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hUMSCs Transplantation Regulates AMPK/NR4A1 Signaling Axis to Inhibit Ovarian Fibrosis in POI Rats

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Abstract

Background

The mechanism of human Umbilical Cord Mesenchymal Stem Cells (hUMSCs) transplantation to improve ovarian function in the rats with Premature Ovarian Insufficiency (POI) is still unclear. The aim of this study is to investigate the signal axis mechanism that is involved in the ovarian function recovery of POI rats following hUMSCs transplantation.

Methods

The rat model with POI was established by intraperitoneal injection of cisplatin. The hUMSCs were transplanted by caudal vein injection into POI rats. Hematoxylin–eosin (H&E) staining was performed to examine the morphology of rat ovarian tissue. Masson staining, Sirus red staining and immunofluorescence were used to observe the fibrosis extent of ovarian tissue. The levels of serum sex hormones and the expression of fibrosis related markers in ovarian tissues were measured by enzyme-linked immunosorbent assay (ELISA). The expression of NR4A1, Phospho-NR4A1 and AMP-activated protein kinase (AMPK) signaling in rat ovarian tissues was measured by immunohistochemistry and immunofluorescence. The role of AMPK/NR4A1 signaling axis in the regulation of ovarian function recovery in POI rats following hUMSCs transplantation was further investigated by adenovirus and siRNA intervention in isolated stromal cells.

Results

The results showed that the hUMSCs transplantation significantly inhibited ovarian tissue fibrosis and restored the ovarian function in POI rats. The level of NR4A1 and AMPK expression in ovarian tissue of POI rats after hUMSCs transplantation was significantly increased compared with the control group. In the cultured ovarian stromal cells, the similar results were obtained on the expression of NR4A1 and its regulation on fibrosis related molecular markers in Cisplatin (CDDP) damaged stromal cells following hUMSCs supernatant treatment. Both hUMSCs supernatant treatment and the addition of AMPK inhibitors increased NR4A1 expression in stromal cells. And after NR4A1 molecular intervention, fibrosis-related indicators in stromal cells changed. The data suggests that the AMPK/NR4A1 signaling axis is involved in the ovarian function changes in POI rats following hUMSCs transplantation.

Conclusion

The data from this study indicate that the inhibition of tissue fibrosis and recovery of ovarian function is regulated by AMPK/NR4A1 signaling axis in POI rats following hUMSCs transplantation.

Graphical Abstract

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Data Availability

All data generated and/or analyzed during this study are included in this published article.

Abbreviations

hUMSCs :

Human umbilical cord mesenchymal stem cells

POI :

Premature ovarian insufficiency

H&E :

Hematoxylin-eosin

ELISA :

Enzyme-linked immunosorbent assay

AMPK :

AMP-activated protein kinase

CDDP :

Cisplatin

E2 :

Estradiol

HRT :

Hormone replacement therapy

Collagen I :

Collagen Type I

Collagen III :

Collagen Type III

NR4A1 :

Nuclear receptor 4A1

FSH :

Follicle stimulating hormone

LH :

Luteinizing hormone

P :

Progesterone

DAB :

Diaminobenzidine

IRS :

Immune response score

SD :

Standard deviation

ANOVA :

Analysis of variance

CTGF :

Connective tissue growth factor

MSCs :

Mesenchymal stem cells

FN :

Fibronectin

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Acknowledgements

All authors are acknowledged for their contribution to the study.

Funding

This research was supported by Scientific Research Start-up funding Project of Binzhou Medical University (NO. BY2021KYQD12), the Natural Science Foundation of Shandong Province (NO. ZR2021QH149), the University and Locality Collaborative Development Program of Yantai (NO. 2021XDHZ082) and Yantai Double Hundred Program to Q.F.

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Author notes

  1. Linlu Cui and Hongchu Bao contributed equally to this work

Authors and Affiliations

  1. Xu Rongxiang Regenerative Medicine Research Center, Binzhou Medical University, Yantai, 264003, Shandong, China

    Linlu Cui, Yu Tang & Zhonglin Jiang

  2. College of Basic Medicine, Binzhou Medical University, Yantai, 264003, Shandong, China

    Linlu Cui, Yu Tang, Qianqian Luo, Yaru Si & Zhonglin Jiang

  3. Department of Clinical Medicine, Yantai Yuhuangding Hospital, Yantai, 264000, Shandong, China

    Hongchu Bao

  4. The Second Hospital of Shandong University, Jinan, 250200, Shandong, China

    Wenqian Zhu

  5. School of Pharmacology, Institute of Aging Medicine, Binzhou Medical University, Yantai, 264003, Shandong, China

    Qiang Fu

  6. Shandong Cellogene Pharmaceutics Co.LTD, Yantai, 264003, Shandong, China

    Qiang Fu

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Contributions

CLL, BHC and JZL were in charge of experimental design and literature research. CLL, ZWQ and TY were in charge of experimental studies. BHC and LQQ were in charge of data analysis and interpretation. SYR assisted with the experiments. FQ and JZL were in charge of the manuscript editing. All authors read and approved the ending version of the final manuscript.

Corresponding authors

Correspondence to Qiang Fu or Zhonglin Jiang.

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Ethics Approval and Consent to Participate

Animals were treated in accordance with the Basel Declaration in the context of phase experimental animals. The use of animals was approved by the Ethics Committee of Binzhou Medical University. We have obtained the participants’ informed consent to donate the tissue for research use. The use of human tissue was approved by the Ethics committee of the Yantai Yuhuangding Hospital.

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Supplementary Information

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12015_2022_10469_MOESM1_ESM.png

Supplementary file1 Supplementary Figure 1. hUMSCs identification. [A-a]: Microscopic morphology of hUMSCs (Scale bar=560 µm). [A-b]: hUMSCs stained with Alizarin red S to demonstrate their differentiation into osteoblasts (Scale bar=230 µm). [A-c]: hUMSCs stained with Oil Red O to demonstrate their differentiation into adipocytes (Scale bar=50 µm). [B]: The following surface markers of hUMSCs were identified: CD44, CD90, CD73, CD105, CD34, CD45 and HLA–DR. (PNG 6496 kb)

12015_2022_10469_MOESM2_ESM.png

Supplementary file2 Supplementary Figure 2. Stromal cell identification. Stromal cells were characterized by expression of cell surface markers. [A]: The isolated cells were characterized with immunofluorescence staining. The mesenchymal cells were stained by Vimentin as green, the endothelial cells were stained with Factor VIII as red and the epithelial cells were stained with cytokeratin as red. NR4A1 is stained green. The cell nucleus was stained by DAPI as blue (Scale bar=60 µm). [B]: The morphology of stromal cells under microscope(Scale bar=560 µm). [C]: Cell viability of stromal cells were measured after different concentrations of NR4A1 agonist, DIM-C-pPhOCH3 treatment. Data are expressed as the mean ± SD. *: P<0.05, **: P<0.01 and ***: P<0.001. (PNG 4197 kb)

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Cui, L., Bao, H., Zhu, W. et al. hUMSCs Transplantation Regulates AMPK/NR4A1 Signaling Axis to Inhibit Ovarian Fibrosis in POI Rats. Stem Cell Rev and Rep 19, 1449–1465 (2023). https://doi.org/10.1007/s12015-022-10469-y

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