Objective: To evaluate the changes of macrophages and expression of Rac1 in the inflammatory site of Crohn's disease, and to investigate the effects of 6-thioguanine (6-TG) and peptidoglycan on apoptosis of human peripheral blood monocyte-macrophage by regulating Rac1 signaling pathway. Methods: Ten patients with Crohn's disease and eight healthy controls diagnosed were enrolled at Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital from January 2013 to January 2014. The number of macrophages, apoptosis and expression of Rac1 in the inflammation sites and non-inflammation sites of intestinal mucosa were detected in both patients and controls. Peripheral blood mononuclear cells (PBMCs) were sorted by CD14 immunomagnetic beads. The apoptosis of monocytes, expression of Rac1 and related apoptosis signaling molecules were detected in patients treated with peptidoglycan, 6-TG and Rac1 inhibitor NSC23766 and another 15 healthy donors. Results: The number of macrophages and apoptotic cells significantly increased in the inflammatory group of Crohn's disease patients compared with the non-inflammatory group. The expression of PAK1, downstream molecular of Rac1 signaling pathway of macrophages was also significantly higher in the inflammatory group of Crohn's disease patients than that in healthy controls and non-inflammatory group. Compared with control group, anti-apoptotic signals (NF-κB, Bcl-xL and STAT-3) in PBMCs increased in the peptidoglycan group, while slightly decreased in 6-TG group. 6-TG and NSC23766 significantly promoted peptidoglycan-related anti-apoptosis [peptidoglycan group (8.6±3.7)%, peptidoglycan+ 6-TG group (42.0±2.7)%, peptidoglycan+ NSC23766 group (58.5±6.9)%, P<0.05]. Conclusions: Peptidoglycan plays a role in the pathogenesis of Crohn's disease by recruiting macrophages. However, 6-TG inhibits peptidoglycan-induced activation of Rac1 signaling pathway leading to macrophage apoptosis.
目的： 观察克罗恩病患者肠黏膜炎症部位巨噬细胞数量变化和Rac1信号表达情况，并探讨硫唑嘌呤活性成分6-硫代鸟嘌呤（6-TG）和细菌成分肽聚糖通过调控Rac1信号通路对人外周血单核-巨噬细胞凋亡的影响及机制。 方法： 选取2013年1月—2014年1月天津医科大学总医院消化科诊断为克罗恩病的10例患者及8例健康对照者，检测健康对照者、克罗恩病肠黏膜炎症部位及非炎症部位的肠黏膜活检组织中巨噬细胞的数量、凋亡和Rac1信号表达情况。另招募15例健康志愿者，抽取外周血，CD14免疫磁珠法分选外周血中单核细胞，以肽聚糖和/或6-TG、Rac1抑制剂NSC23766分组处理后检测单核细胞凋亡情况，并检测Rac1及相关凋亡信号分子的表达水平。 结果： 免疫荧光染色结果显示，在克罗恩病肠黏膜炎症组的肠黏膜中，巨噬细胞数量多于健康对照组和克罗恩病肠黏膜非炎症组，但凋亡的巨噬细胞数量无明显差异，巨噬细胞Rac1信号通路下游分子PAK1的表达水平亦显著高于健康对照组和克罗恩病肠黏膜非炎症组；在外周血单核细胞中，与空白对照组相比，肽聚糖组抗凋亡信号（NF-κB、Bcl-xL和STAT-3）水平升高，而6-TG组则略降低；6-TG和NSC23766可显著抑制肽聚糖诱导的抗细胞凋亡作用[肽聚糖组、肽聚糖+6-TG组、肽聚糖+NSC23766组凋亡率分别为（8.6±3.7）%、（42.0±2.7）%和（58.5±6.9）%，P<0.05]。 结论： 肽聚糖通过活化克罗恩病患者单核-巨噬细胞中的Rac1信号通路，发挥抗凋亡作用，而6-TG可改善克罗恩病Rac1信号的活化失衡，促进巨噬细胞凋亡，维持免疫稳态。.
Keywords: Apoptosis; Azathioprine; Crohn disease; Macrophages; Peptidoglycan; Rac1 signaling.