Mesenchymal Stem Cells Improve Ovarian Function by Suppressing Fibrosis Through CTGF/FAK Signaling in Systemic Lupus Erythematosus

Abstract

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic inflammation and multiple organ damage, including in the female reproductive system. Mesenchymal stem cells (MSCs) have been widely studied for their anti-inflammatory and regenerative effects and have been shown to alleviate SLE. However, the mechanism underlying how MSC transplantation in SLE can improve ovarian function is still not clear. The purpose of this study was to investigate the effect of human umbilical cord-derived MSCs (UC-MSCs) on ovarian function in a lupus mouse model and its underlying mechanism in vivo and in vitro.

Methods: LPR and MPJ mice were used for the test group and control group, respectively, in this study. The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The number of ovarian follicles was detected by H&E staining. The tissue fibrosis in the ovary was examined using Masson staining. Quantitative reverse-transcription polymerase chain reaction (RT‒qPCR) was used to detect the mRNA expression levels of various inflammatory cytokines, fibrosis-related factors and ovarian hormone function receptors. Western blotting was used to detect the protein expression levels of CTGF, COLLAGENI and α-SMA in ovarian tissue. Immunofluorescence and immunohistochemistry were used to examine the deposition of fibrosisrelated proteins and immune complexes and their expression levels in T-cell subsets. Primary granulosa cells were isolated from 22-week-old LPR murine ovaries to coculture with MSCs. Additionally, the protein levels of CTGF, COLLAGENI and α-SMA in ovarian granulosa cells were examined by RT‒qPCR and Western blot. A human granulosa cell line (KGN) was used to further investigate the relationship between CTGF, FAK/FAK-Tyr576/577 phosphorylation and fibrosis.

Findings: The experimental results showed that compared with the healthy control group, LPR mice had higher expression levels of inflammatory- and fibrosis-related factors, accompanied by downregulated expression of ovarian function hormone receptors. However, the expression of inflammatory factors (TNF-α, IL-1β, IL-6, IL-18) and fibrosis factors (CTGF, Collagen 1, α-SMA) in LPR mice’s ovary was decreased, while the expression of ovarian hormone receptors (AMH, ESR1, ESR2) was increased after UC-MSC transplantation. In addition, downregulation of CD3 T cells, CD4 T cells, complement C3, and IgG and upregulation of the Treg-cell ratio were also detected in the ovaries of lupus mice treated with UC-MSC transplantation. Further results demonstrated that the protein expression of CTGF, collagen type I and α-SMA was significantly decreased in primary ovarian granulosa cells (GCs) after MSC treatment, as well as FAKTyr576/577 phosphorylation. In contrast, the addition of recombinant CTGF in vitro stimulated the fibrogenesis of human granulosa cells (KGN), whereas UC-MSC treatment inhibited the phosphorylation of FAK-Tyr576/577 and downregulated the expression of Collagen 1 and α-SMA, presumably alleviating the development of ovarian fibrosis by inhibiting the phosphorylation of theFAK-Tyr576/577 pathway.

Interpretation: This study demonstrated that UC-MSC treatment improved ovarian function and reduced ovarian fibrosis in MRL/lpr mice through CTGF/FAK-Tyr576/577 phosphorylation.

Funding: This work was supported by the National Key R&D Program of China (2020YFA0710804), the Key Program of National Natural Science Foundation of China (81930043), and Jiangsu Provincial Key Research and Development Program (BE2020621).

Declaration of Interest: The authors have declared that no conflict of interest exists.

Ethical Approval: All animal experiments followed the institutional guidelines of the Affiliated Drum Tower Hospital of Medical School of Nanjing University. The study was conducted in accordance with the National Research Council Guidance for Care and Use of Laboratory Animals and approved by the Committee of Experimental Animal Administration of the Affiliated Drum Tower Hospital of Medical School of Nanjing University (Approval number: 2022AE01012).

Keywords: Mesenchymal stem cells, Systemic lupus erythematosus, Ovarian insufficiency, Fibrosis, Focal adhesion kinase (FAK)