GNMT inhibits intrauterine adhesion fibrosis through TGF-β1/Smad3 signaling pathway and its mechanism

Abstract

Objective To investigate the effect of glycine N-methyl transferase (GNMT) on intrauterine adhesion (IUA) fibrosis and its related mechanism. Methods In vivo experiment: A total of 36 healthy female SD rats (SPF grade, 6~8 weeks old and weighing from 180~220 g) were subjected in this study. IUA model of SD rats and IUA model of GNMT overexpressed rats were established. RT-qPCR and immunofluorescence assay were applied to detect GNMT expression level in normal uterus and model group. RT-qPCR and Western blotting were used to detect the mRNA and protein levels of fibrosis-related molecules and the activation of TGF-β1/Smad3 signaling pathway in each group. The number of endometrial glands in each group was observed by HE staining. Masson staining was used to analyze the severity of endometrial fibrosis in each group. In vitro experiment: transformed human endometrial stromal cells (THESCs) fibrotic phenotype model was constructed using TGF-β1, and THESCs stably transfected with GNMT overexpression lentvirus were treated with TGF-β1. RT-qPCR and Western blotting were used to detect the mRNA and protein expression of fibrosis-related molecules. The expression of TGF-β1/Smad3 signaling pathway was detected by Western blotting. TGF-β1/Smad3 signaling pathway was activated by TGF-β1/Smad signaling pathway activator (SRI-011381), and the expression of TGF-β1/Smad3 signaling pathway and key molecular proteins of fibrosis phenotype was measured with Western blotting. Results In vivo experiment, the mRNA and protein expression levels of GNMT were significantly decreased in the IUA rats than the control rats (P<0.05). Overexpression of GNMT decreased the mRNA and protein levels of fibrosis related molecules, Collagen Ⅰ, Collagen Ⅲ and FN in the IUA rats (P<0.05), and decreased the phosphorylation levels of TGF-β1 and its downstream Smad3 protein (P<0.05). HE and Masson staining showed that overexpression of GNMT could increase the number of endometrial glands and reduce the severity of fibrosis in the IUA rats (P<0.05). In vitro experiments: overexpression of GNMT decreased the mRNA and protein levels of Collagen Ⅰ, Collagen Ⅲ and FN associated with fibrotic phenotype of THESCs (P<0.05), and reduced the phosphorylation level of Smad3 protein, downstream of TGF-β1 (P<0.05). After activation of TGF-β1/Smad3 signaling pathway, the protein levels of TGF-β1/Smad3 signaling pathway and downstream fibrosis phenotype molecules, Collagen Ⅲ and FN, were significantly decreased in the LV-GNMT+SRI-011381 group. Conclusion Overexpression of GNMT can inhibit endometrial fibrosis by regulating TGF-β1/Smad3 signaling pathway, thus achieving therapeutic effect on IUA.

Keywords

• glycine n-methyl transferase
• intrauterine adhesion
• fibrosis
• tgf-β1/smad3