Rac1活性变化对炎症性肠病巨噬细胞清除细菌能力的影响及机制研究

Though the pathogenesis of Inflammatory Bowel Disease (IBD) remainns unclear, the impaired bacetrial clearance of macrophages is one of the key mechanisms. Rac1, whose active form is Rac1-GTP, is an important signaling molecule mediating the ability of bacterial clearance of macrophages. Recent research identified Rac1 as an IBD susceptibility gene,the expression of which is increased in IBD patients. We have found that the immunosuppresant Azathioprine significantly inhibited the levels of Rac1-GTP and the activities of its downstream effectors in macrophages, whereas consequently increased bacterial phagocytosis of macrophages. However, the activation status of Rac1 signaling pathway of macrophages in IBD, and its effects on bacterial clearance and the disease treatment are still unknow. In the present study, by the usage of GLISA? and immunofluoresent methods, we will study the Rac1-GTP levels and the cytoskeletal morphology of monocyte-derived macrophages from peripheral blood of IBD patients, getting to know the Rac1 activation status of these cells. Furthermore, by performing in vitro and in vivo experiments, we will study how the Rac1 activation statuses of IBD macrophages influence bacterial clearance with respect to phagocytosis, degradation and killing. Moreover, we will analyze how the IBD susceptibilith gene NOD2 regulate Rac1 activation of macrophages and also the bacterial clearance. Our data will provide valuable information for clarifying the pathogenesis of IBD and for developing the strategies of personalized therapy.

炎症性肠病(IBD)发生机制未明，但巨噬细胞对细菌清除障碍是其关键机制之一。Rac1是调控巨噬细胞清除细菌能力的重要信号分子，Rac1-GTP是其活性形式，最近研究发现Rac1是IBD易感基因，在IBD患者中表达增加。我们发现，免疫抑制剂硫唑嘌呤显著抑制巨噬细胞中Rac1-GTP水平和下游信号活性，并增强单核细胞吞噬细菌的能力。然而，关于IBD患者巨噬细胞中Rac1信号通路的活性，及其在细菌清除和疾病治疗中的作用尚未见报道。本研究应用GLISA?和免疫荧光方法检测IBD患者外周血单核衍生的巨噬细胞Rac1-GTP和细胞骨架形态，分析Rac1活化状态；通过体外实验和动物实验观察IBD巨噬细胞Rac1信号通路活性改变如何影响细菌清除过程中的吞噬、降解和杀菌；探讨IBD易感基因NOD2如何影响巨噬细胞Rac1活性和细菌清除功能。为阐释IBD发病机制和探索个体化治疗策略奠定基础。

本研究旨在证实炎症性肠病（inflammatory bowel disease, IBD) 患者巨噬细胞Rac1活性改变是其清除细菌能力下降的分子机制，对Rac1活性进行有效的调控可改变巨噬细胞清除细菌的能力和减少粘膜炎症的发生。IBD易感基因NOD2突变是巨噬细胞Rac1活性失调的原因之一。该目标的实现为阐明IBD的病因和进行个体化提供理论依据。. 我们研究发现，IBD患者肠粘膜炎症部位较非炎症部位和健康对照者肠粘膜的巨噬细胞数量增多，同时这些细胞中Rac1活性显著增强。在体外实验中，细菌成分PGN显著增加Rac1的活性，且硫唑嘌呤显著抑制Rac1的活性，其促进和抑制的作用均呈剂量依赖性和时间依赖性。重要的是，Rac1活化后细胞的吞噬作用减弱，抑制Rac1活性可以增加细胞的吞噬功能。细菌成分PGN表现抗凋亡作用而硫唑嘌呤活性成分6-TG表现促凋亡作用（对照组、PGN组和6-TG组早期凋亡率分别为12.7±2.2%、8.6±3.7%和22.3±5.6%, P＜0.05）。重要的是，6-TG显著增加PGN相关的细胞凋亡 (42.0±2.7)%。同样地，Rac1抑制剂NSC23766显著增加PGN相关的细胞凋亡率(58.5±6.9) %。在外周血单核细胞中，PGN上调凋亡信号NF-κB、Bcl-xL和STAT-3、下调细胞Caspase-3水平，而6-TG显著下调这些信号分子活性，其作用在PGN存在时更加显著。NSC23766与6-TG对上述通路的调控作用具有一致性。