and AMH (E-EL-M3015) levels were determined following the instructions of ELISA kits (all from Elabscience Biotechnology; Wuhan, China) and data were acquired with a microplate reader (Bio-Rad 680, Bio-Rad, Hercules, CA, USA).

H&E staining

Ovarian tissues were routinely fixed, dehydrated, embedded, and cut into 5 μm-thick sections. Staining was performed using the H&E kit (Solarbio Science & Technology Co., Ltd, Beijing, China) according to the manufacturer’s instructions. The pathological changes of mouse ovarian tissues were observed with an Olympus optical microscope, and the follicles observed were counted. The follicles were counted as hereinafter: the continuous cross-section slices of mouse ovarian tissues were collected. Subsequently, one every 12 slices were selected, with the numbers of primordial, primary, secondary, mature and atretic follicles documented, respectively. The morphological characteristics of the follicles at all levels were as follows: primordial follicles contained a layer of flat GCs surrounding the oocyte; primary follicles contained a layer of cubic GCs surrounding the oocyte; secondary follicles contained multiple layers of cubic GCs surrounding oocytes, without the formation of follicular lumen; mature follicles had the formation of a follicular lumen; and atretic follicles had the oocyte disappeared, with the GCs arranged irregularly and a pyknosis. Five sections from each mouse were taken for counting (6 mice/group). The results were signified as mean values.

Immunohistochemistry

After conventional dewaxing, rehydration, antigen recovery, and inactivation of endogenous peroxidase, the ovarian tissue sections were incubated with antibodies against IGF-1 (ab223567, 1:100, Abcam, Cambridge, MA, USA), FSH receptor (FSHR) (a specific marker for GCs) (ab113421, 1:100, Abcam), LC3 (ab192890, 1:2000, Abcam), and Beclin-1 (ab62557, 1:100, Abcam) at 4ºC overnight. The tissue sections were then washed and subject to incubation with goat anti-rabbit IgG H&L (horse radish peroxidase, HRP) (ab6721, 1:1000, Abcam) for 30 min. The nuclear was stained with diaminobenzidine (Sigma Aldrich), and sections were counterstained with hematoxylin. The sections were observed with an optical microscope (Olympus) and quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).

In-vitro cell culture

Mouse ovarian GCs were obtained from Pricella (CP-M050, Pricella). The mouse ovarian tissues were mechanically separated, and then detached by collagenase, with the mouse GCs prepared by differential adhesion method. The GCs were kept in specialized complete medium (CM-M050, Pricella), and were detected for the GC-specific marker FSHR by immunofluorescence, which showed a cell purity of more than 98%, good cellular vitality, and free of HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi (Supplementary Fig. 1). The cultures were placed in a humidified environment at 37 °C with 5% CO2 and 95% air, and the medium was replaced every 2 days.

Treatment and groups of mouse GCs

Mouse GCs were divided into the blank, CTX (GCs treated with 30 µM CTX for 24 h) [27], CTX + EVs and CTX + PBS (GCs treated with 30 µM CTX and 30 µg/mL hUC-MSCs-EVs or equivalent PBS for 24 h) [29], CTX + EVs-sh-IGF-1 and CTX + EVs-sh-NC (GCs treated with 30 µM CTX and 30 µg/mL EVs-sh-IGF-1 or EVs-sh-NC for 24 h), as well as CTX + EVs + ML385 groups [GCs pre-treated with 10 µM ML385 (a Nrf2 inhibitor, MedChemExpress, Monmouth Junction, NJ, USA) for 2 h followed by treatment with 30 µM CTX and 30 µg/mL hUC-MSCs-EVs for 24 h, the concentration of ML385 was selected referring to a previous similar study amd product instructions [30]].

Uptake of EVs by GCs

EVs were labeled with PKH26 red fluorescent dye (MedChemExpress) and cultured with GCs for 24 h. GC slides were fixed in 4% paraformaldehyde (Beyotime) for 20 min, washed three times with PBS, stained with 4 -6-diamidino- 2-phenylindole (DAPI) (Beyotime) for 1 h, and the uptake of EVs by GCs was observed under a confocal fluorescence microscope (Carl Zeiss, Jena, Germany).

