Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models.
We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvβ5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo.
MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.
方法:使用三种不同的方案测试去细胞化全厚度肠组织平台,通过蛋白质组学研究不同组织表型的ECM组成,并通过定量PCR(qPCR)和免疫组织化学进行验证。评估了用乳脂球-表皮生长因子8(MFGE8)处理的原代人肠肌成纤维细胞(HIMFs)的抗纤维化反应机制,在两个实验性肠纤维化模型中测试了MFGE8的作用。
结果:我们建立并验证了肠道IBD组织的最佳去细胞化方案。Matrisome分析显示在CD狭窄(CD)组织中MFGE8表达升高,这在mRNA和蛋白质水平得到证实。用MFGE8处理抑制正常对照HIMF而不是CDHIMF中的ECM产生。下一代测序揭示了功能相关的整合素介导的信号通路,整合素αvβ5和粘着斑激酶的阻断使HIMF对MFGE8无反应。MFGE8在体外和体内预防和逆转实验性肠纤维化。
结论:MFGE8具有抗纤维化作用,并且其给药可能代表了预防IBD引起的肠狭窄的未来方法。