[Mechanism of intestinal injury induced by WNT2B high-expressed fibroblasts in Crohn's disease].

Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3β phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.目的： 探讨WNT2B基因高表达成纤维细胞通过激活巨噬细胞促进克罗恩病肠道组织损伤的机制。 方法： 生物信息分析、病理组织研究及细胞实验研究。生物信息分析方面，收集课题组前期炎症性肠病（IBD）患儿结肠组织细胞生物信息研究数据，再次进行单细胞测序分析。病理组织研究方面，以2022年7至9月于广州市妇女儿童医疗中心消化科住院行结肠镜检查确诊为克罗恩病的10例患儿为研究对象，每例患儿均分别取结肠炎症明显或溃疡部位组织及邻旁炎症轻微或正常部位组织，根据结肠镜下外观显示炎症明显或溃疡部位组织为炎症组，炎症轻且无溃疡部位组织为非炎症组。结肠组织行HE染色观察其病理情况，组织免疫荧光检测巨噬细胞浸润和CXCL12的表达情况。细胞实验研究方面，转染了WNT2B质粒或空载质粒的成纤维细胞分别与有或无经盐霉素处理的巨噬细胞共培养，蛋白质印迹法检测Wnt经典通路蛋白表达情况；使用SKL2001处理的巨噬细胞作为实验组，磷酸盐缓冲液处理的巨噬细胞为对照组，采用实时荧光定量PCR（qPCR）反应、酶联免疫吸附试验检测巨噬细胞CXCL12的表达及分泌情况。组间比较采用t检验或秩和检验。 结果： 单细胞测序分析提示巨噬细胞是IBD结肠组织的主要细胞，高表达WNT2B基因的成纤维细胞和巨噬细胞存在相互作用关系。病理组织研究中，10例患儿［年龄（9.3±3.8）岁，男7例、女3例］的结肠组织HE染色显示，炎症组结肠组织病理评分高于非炎症组［4（3，4）比2（1，2）分，Z=3.05，P=0.002］，组织免疫荧光提示每高倍镜视野中炎症组巨噬细胞浸润数明显高于非炎症组［（72.8±10.4）比（8.4±3.5）个，t=25.10，P<0.001］，炎症组每高倍镜视野中表达CXCL12的细胞明显多于非炎症组［（14.0±3.5）比（4.7±1.9）个，t=14.68，P<0.001］。细胞实验中，蛋白质印迹法提示与转染了WNT2B质粒的成纤维细胞共培养的巨噬细胞磷酸化的糖原合成酶激酶3β（p-GSK3β）水平升高，而盐霉素可逆转这一现象；qPCR提示实验组中CXCL12的转录水平高于对照组（6.42±0.04比1.00±0.03，t=183.00，P<0.001），酶联免疫吸附试验显示实验组中CXCL12的表达及分泌水平高于对照组［（465±34）比（77±9）ng/L，t=13.21，P=0.006］。 结论： WNT2B基因高表达成纤维细胞分泌WNT2B蛋白，激活巨噬细胞Wnt经典信号通路，从而促进巨噬细胞CXCL12的表达和分泌，诱导克罗恩病肠道炎症的发展。.