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

EVs and total cellular RNA were extracted by TRIzol regent (Invitrogen Inc., Carlsbad, CA, USA) and transcribed into cDNA with the PrimeScript RT kit (Takara Biotechnology Co., Ltd., Tokyo, Japan). PCR was conducted on an ABI 7900HT Rapid PCR Real-Time System (Applied Biosystems, Foster City, CA, USA) using SYBR® Premix Ex Taq™II (Takara) under the conditions of pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 34 s for a total of 40 cycles. The experiment was repeated three times. Target gene expression was quantified using the 2−ΔΔCt method with β-actin as the housekeeping gene. See the primer sequences in Table 1.

Table 1 Primer sequences
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Western blot

The hUC-MSCs, EVs, ovarian tissues and GCs were lysed on ice by RIPA lysis solution (Beyotime) to isolate nuclear and cytoplasmic proteins as per the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Proteins were routinely isolated, transferred to a membrane, and blocked before incubation with primary antibodies against CD9 (ab236630, 1:1000, Abcam), CD63 (ab134045, 1:1000, Abcam), CD81 (ab109201, 1:1000, Abcam), Calnexin (ab22595, 1:1000, Abcam), IGF-1 (ab223567, 1:1000, Abcam), Nrf2 (GTX103322, 1:1000, GeneTex), HO-1 (GTX101147, 1:1000, GeneTex), LC3 (ab192890, 1:2000, Abcam), Beclin-1 (ab62557, 1:1000, Abcam), β-actin (ab8227, 1:2000, Abcam) and Lamin B1 (GTX103292, 1:1000, GeneTex) at 4 °C for 12 h. The membrane was washed in Tris-buffered saline with Tween (Solarbio) and incubated with goat anti-rabbit IgG H&L (HRP) (ab6721, 1:1000, Abcam) for 2 h at room temperature. Following development with ECL working solution (EMD Millipore, USA), the gray value of bands in the western blot images was quantified through ImageJ software (version 1.61; NIH Image), with β-actin as an internal control of cytoplasmic protein and Lamin B1 as an internal control of nuclear protein. Each experiment was repeated three times.

Cell counting kit-8 (CCK-8) for GC proliferation

GCs were seeded onto a 96-well plate with a density of 1 × 104 cells/well and cultured for 0, 6, 12, and 24 h to detect their proliferation ability with the CCK-8 kit (Beyotime).

Oxidative stress-related indicators

ROS (S0033M), glutathione (GSH; S0052) and malondialdehyde (MDA; S0131) contents in mouse ovarian tissues or GCs were determined as per the manufacturer’s instructions.

Lactate dehydrogenase (LDH) for GC death

Cell death was assessed by detecting LDH release using the LDH assay kit (Beyotime). The absorbance of the samples was measured at 490 nm using a microplate reader to calculate the amount of LDH release. LDH release (%) = (OD value of the experimental group - OD value of the blank group)/(OD value of the control group - OD value of the blank group) × 100%.

Immunofluorescence

Mouse GCs were fixed in PBS containing 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 15 min. After being blocking with 2% BSA, the GCs were incubated overnight in a humidified chamber at 4 °C with primary antibodies against Nrf2 (GTX103322, 1:100, GeneTex) and LC3 (ab192890, 1:100 Abcam). Subsequently, incubation with secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077, 1:200, Abcam) was performed for 2 h at room temperature. Nuclei were counterstained with DAPI (Beyotime) for 5 min. The stained sections were visualized with an Olympus BX53 microscope and quantified with ImageJ software (version 1.61; NIH Image) for co-localization.

