Abstract:

Objective To discuss the effect of Wnt/β-catenin signaling pathway inhibitor methyl 3-｛［（4-methyl- phenyl）sulfonyl］ amino｝ benzoate（MSAB）on the fibrogenic response of the human endometrial stromal cells （HESCs）， and to provide the foundation for the application of MSAB in the target therapy of intrauteriue adhesion （IUA）. Methods The normal HESCs were cultured in vitro and divided into two groups： control group and transforming growth factor β1 （TGF-β1） group； the HESCs from the adhesion part of the IUA patients were cultured in vitro， regarded as IUA group. Western blotting method was used to detect the expression levels of fibrotic marker protein type Ⅰ collagen α1 （COL1A1） in the cells in various groups at different time points （0， 12， 24， 48， and 60 h） after treated with TGF-β1. MTT assay was used to detect the proliferation activities of the cells in various groups. Western blotting method was used to detect the expression levels of the fibrotic marker protein COL1A1， stromal marker proteins such as N-cadherin and α-smooth muscle actin （α-SMA）， and Wnt/β-catenin signaling pathway-related protein β-catenin in the cells in control and IUA groups. Based on the MSAB concentrations， the normal HESCs were divided into 0 （control）， 0.25， 0.50， 0.75， and 1.00 μmol·L^-1 MSAB groups， and MTT assay was used to detect the survival rates of the cells in various groups. After treated with MSAB， the normal HESCs were divided into control group （normal HESCs）， TGF-β1 group （10 μg·L^-1 TGF-β1 induced normal HESCs for 24 h then the drug was withdrawn， replaced with complete culture medium， and the cells continued to be cultured for 24 h）， and MSAB group （10 μg·L^-1 TGF-β1 induced normal HESCs for 24 h then the drug was withdrawn， replaced with a complete medium containing 0.75 μmol·L^-1 MSAB and the cells continued to be cultured for 24 h）. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of epithelial-mesenchymal transition （EMT）-related transcription factors Snail， Slug， Smuc， ZEB1， and ZEB2， and COL1A1 mRNA in the cells in various groups. Western blotting method was used to detect the expression levels of COL1A1， N-cadherin， α-SMA， β-catenin， and c-myc proteins in the cells in various groups. Results Compared with control group （after treated with TGF-β1 for 0 h）， the expression levels of COL1A1 proteins in the HESCs after treated with TGF-β1 for 12， 24， 48， and 60 h in TGF-β1 group were increased （P<0.05 or P<0.01）.Compared with control group， there was no significant difference in the proliferation activity of the HESCs in IUA and TGF-β1 groups （P>0.05）.Compared with control group， the expression levels of COL1A1， β-catenin， N-cadherin， and α-SMA proteins in the cells in IUA group were increased （P<0.05 or P<0.01）. Compared with control group， the survival rates of the cells in 0.75 and 1.00 μmol·L^-1 MSAB groups were decreased （P<0.05 or P<0.01）. Compared with control group， the expression levels of Snail， Slug， and COL1A1 mRNA in the cells in TGF-β1 group were increased （P<0.05 or P<0.01）； compared with TGF-β1 group， the expression levels of Snail， Slug， and COL1A1 mRNA in the cells in MSAB group were decreased （P<0.05 or P<0.01）. Compared with control group， after treated with TGF-β1 for 24 h， the expression levels of COL1A1， N-cadherin， α-SMA， β-catenin， and c-myc proteins in the cells in TGF-β1 group were increased （P<0.01）； compared with TGF-β1 group， the expression levels of COL1A1， N-cadherin， α-SMA， β-catenin， and c-myc proteins in the cells in MSAB group were decreased （P<0.05 or P<0.01）. Conclusion MSAB can inhibit the fibrogenic responses of the HESCs in vitro，and the results provide the theoretical basis for the application of MSAB in the target therapy of IUA.

Key words: Endometrial fibrosis, Transforming growth factor-β1, Epithelial-mesenchymal transition, Asherman syndrome, Intrauterine adhesion