吉林大学学报(医学版) ›› 2024, Vol. 50 ›› Issue (5): 1266-1274.doi: 10.13481/j.1671-587X.20240510

• 基础研究 • 上一篇    

Wnt/β-catenin信号通路抑制剂MSAB对人子宫内膜基质细胞纤维化反应的影响

王飞娜1,米旭光2,林秀英2,付建华2,刘磊2,于歆悦1,臧欢欢1,刘霖君1,陈士玲2,方艳秋1,2()   

  1. 1.长春中医药大学临床医学院, 吉林 长春 130021
    2.吉林省人民医院生殖医学中心, 吉林 长春 130021
  • 收稿日期:2023-10-26 出版日期:2024-09-28 发布日期:2024-10-28
  • 通讯作者: 方艳秋 E-mail:yq.fang@163.com
  • 作者简介:王飞娜(1996-),女,陕西省咸阳市人,在读硕士研究生,主要从事子宫内膜纤维化及其治疗方面的研究。
  • 基金资助:
    吉林省科技厅科技发展计划项目(20230204036YY);吉林省卫健委卫生健康技术创新项目(2021lc059)

Effect of Wnt/β-catenin signaling pathway inhibitor MSAB on fibrogenic responses of human endometrial stromal cells

Feina WANG1,Xuguang MI2,Xiuying LIN2,Jianhua FU2,Lei LIU2,Xinyue YU1,Huanhuan ZANG1,Linjun LIU1,Shiling CHEN2,Yanqiu FANG1,2()   

  1. 1.School of Clinical Medicine, Changchun University of Chinese Medicine, Changchun 130021, China
    2.Reproductive Medicine Center, Jilin Provincial People’s Hospital, Changchun 130021, China
  • Received:2023-10-26 Online:2024-09-28 Published:2024-10-28
  • Contact: Yanqiu FANG E-mail:yq.fang@163.com

摘要:

目的 探讨Wnt/β-连环蛋白(β-catenin)信号通路抑制剂3-(4-甲基苯基磺酰胺基)苯甲酸甲酯(MSAB)对人子宫内膜基质细胞(HESCs)纤维化反应的影响,为MSAB应用于宫腔粘连(IUA)靶向治疗提供依据。 方法 体外培养正常HESCs,分为对照组和转化生长因子β1(TGF-β1)组;体外培养IUA患者粘连部分的HESCs,作为IUA组。采用Western blotting法检测TGF-β1作用不同时间(0、12、24、48和60 h)后各组细胞中纤维化标志蛋白Ⅰ型胶原α1(COL1A1)蛋白表达水平。采用MTT实验检测各组细胞增殖活性。采用Western blotting法检测对照组和IUA组细胞中细胞COL1A1、间质标志蛋白[N-钙黏蛋白和α-平滑肌肌动蛋白(α-SMA)]以及Wnt/β-catenin信号通路相关蛋白β-catenin表达水平。根据MSAB浓度,将正常HESCs分为0(对照组)、0.25、0.50、0.75和1.00 μmol·L-1 MSAB组,MTT实验检测各组细胞存活率。MSAB作用后,将正常HESCs分为对照组(正常HESCs)、TGF-β1组(10 μg·L-1 TGF-β1诱导正常HESCs 24 h后撤药,更换为完全养基并继续培养24 h)和MSAB组(10 μg·L-1 TGF-β1诱导正常HESCs 24 h后撤药,更换为含0.75 μmol·L-1 MSAB的完全培养基并继续培养24 h)。采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中上皮-间质转化(EMT)相关转录因子Snail、Slug、Smuc、ZEB1和ZEB2和COL1A1 mRNA表达水平,采用Western blotting法检测各组细胞中COL1A1蛋白、N-钙黏蛋白、α-SMA蛋白、β-catenin和c-myc蛋白表达水平。 结果 与对照组(TGF-β1作用0 h)比较,TGF-β1组作用12、24、48和60 h时HESCs 中COL1A1蛋白表达水平升高(P<0.05或P<0.01)。与对照组比较,IUA组和TGF-β1组HESCs增殖活性差异无统计学意义(P>0.05)。与对照组比较,IUA组HESCs 中COL1A1、β-连环蛋白、N-钙黏蛋白和α-SMA蛋白表达水平升高(P<0.05或P<0.01)。与对照组比较,0.75和1.00 μmol·L-1 MSAB组细胞存活率降低(P<0.05或P<0.01)。与对照组比较,TGF-β1组细胞中Snail、Slug和COL1A1 mRNA表达水平升高(P<0.05或P<0.01);与TGF-β1组比较,MSAB组细胞中Snail、Slug和COL1A1 mRNA表达水平降低(P<0.05或P<0.01)。与对照组比较,作用24 h时TGF-β1组细胞中COL1A1蛋白、N-钙黏蛋白、α-SMA蛋白、β-catenin和c-myc蛋白表达水平升高(P<0.01);与TGF-β1组比较,MSAB组细胞中COL1A1蛋白、N-钙黏蛋白、α-SMA蛋白、β-catenin和c-myc蛋白表达水平降低(P<0.05或P<0.01)。 结论 MSAB可抑制体外培养HESCs的纤维化反应,该结果为MASB应用于IUA靶向治疗提供了理论基础。

关键词: 子宫内膜纤维化, 转化生长因子β1, 上皮-间质转化, Asherman综合征, 宫腔粘连

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 vitroand 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

中图分类号: 

  • R711