Statistical analysis

Statistical analysis and graph drawing for all data were carried out by GraphPad Prism 8.01 (GraphPad Software Inc). Outlier detection was performed by Grubbs’ test and no data point was excluded by the test. Measurement data, expressed as mean ± standard deviation, were subject to independent t test in case of two groups, and one-way analysis of variance in case of multiple groups, followed by Tukey’s multiple comparisons test. Two-sided P value of < 0.05 was deeded to have statistical significance.

Results

Characterization of hUC-MSCs-EVs

The purchased hUC-MSCs were characterized firstly. Microscopically, hUC-MSCs were spindle-shaped, grew in monolayers, and showed fibroblast-like features (Fig. 1A). Detected by flow cytometry for the surface antigens of hUC-MSCs, CD29, CD44 and CD90 were positively expressed, and CD43, CD14 and HLA-DR were negatively expressed (Fig. 1B). Subsequently, EVs were isolated and collected from hUC-MSCs by ultracentrifugation. Morphological observation using TEM showed a typical cup-shaped structure (Fig. 1C). NTA showed that the diameter of EVs ranged from 50 to 100 nm, and the particle concentration was 8.52 × 1010 (Fig. 1D). As shown by western blot, the EVs group had significant CD9, CD81 and CD63 expression, whereas Calnexin was not expressed, compared to the GW group (Fig. 1E). The above results indicated the successful isolation of hUC-MSCs-EVs.

Fig. 1
figure 1

Isolation and characterization of hUC-MSCs-EVs

A: Observation of hUC-MSCs morphology by an inverted microscope; B: Flow cytometry evaluated hUC-MSCs surface markers CD29, CD44, CD90, CD43, CD14, and HLA-DR; C: The structure of extracted hUC-MSCs-EVs was visualized by transmission electron microscope; D: Nanoparticle size analysis was used to determine the distribution and concentration of hUC-MSCs-EVs diameter; E: western blot quantified hUC-MSCs-EVs surface markers CD9, CD63, CD81 and Calnexin. hUC-MSCs-EV, human umbilical cord mesenchymal stem cell-derived extracellular vesicles

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hUC-MSCs-EVs restore ovarian function in mice suffering from POI by upregulating IGF-1

CTX was applied to induce the POI mouse model. H&E results showed that mice in the control group had normal ovarian structure and GC arrangement; while mice in the POI group had disorganized GCs, thinner GCs layer, reduced number of GCs and follicles (Fig. 2A, P < 0.001), and decreased numbers of follicles at all levels (Fig. 2B, P < 0.01). Compared to the control mice, the CTX-treated mice had lower serum E2 and AMH, as well as higher FSH and LH (Fig. 2C, all P < 0.001). In contrast, after treatment with hUC-MSCs-EVs, the CTX-induced mice had thickened GC layer and more GCs and numbers of total follicles and follicles at all levels along with elevated serum E2 and AMH levels, and decreased FSH and LH level (Fig. 2A-C, all P < 0.01), indicating that hUC-MSCs-EVs improved ovarian function in CTX-induced mice.

Fig. 2
figure 2

hUC-MSCs-EVs upregulate IGF-1 to improve mouse ovarian function in CTX-stimulated POI

A-B: H&E staining displayed ovarian histopathological changes, and the numbers of total follicles and follicles at all levels (including secondary, primordial, mature, primary and atretic follicles) were statistically analyzed; C: serum E2, FSH, LH, and AMH levels were detected by ELISA; D: immunohistochemistry was used to detect the expression of IGF-1. N = 6, and the data were expressed as mean ± standard deviation. Data comparisons among multiple groups were performed by one-way analysis of variance, followed by Tukey’s multiple comparisons test, **, P < 0.01; ***, P < 0.001. hUC-MSCs-EV, human umbilical cord mesenchymal stem cell-derived extracellular vesicles; E2, estradiol; FSH, follicle stimulating hormone; LH, luteinizing hormone; AMH, anti-mullerian tubular hormone

