南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 206-212.doi: 10.12122/j.issn.1673-4254.2023.02.07

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WNT2b高表达的成纤维细胞破坏肠道黏膜屏障

肖姝喆，成燕玲，朱 云，唐 芮，古建标，兰 林，何志华，刘丹琼，耿岚岚，程 旸，龚四堂   

  1. 广州医科大学附属广州市妇女儿童医疗中心，广东省儿童健康与疾病临床医学研究中心，消化科，广东 广州 510120；器官衰竭防治国家重点实验室，广东省病毒性肝炎研究重点实验室，南方医科大学南方医院感染内科，广东 广州 510515；南方医科大学第一临床医学院，广东 广州 510515
  • 出版日期:2023-02-20 发布日期:2023-03-16

Fibroblasts overpressing WNT2b cause impairment of intestinal mucosal barrier

XIAO Shuzhe, CHENG Yanling, ZHU Yun, TANG Rui, GU Jianbiao, LAN Lin, HE Zhihua, LIU Danqiong, GENG Lanlan, CHENG Yang, GONG Sitang   

  1. Department of Digestive Diseases, Guangzhou Women and Children's Medical Center,Guangdong Provincial Clinical Research Center for Child Health, Guangzhou Medical University,Guangzhou 510623,China; State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 探讨WNT2b高表达成纤维细胞破坏肠黏膜，促进炎症性肠病（IBD）进展的机制。方法 通过体外细胞共培养及条件培养，构建成纤维细胞对Caco-2细胞的作用模型。实验组为加入20%成纤维细胞条件培养基或与WNT2b高表达成纤维细胞共培养，对照组为不含条件培养基或与野生型成纤维细胞共培养，通过跨膜电阻和荧光黄渗透率测定，评估Caco-2细胞屏障通透性的变化。与WNT2b高表达或对照肠道成纤维细胞共培养后，使用免疫荧光法检测Caco-2细胞β-catenin入核情况，使用Western blot法检测ZO-1、E-Cadherin等紧密连接蛋白表达情况。采用DSS（葡聚糖硫酸钠）对C57小鼠进行类IBD肠炎造模，实验组使用Salinomycin（5 mg/kg，腹腔注射），对照组为对应溶剂生理盐水，分别对小鼠进行处理，评价该抑制剂对肠炎的治疗作用。结果 与对照组相比，过表达WNT2b基因可致成纤维细胞分泌WNT2b蛋白显著增加，并促进Caco-2细胞β-catenin入核（P<0.01），紧密连接蛋白表达下降；Caco-2细胞中敲低FZD4表达可逆转这一作用。Salinomycin可明显减轻对DSS小鼠肠道炎症并使肠黏膜紧密连接蛋白表达增多。结论 WNT2b高表达成纤维细胞破坏肠黏膜屏障功能，是治疗IBD的潜在靶点。

关键词: 炎症性肠病；成纤维细胞；WNT2b；WNT/β-catenin信号通路

Abstract: Objective To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). Methods Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild- type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E- cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. Results In the co-culture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P<0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS- treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. Conclusion Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.

Key words: inflammatory bowel disease; fibroblasts; WNT2b; WNT/β-catenin signaling pathway