Vitali Roberta, Palone Francesca, Armuzzi Alessandro, Fulci Valerio, Negroni Anna, Carissimi Claudia, Cucchiara Salvatore, Stronati Laura
Division of Health Protection Technologies, Territorial and Production Systems Sustainability Department, ENEA, Rome, Italy.
IBD Center, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.
J Crohns Colitis. 2023 Jan 27;17(1):92-102. doi: 10.1093/ecco-jcc/jjac110.
Faecal biomarkers have emerged as important tools in managing of inflammatory bowel disease [IBD], which includes Crohn's disease [CD] and ulcerative colitis [UC].
To identify new biomarkers of gut inflammation in the stools of IBD patients using a proteomic approach.
Proteomic analysis of stools was performed in patients with both active CD and CD in remission and in controls by 2-DIGE and MALDI-TOF/TOF MS. An ELISA was used to confirm results in a second cohort of IBD patients and controls.
2-DIGE analysis detected 70 spots in the stools of patients with active CD or patients in remission CD and in controls. MALDI-TOF/TOF MS analysis identified 21 proteins with Chymotrypsin C, Gelsolin and Rho GDP-dissociation inhibitor 2 [RhoGDI2] best correlating with the levels of intestinal inflammation. Results were confirmed in a second cohort of IBD patients and controls [57 CD, 60 UC, 31 controls]. The identified faecal markers significantly correlated with the severity of intestinal inflammation in IBD patients [SES-CD in CD, Mayo endoscopic subscore in UC] [CD; Chymotrypsin-C: r = 0.64, p < 0.001; Gelsolin: r = 0.82, p < 0.001; RhoGDI2: r = 0.64, p < 0.001; UC; Chymotrypsin-C: r = 0.76, p < 0.001; Gelsolin: r = 0.75, p < 0.001; RhoGDI2: r = 0.63, p < 0.001]. Moreover, ROC analysis showed that Gelsolin [p < 0.0002] and RhoGDI2 [p < 0.0001] in CD, and RhoGDI2 [p = 0.0004] in UC, have higher sensitivity and specificity than faecal calprotectin in discriminating between patients and controls.
We show for the first time that 2-DIGE is a reliable method to detect proteins in human stools. Three novel faecal biomarkers of gut inflammation have been identified that display good specificity and sensitivity for identifying IBD and significantly correlate with IBD severity.
粪便生物标志物已成为炎症性肠病（IBD，包括克罗恩病（CD）和溃疡性结肠炎（UC））管理中的重要工具。
采用蛋白质组学方法鉴定IBD患者粪便中肠道炎症的新生物标志物。
通过二维差异凝胶电泳（2-DIGE）和基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF/TOF MS）对活动期CD患者、缓解期CD患者及对照者的粪便进行蛋白质组分析。采用酶联免疫吸附测定（ELISA）在另一组IBD患者和对照者中验证结果。
二维差异凝胶电泳分析在活动期CD患者、缓解期CD患者及对照者的粪便中检测到70个斑点。基质辅助激光解吸电离飞行时间串联质谱分析鉴定出21种蛋白质，其中胰凝乳蛋白酶C、凝溶胶蛋白和Rho GDP解离抑制剂2（RhoGDI2）与肠道炎症水平相关性最佳。在另一组IBD患者和对照者（57例CD患者、60例UC患者、31例对照者）中验证了结果。所鉴定的粪便标志物与IBD患者肠道炎症的严重程度显著相关（CD患者的简化内镜评分（SES-CD）、UC患者的梅奥内镜亚评分）（CD患者中，胰凝乳蛋白酶C：r = 0.64，p < 0.001；凝溶胶蛋白：r = 0.82，p < 0.001；RhoGDI2：r = 0.64，p < 0.001；UC患者中，胰凝乳蛋白酶C：r = 0.76，p < 0.001；凝溶胶蛋白：r = 0.75，p < 0.001；RhoGDI2：r = 0.63，p < 0.001）。此外，受试者工作特征（ROC）分析显示，在区分患者和对照者方面，CD患者中的凝溶胶蛋白（p < 0.0002）和RhoGDI2（p < 0.0001），以及UC患者中的RhoGDI2（p = 0.0004）比粪便钙卫蛋白具有更高的敏感性和特异性。
我们首次表明二维差异凝胶电泳是检测人粪便中蛋白质的可靠方法。已鉴定出三种新型肠道炎症粪便生物标志物，它们在识别IBD方面具有良好的特异性和敏感性，且与IBD严重程度显著相关。