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Subsequently, IGF-1 expression was quantified in the ovarian tissues of POI mice. Immunohistochemistry results showed that the proportion of IGF-1-positive cells in the ovarian tissues of POI mice was lower than that in the control mice, whereas the proportion of IGF-1-positive cells in the POI + EVs group was remarkably increased compared with that in the POI + PBS group (Fig. 2D, both P < 0.01). The above results suggested that hUC-MSCs-EVs upregulated IGF-1 to improve ovarian function of mice with POI.

hUC-MSCs-EVs carry IGF-1 to improve CTX-triggered injury in GCs

hUC-MSCs-EVs were treated with proteinase K and Triton X-100 to detect IGF-1 expression in hUC-MSCs-EVs by western blot, which showed that there was no significant change in the IGF-1 expression in the EVs after treatment with Proteinase K (Fig. 3A, P > 0.05); however, the IGF-1 expression was suppressed by the co-treatment of proteinase K and Triton X-100 (Fig. 3A, P < 0.001). The above results indicated that IGF-1 was encapsulated within hUC-MSCs-EVs. In addition, the 3rd generation of the hUC-MSCs was transfected with sh-IGF-1 before EVs extraction, and the expression of IGF-1 in hUC-MSCs was determined by RT-qPCR and western blot for verifying the transfection efficiency, which indicated that the expression of IGF-1 was diminished (Fig. 3B, both P < 0.01). The IGF-1 protein level in the EVs-sh-IGF-1 group was tampered than that in the EVs-sh-NC group (Fig. 3A, P < 0.001).

Fig. 3
figure 3

hUC-MSCs-EVs carrying IGF-1 ameliorates CTX-induced damage of GCs

A: Western blot quantified the protein levels of IGF-1 carried in differently-treated EVs; B: RT-qPCR and western blot verified IGF-1 expression in hUC-MSCs; C: PKH26 staining was used to observe the uptake of hUC-MSCs-EVs by GCs; D: Western blot quantified IGF-1 expression in GCs; E: CCK-8 assay was used to evaluate GC proliferation; F: LDH determined GC death. The cellular experiments were independently repeated three times, and the data were expressed as mean ± standard deviation. One-way analysis of variance was used for data comparison among multiple groups, followed by Tukey’s multiple comparisons test. **, P < 0.01; ***, P < 0.001. hUC-MSCs-EV, human umbilical cord mesenchymal stem cell-derived extracellular vesicles; IGF-1, insulin-like growth factor 1; GCs, granulosa cells; LDH, lactate dehydrogenase

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Purchased mouse GCs were identified by immunofluorescence detection of FSHR, a GC-specific marker, to have more than 98% purification and be free of contamination (Supplementary Fig. 1). GCs were treated with hUC-MSCs-EVs, which could be internalized by GCs (Fig. 3C). The IGF-1 expression in GCs was lowered by CTX exposure than blank treatment; compared to the CTX + PBS group, the CTX + EVs group had enhanced IGF-1 expression; compared with CTX + EV-sh-NC group, the CTX + EV-sh-IGF-1 group had decreased IGF-1 level (Fig. 3D, P < 0.01). CCK-8 and LDH results showed that CTX treatment substantially impaired GC proliferation and aggravated GC deaths compared to blank treatment, while hUC-MSCs-EVs treatment partially reversed these changes; the CTX + EV-sh-IGF-1 group showed weakened GC proliferation and increased GC deaths than the CTX + EV-sh-NC group (Fig. 3E-F, P < 0.01). The above results indicated that hUC-MSCs-EVs harboring IGF-1 ameliorated CTX-induced injury of GCs.

hUC-MSCs-EVs harboring IGF-1 suppress CTX-induced excessive autophagy by activating Hrf2/HO-1 pathway

