Growth hormone combined with estrogen improves intrauterine adhesion fibrosis by downregulating endometrial microbial citraconic acid to target β-catenin protein

Abstract

Intrauterine adhesions (IUAs) are a major cause of secondary infertility. This study aimed to investigate the therapeutic effects and mechanisms of combined recombinant rat growth hormone (rrGH) and estrogen therapy on endometrial fibrosis in IUA rats. Our findings revealed that IUA rats exhibited severe endometrial damage, heightened inflammatory responses, significant collagen deposition, and imbalances in various inflammatory and growth factors. However, these pathological changes were markedly improved following combined rrGH and estrogen treatment. Additionally, the endometrial microbial diversity in IUA rats was significantly reduced, and the combined therapy effectively promoted its restoration. Biochemical serum analysis showed that the combined therapy upregulated key reproductive hormone levels. Notably, the combined application of rrGH and estrogen partially restored GH receptor levels. TGF-β1, MMP9, and β-catenin were upregulated in the endometria of IUA rats, while the p-smad3/smad3 ratio was downregulated, and these key indicators were reversed after combined therapy. Furthermore, antibiotic treatment weakened the effects of combined therapy, indicating the role of endometrial microbiota in IUA. Molecular docking results revealed a high affinity between β-catenin and differential metabolites such as citraconic acid, suggesting their potential importance in regulating the β-catenin signaling pathway. In a TGF-β1-induced IUA cell model, we found that TGF-β1 treatment upregulated fibrosis-related protein expression but decreased β-catenin protein levels and stability. Citraconic acid intervention enhanced the effects of TGF-β1, while β-catenin overexpression inhibited these changes. In summary, the combined therapy targeting the β-catenin pathway through citraconic acid regulation alleviated endometrial fibrosis, offering a new approach to treating IUA.IMPORTANCEIntrauterine adhesions (IUAs) are an important endometrial disease. Our study highlights the importance of the combination of recombinant rat growth hormone (rrGH) and estrogen in ameliorating endometrial damage and fibrosis, as well as promoting endometrial regeneration in IUA rats. In addition, our study emphasizes their important role in ameliorating microecological disturbances in the intrauterine environment and regulating serum metabolism. Our experiments also revealed for the first time that the combination of rrGH and estrogen may modulate endometrial microbes or influence the progression of IUA by promoting β-catenin expression, which is important for understanding the treatment of IUA disease. Our study provides new and important insights into the understanding and treatment of IUA disease.

Keywords: citraconic acid; endometrial microbiota; estrogen; growth hormone; intrauterine adhesions; β-catenin pathway.

Fig 1

rrGH combined with estrogen improves endometrial injury and fibrosis in IUA rats. (A and B) HE and Masson staining of the uterus before (above) and after (below) treatment (scale bar: 250 µm). (C) The thickness of the endometrium and epithelium before (left) or after (right) treatment. (D and E) Immunohistochemistry staining analysis of collagen I expression in the endometrial tissue of IUA rats (scale bar: 100 µm). *P < 0.05 vs Control group, ^#P < 0.05 vs IUA group, and ^&P < 0.05 vs Estrogen group; n = 3.

Fig 2

rrGH combined with estrogen inhibits endometrial inflammation and promotes endometrial regeneration in IUA rats. (A and B) IHC staining of CK-18 and vimentin in endometrium before (above) and after (below) treatment (scale bar: 100 µm). (C) The mRNA expression of TNF-α, IL-4, IL-6, and IL-10 was assessed by quantitative real-time PCR (qRT-PCR). (D) The concentration of TNF-α, IL-4, IL-6, and IL-10 was detected by ELISA. (E) The mRNA expression of IGF-1, SDF-1, and TSP-1 was tested by qRT-PCR. *P < 0.05 vs Control group, ^#P < 0.05 vs IUA group, and ^&P < 0.05 vs Estrogen group; n = 3.

Fig 3

rrGH combined with estrogen regulates microbial diversity in the endometrium of IUA rats. (A) Alpha analysis of microbial changes. (B) Venn plot showing changes in microbial numbers. (C) Lefse analysis of microbiota at the genus level in the endometrium of rats.

Fig 4

Effects of rrGH combined with estrogen on peripheral blood metabolism in IUA Rats. (A) The levels of FSH, LH, E2, P, T, and PRL in the peripheral blood of rats were measured by ELISA. (B) PCA grade outcomes of rat serum samples. (C and D) Volcano map showing differential metabolites. (E) Abundance analysis of differential metabolites. *P < 0.05 vs Control group, ^#P < 0.05 vs IUA group, and ^&P < 0.05 vs Estrogen group.

Fig 5

rrGH combined with estrogen to alleviate endometrial fibrosis and promote β-catenin expression in IUA rats. (A) The level of GHR in the peripheral blood and endometrium of rats was detected by ELISA. (B and C) IHC staining of TGF-β1 in the endometrium of rats (scale bar: 100 µm). (D and E) The expressions of p-smad3, smad3, MMP9, and β-catenin were assessed by WB. *P < 0.05 vs Control group, ^#P < 0.05 vs IUA group, and ^&P < 0.05 vs Estrogen group; n = 3.

Fig 6

rrGH combined with estrogen to ameliorate IUA through the modulation of endometrial microbiota. (A and B) HE and Masson staining of the uterus before and after treatment (scale bar: 250 µm). (C) The thickness of the endometrium and epithelium before and after treatment. (D and E) IHC staining of collagen I expression in the endometrial tissue of IUA rats (scale bar: 100 µm). (F and G) IHC staining of CK-18 and vimentin in the endometrial tissue of IUA rats (scale bar: 100 µm). (H) The concentration of TNF-α, IL-4, IL-6, and IL-10 was detected by ELISA. (I) The mRNA expression of IGF-1, SDF-1, and TSP-1 was tested by quantitative real-time PCR. (J) The level of GHR in peripheral blood and endometrium of rats was detected by ELISA. (K) The expressions of p-smad3, smad3, MMP9, and β-catenin were assessed by WB. *P < 0.05 vs Control group, ^#P < 0.05 vs IUA group, and ^&P < 0.05 vs Estrogen + rrGH group; n = 3.

Fig 7

Citraconic acid targets β-catenin to regulate TGF-β1-induced IUA model in vitro. (A) The level of citraconic acid was determined by high-performance liquid chromatography-tandem mass spectrometry. (B and C) IF staining of α-SMA and collagen I in ESCs (scale bar: 25 µm). (D) The expressions of β-catenin were evaluated by WB. (E) The stability of β-catenin protein was detected by WB. *P < 0.05 vs Control group, ^#P < 0.05 vs IUA group. (F) Co-immunoprecipitation (CO-IP) was used to detect the binding of β-catenin with AXIN, APC, CK1, GSK-3β, and β-TrCP. (G) CO-IP was used to detect the ubiquitination level of β-catenin. (H) Cell proliferation was detected by CCK-8. (I and J) IF staining of α-SMA and collagen I in ESCs. (K) The expressions of β-catenin, GSK-3β, and cyclin D1 were evaluated by WB. *P < 0.05 vs oe-NC group, ^#P < 0.05 vs oe-β-catenin group; n = 3.