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hUMSCs regulate the differentiation of ovarian stromal cells via TGF-β1 /Smad3 signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats
Hongqin Zhang
Binzhou Medical University - Yantai Campus
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1440-7365
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Stem Cell Research & Therapy.
Objective: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1 ) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis.
Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smad3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.
Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats were significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smad3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process.
Conclusion : Our study demonstrated that the TGF-β1 /Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.
Keywords
primary ovarian insufficiency, human umbilical cord - derived mesenchymal stem cells, ovarian stromal cells, TGF-β 1 signaling pathway, fibrosis
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- Table1..jpg
Table 1. Effect of hUMSCs transplantation on pregnancy rate of POI rats. hUMSCs human umbilical cord - derived mesenchymal stem cells
- SupplementaryFigure1..jpg
Supplementary Figure1. Flowcytometria analysis of cell markers in hUMSCs. The hUMSCs were characterized by its cell surface marker expression to confirm its phenotype. [a-g]: The following surface markers were identified: CD44, CD90, CD73, CD45, CD34, HLA - DR and CD105. [h-i]: The hUMSCs staining with Alizarin red S and Oil Red O to confirm its differentiation to Osteoblasts (100×) and adipoblasts (200×). [j, 400×]: The hUMSCs shows fibroblast - like morphology under light microscopy examination. hUMSCs human umbilical cord-derived mesenchymal stem cells
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CURRENT STATUS: Published
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On 20 Aug, 2020
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Editorial decision: Major Revision
On 07 Jul, 2020
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Received 05 Jul, 2020
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Received 05 Jul, 2020
Reviewer #5 agreed
On 03 Jul, 2020
Reviewer #4 agreed
On 02 Jul, 2020
Reviewer #3 agreed
On 01 Jul, 2020
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Reviewer #2 agreed
On 30 Jun, 2020
Editor assigned
On 30 Jun, 2020
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This preprint is under consideration at Stem Cell Research & Therapy. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
hUMSCs regulate the differentiation of ovarian stromal cells via TGF-β1 /Smad3 signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats
Hongqin Zhang
Binzhou Medical University - Yantai Campus
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1440-7365
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 3
Posted 20 Aug, 2020
Published
On 07 Sep, 2020
No community comments so far
Editorial decision: Accept
On 20 Aug, 2020
Editor assigned
On 18 Aug, 2020
Submission checks complete
On 17 Aug, 2020
Editor invited
On 17 Aug, 2020
No comments provided
Review #2 received
Received 14 Aug, 2020
Editorial decision: Minor Revision
On 14 Aug, 2020
Review #1 received
Received 08 Aug, 2020
Reviewer #2 agreed
On 07 Aug, 2020
Reviewers invited
Invitations sent on 05 Aug, 2020
Reviewer #1 agreed
On 05 Aug, 2020
Editor assigned
On 04 Aug, 2020
Submission checks complete
On 03 Aug, 2020
Editor invited
On 03 Aug, 2020
No comments provided
Review #2 received
Received 07 Jul, 2020
Editorial decision: Major Revision
On 07 Jul, 2020
Review #4 received
Received 05 Jul, 2020
Review #3 received
Received 05 Jul, 2020
Reviewer #5 agreed
On 03 Jul, 2020
Reviewer #4 agreed
On 02 Jul, 2020
Reviewer #3 agreed
On 01 Jul, 2020
Review #1 received
Received 01 Jul, 2020
Reviewer #2 agreed
On 30 Jun, 2020
Editor assigned
On 30 Jun, 2020
Reviewers invited
Invitations sent on 30 Jun, 2020
Reviewer #1 agreed
On 30 Jun, 2020
Editor invited
On 29 Jun, 2020
Submission checks complete
On 25 Jun, 2020
First submitted
On 22 Jun, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Stem Cell Research & Therapy.
Objective: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1 ) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis.
Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smad3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.
Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats were significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smad3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process.
Conclusion : Our study demonstrated that the TGF-β1 /Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.
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