The study further addressed the role of hUC-MSCs-EVs carrying IGF-1 in activating Nrf2/HO-1 signaling pathway and CTX-induced excessive autophagy. As revealed by western blot, the CTX group showed remarkable decreases in Nrf2 (nuclear) and HO-1 protein levels and a significant increase in Nrf2 (cytoplasm) protein level compared with the blank group, whereas the CTX + EVs group showed a substantial increase in Nrf2 (nuclear) and HO-1 protein levels and a decrease in Nrf2 (cytoplasm) protein level than the CTX + PBS group, and the CTX + EVs-sh-IGF-1 group showed prominent decreases in Nrf2 (nuclear) and HO-1 protein levels, and an increase in Nrf2 (cytoplasm) protein level than the CTX + EVs- sh-NC group (Fig. 4A, all P < 0.01). Furthermore, the levels of ROS, MDA, LC3II/I, Beclin-1, and LC3-positive cell number were enhanced, and GSH content was suppressed in the CTX group than those in the blank group; both GC oxidative stress and autophagy were suppressed after treatment with hUC-MSCs-EVs, but inhibiting IGF-1 in hUC-MSCs-EVs exacerbated oxidative stress and autophagy of GCs (Fig. 4B-D, both P < 0.05). Moreover, immunofluorescence detected decreased Nrf2 nuclear translocation in the CTX group than in the blank group and enhanced Nrf2 nuclear translocation in the CTX + EVs group relative to the CTX + PBS group; at the same time, Nrf2 nuclear translocation was decreased in the CTX + EVs-sh-IGF-1 group than in the CTX + EVs-sh-NC group (Fig. 4D, both P < 0.01). Overall, hUC-MSCs-EVs carrying IGF-1 activated Nrf2/HO-1 signaling to inhibit CTX-induced excessive autophagy in GCs.

Fig. 4
figure 4

hUC-MSCs-EVs carrying IGF-1 activate Nrf2/HO-1 signaling to inhibit CTX-induced excessive autophagy in GCs

A: Western blot evaluated Nrf2 (nuclear), Nrf2 (plasma), and HO-1 protein expression; B: ROS, MDA, and GSH levels were assayed by kits; C: Autophagy-related proteins LC3II/I and Beclin-1 were quantified by western blot; D: Immunofluorescence displayed Nrf2 nuclear translocation and LC3 positive cells. The cellular experiments were independently repeated three times, and the data were expressed as mean ± standard deviation. One-way analysis of variance was used for data comparison among multiple groups, followed by Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. hUC-MSCs-EV, human umbilical cord mesenchymal stem cell-derived extracellular vesicles; IGF-1, insulin-like growth factor 1; GCs, granulosa cells; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; Nrf2/HO-1, nuclear factor E2 related factor 2/heme oxygenase-1

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Inhibiting Nrf2/HO-1 partially nullifies the suppression of hUC-MSCs-EVs on CXT-induced excessive autophagy and injury of GCs

The role of Nrf2/HO-1 signaling in the amelioration of hUC-MSCs-EVs on CTX-induced excessive autophagy and injury in GCs was investigated by treating GCs with ML385 (a Nrf2 inhibitor) for 2 h before exposure to 30 µM CTX and 30 µg/mL hUC-MSCs-EVs. Through western blot and immunofluorescence, compared to the CTX + EVs group, the protein levels of Nrf2 (nuclear) and HO-1 and Nrf2 nuclear translocation were decreased and Nrf2 (cytoplasm) expression was upregulated in the CTX + EVs + ML385 group (Fig. 5A, F, all P < 0.05), which indicated inhibition of Nrf2/HO-1 signaling. Meanwhile, relative to the CTX + EVs group, the CTX + EVs + ML385 group showed diminished cell proliferation, increased cell deaths, and deteriorated oxidative stress and autophagy (Fig. 5B-F, both P < 0.05). The above revealed that inhibition of Nrf2/HO-1 signaling partially reversed the ameliorative effect of hUC-MSCs-EVs on CTX-induced excessive autophagy and cell damage in GCs.

Fig. 5
figure 5

Inhibition of Nrf2/HO-1 signaling partially reverses the ameliorative effect of hUC-MSCs-EVs on CTX-induced excessive autophagy injury in GCs

A: Western blot quantified Nrf2 (nuclear), Nrf2 (cytoplasm), and HO-1 protein expression; B: CCK-8 assay determined GC proliferation; C: LDH detection was used to indicate GC death; D: ROS, MDA, and GSH levels were assayed by kits; E: Autophagy-related proteins LC3II/I and Beclin-1 were quantified by western blot; F: Immunofluorescence displayed Nrf2 nuclear translocation and LC3 positive cells. The cellular experiments were independently repeated three times, and the data were expressed as mean ± standard deviation. Comparison between the two groups was performed by independent sample t-test. *, P < 0.05. hUC-MSCs-EV, human umbilical cord mesenchymal stem cell-derived extracellular vesicles; IGF-1, insulin-like growth factor 1; GCs, granulosa cells; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; LDH, lactate dehydrogenase; Nrf2/HO-1, nuclear factor E2 related factor 2/heme oxygenase-1

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hUC-MSCs-EVs block excessive autophagy and injury of GCs through the IGF-1/Nrf2/HO-1 signaling to alleviate CTX-induced ovarian dysfunction in POI mice

Following injection of EVs-sh-NC or EVs-sh-IGF-1, mice in the POI + EVs-sh-IGF-1 group were found to have less IGF-1-positive cells in the ovarian tissue, thinner GC layer, decreased numbers of GCs and total and all levels of follicles (Fig. 6A-C, all P < 0.05), lower serum E2 and AMH level, and higher FSH and LH level, compared to those in the POI + EVs-sh-NC group (Fig. 6D, all P < 0.05). In addition, POI mice appeared to have reduced Nrf2 (nuclear) and HO-1 protein levels, upregulated Nrf2 (cytoplasm) expression, enhanced ROS, MDA and Beclin-1 levels, more LC3-positive cells, and downregulated GSH content than the control mice, suggesting that the Nrf2/HO-1 pathway was inhibited and oxidative stress and autophagy were arisen in the ovarian tissues of POI mice (Fig. 6A-G, all P < 0.05). CTX-induced inhibition on Nrf2/HO-1 pathway and increases in excessive oxidative stress and autophagy were weakened by administration of hUC-MSCs-EVs, and further suppressing IGF-1 expression impeded the activation of Nrf2/HO-1 pathway and enhanced oxidative stress and autophagy in mice (Fig. 6A-G, all P < 0.05). Therefore, hUC-MSCs-EVs inhibited excessive autophagy and damage in GCs through the IGF-1/Nrf2/HO-1 pathway and improved the ovarian function in CTX-induced mice.

Fig. 6
figure 6

hUC-MSCs-EVs suppress excessive autophagy and injury of GCs and improved ovarian function in mice suffering from POI through the IGF-1/Nrf2/HO-1 pathway

A: Immunohistochemistry detected GCs positive to IGF-1, FSHR, LC3 and Beclin-1; B: H&E staining displayed ovarian histopathological changes, with the numbers of total follicles and follicles at all levels (comprising primary, secondary, primordial, atretic and mature follicles) statistically analyzed; D: serum E2, FSH, LH, and AMH levels were detected by ELISA; E: Western blot quantified Nrf2 (nuclear), Nrf2 (cytoplasm), and HO-1 protein expression; F: ROS, MDA, and GSH levels were assayed by kits; G: LDH detection was used to indicate GC death. N = 6, and the data were expressed as mean ± standard deviation. Data comparisons among multiple groups were performed by one-way analysis of variance, followed by Tukey’s multiple comparisons test, *, P < 0.05; **, P < 0.01; ***, P < 0.001. hUC-MSCs-EV, human umbilical cord mesenchymal stem cell-derived extracellular vesicles; E2, estradiol; FSH, follicle stimulating hormone; LH, luteinizing hormone; AMH, anti-mullerian tubular hormone; IGF-1, insulin-like growth factor 1; GCs, granulosa cells; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; LDH, lactate dehydrogenase; Nrf2/HO-1, nuclear factor E2 related factor 2/heme oxygenase-1

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Fig. 6
figure a

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Discussion

A previous study makes clear that hUC-MSCs-EVs enhanced ovarian function and GC proliferation in mice with POI by mediating the Hippo signaling pathway [27]. However, the specific and underlying therapeutic mechanisms of hUC-MSC-EVs have not been fully understood. This study firstly demonstrated that hUC-MSCs-EVs harboring IGF-1 activated the Nrf2-HO pathway to suppress excessive autophagy and injury of GCs, thus alleviating CTX-induced ovarian dysfunction in mice.

hUC-MSCs-EVs is feasible to restore ovarian function, protect the fertility, and improve local microenvironment of ovarian tissues by regulating immunity, cellular vitality, inflammation, and other related signaling pathways, in rats with chemotherapy-induced POI [31]. hUC-MSCs-derived exosomes act as carriers and transfer microRNA-126-3p to facilitate angiogenesis and attenuate GC apoptosis in POI [32]. IGF-1 and its receptor IGF-1R are essential for follicular development and ovulation, which can stimulate the differentiation and proliferation of GCs and follicular membrane cells [33, 34]. Thus, IGF-1 is a crutial target for GCs repair. It has been demonstrated that recombinant IGF-1 can supplement human IGF-1 to ameliorate IGF-1 deficiency-related diseases, comprising primary IGF-1 deficiency-related childhood dwarfism and type 2 diabetes mellitus [35,36,37]. Thus, recombinant IGF-1 may produce a reparative effect on GCs in ovarian premature failure. However, it has not been reported regarding the treatment and application of recombinant IGF-1 in premature ovarian failure. Relative to recombinant IGF-1, exosomal IGF-1 possesses the following advantages: (1) exosomes have high affinity and low immunogenicity, with high absorption and utilization by the body and low immune rejection. (2) exosomes can keep the structure and physiological function of IGF-1 stable, which is convenient for long-term preservation and stabilisation of its physiological function. (3) exosomes carry a large number of biologically active substances such as lipids, proteins and nucleic acids, which may play a protective role in POI via mutiple pathways and therapeutic targets [38, 39]. (4) exosomes can serve as drug carriers [40] to carry a huge amount of recombinant IGF-1, so that the recombinant IGF-1 can play a better therapeutic role. Therefore, we believe that exosomes have more advantages than recombinant IGF-1 for the treatment of POI. This study observed that hUC-MSCs-EVs restored the number of GCs and follicles, promoted serum E2 and AMH level, and blocked GC deaths in CTX-induced mice. Additionally, ovarian IGF-1 was detected to be downregulated in response to CTX exposure and hUC-MSCs-EVs treatment elevated IGF-1 expression in mice with experimental POI. IGF-1 in synergy with balanced concentration of androgen and FSH boosted the function of early-stage ovarian follicles with chronic stress [41]. Human amnion-derived MSCs can secrete IGF-1, VEGF and other growth factors through a paracrine mechanism to relief chemotherapy-induced POI [42]. After suppressing IGF-1 expression in the hUC-MSCs-EVs, we found the therapeutic effect of hUC-MSCs-EVs on GC injury and ovarian function disturbance was reduced, indicating that hUC-MSCs-EVs rescued CTX-induced GC injury and ovarian function by carrying IGF-1.

Autophagy is a self-renewal process of eukaryocytes to scavenge dysfunctional proteins and degenerative organelles [43]. Lack of autophagy in GCs appears to inhibit GC differentiation, involving in the onset of POI [44]. However, this study demonstrated that hUC-MSCs-EVs impeded both oxidative stress and autophagy in GCs. Excessive autophagy may cause digestion of essential components for cell survival and induce self-destruction of GCs suffering from oxidative stress [45]. Likewise, melatonin impaired mitochondrial oxidative stress and GC cycle arrest triggered by excessive autophagy by suppressing ERK pathway, thereby rescuing mitochondrial function and ovarian reserve [46]. Estrogen receptor beta increased forkhead transcription factor family 3a (FOXO3a) expression, which is conducive to autophagy activation in GCs and ultimately results in diminished ovarian reserve [47]. In both animal and cellular models, inhibiting IGF-1 in hUC-MSCs-EVs weakened the suppression on oxidative stress and autophagy in ovarian tissues and murine GCs, exacerbating ovarian dysfunction induced by CTX. IGF-1 was noted to regulate follicle survival and atresia by suppressing GC apoptosis, and it could enhance Beclin 1 level and LC3II/LC3I, indicating the induction of autophagy in porcine GCs [48]. Therefore, IGF-1 may exert different regulatory roles in GCs from different sources, which prompts a necessity to further verity the regulation of GC autophagy by IGF-1 in human GCs. Intriguingly, IGF-1 could trigger Nrf2/HO-1 activation to prevent ROS generation [49]. IGF-1 maintained mitochondrial dynamics and turnover through the conserved Glycogen Synthase Kinase-3β/Nrf2/BNIP3 [50]. In CTX-induced POI models in mice and GCs, Nrf2/HO-1 pathway was suppressed. Further treatment with hUC-MSCs-UCs nullified such suppression. Notably, suppression of IGF-1 expression blocked the activation of Nrf2/HO2 pathway. Berberine can block inflammation and oxidative stress by inducing Nrf2 pathway activation, thereby alleviating ovarian functions of mice suffering from POI [51]. Human placental MSCs-secreted epidermal growth factor suppressed oxidative stress through Nrf2-HO-1 activation to mitigate POI [52]. In our study, inhibiting Nrf2/HO-1 signaling weakened hUC-MSCs-EVs-promoted GC proliferation and increased their death, oxidative stress and autophagy.

In CTX-induced experimental POI, hUC-MSCs-EVs could carry IGF-1 to activate the Nrf2/HO-1 pathway, thereby blocking excessive autophagy and injury of GCs and reserving mouse ovarian functions. This study provides a promising target for developing therapeutic approaches for POI, but it only draws a conclusion from mouse and murine GC models, and has not involved with human GCs. In addition, the optimal dosage of hUC-MSCs-EVs treating POI still requires further determination. Our future study will use human GCs to further discuss the mechanism of action of hUC-MSCs-EVs in POI and optimize the dosage of hUC-MSCs-EVs to provide solid evidence for hUC-MSCs-EVs treating POI in clinical.

Data availability

The datasets generated during and analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.

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Acknowledgements

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Funding

This research was supported by grants from Shanxi Provincial Health Commission Key project of scientific and technological medical innovation plan (2021XM38).

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Authors and Affiliations

  1. Department of Reproductive Genetics, Heping Hospital of Changzhi Medical College, Changzhi, 046000, P.R. China

    Hui Miao, Congxiu Miao, Na Li & Jing Han

  2. Institute of Reproduction and Genetics of Changzhi Medical College, Changzhi, 046000, P.R. China

    Hui Miao, Congxiu Miao, Na Li & Jing Han

  3. Key Laboratory of Reproduction and Genetics of Changzhi Medical College, Changzhi, 046000, P.R. China

    Hui Miao, Congxiu Miao, Na Li & Jing Han

  4. Key Laboratory of Reproduction Engineer of Shanxi Health Committee, Changzhi, 046000, P.R. China

    Hui Miao, Congxiu Miao, Na Li & Jing Han

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guarantor of integrity of the entire study: Congxiu Miao; study concepts: Hui Miao; study design: Hui Miao; definition of intellectual content: Hui Miao and Na Li; literature research: Na Li and Jing Han; clinical studies: Hui Miao and Jing Han; experimental studies: Na Li; data acquisition: Na Li; data analysis: Hui Miao and Jing Han; statistical analysis: Jing Han and Na Li; manuscript preparation: Hui Miao and Jing Han; manuscript editing: Hui Miao; manuscript review: Congxiu Miao.

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Miao, H., Miao, C., Li, N. et al. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles harboring IGF-1 improve ovarian function of mice with premature ovarian insufficiency through the Nrf2/HO-1 pathway. J Ovarian Res 17, 224 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13048-024-01536-8

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