普氏栖粪杆菌的肠道黏膜保护作用及机制研究

Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, are chronic, relapsing inflammatory disorders of the intestine in genetically susceptible individuals exposed to environmental risk factors. Previous studies have shown that mutations in the NOD2 gene are strongly associated with susceptibility to IBD, which disturbed the right reconization of pathogen assocaited molecular patterns from the intestine. In addition, evidence has demonstrated that chronic inflammation of the intestinal tract caused by the dysbiosis of intestinal microbiota may be the initiating and durating factors of IBD. Faecalibacterium prausnitzii, an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients, plays vital roles in the developemt of IBD. Our previous preliminary study also shows that F. prausnitzii has the potential protective effects for the intestinal barrier, while its specific mechanisms remain unclear. Whether it could regulate the expression of NOD2 in the intestinal epithelium and NOD2-mediated signaling transduction pathways are still unknown. The aims of our present study are to explore the protective effects from the restoration of intestinal microibota, intestinal inflammation, intestinal physical barrier and immune barrier in a DSS-induce colitis animal models. And we also try to understand the role of F. prausnitzii on the expression of the proven NOD2 key regulatory factor by up-regulating and down-regulating the expression of NOD2 in Caco-2 cells. Then we also explore the changes of key protein or protein kinase in the NOD2 related signaling transduction pathways, and the expression of inflammatory cytokines. According to our present study, we will understand the specifc mechanisms of F. prausnitzii in the intestinal mucosal protection in a deeper level, which will reveal the role of F. prausnitzii in the development of IBD and provide a scientific basis for F. prausnitzii to be developed into probiotics.

炎症性肠病(IBD)是一种肠道慢性炎症性疾病，NOD2基因突变是IBD发生的关键易感因素。有证据显示肠道菌群失衡引起的肠道慢性炎症可能是其发病的始动和持续因素，而普氏栖粪杆菌(Fp)具有显著抗炎活性及益生菌活性，为IBD发生发展中的潜在关键功能菌。本课题组预实验也发现Fp具有潜在肠道黏膜保护功能，但其作用机制仍不明确，其是否通过调控NOD2的表达及其介导的信号转导通路发挥作用尚不可知。本课题组拟采用Fp干预小鼠结肠炎动物模型，从肠道菌群、肠道炎症指标、肠道黏膜机械屏障和免疫屏障等方面观察Fp的肠道黏膜保护作用，并采用正向和反向调控Caco-2细胞上NOD2的表达，探明Fp对NOD2表达关键调控因子的影响，观察相关信号转导通路蛋白或激酶的活化情况及下游炎症因子的表达差异，明确Fp对肠道黏膜保护作用的分子机制，从而揭示Fp在IBD发生发展过程中的作用规律，为开发Fp为肠道益生菌提供科学依据。

目的：肠道共生Faecalibacterium prausnitzii可参与肠道黏膜免疫调控，对维持宿主肠道免疫自稳、促进肠道健康等方面发挥着重要作用，是宿主肠道黏膜防御系统的重要组成部分，但肠道黏膜上皮细胞如何识别肠道共生F. prausnitzii，是否通过NOD2这一关键信号转导通路实现调控从而影响肠道黏膜免疫自稳，这一宿主―微生物相互作用的核心问题尚不清楚。方法 本研究通过NOD2 siRNA技术，观察F. prausnitzii活菌及其组分和发酵滤液对干扰前后HT-29细胞的下游产物表达的影响，同时应用信号蛋白特异性抑制剂对关键信号蛋白进行阻断，研究肠道共生F. prausnitzii发挥调控作用的可能机制，进而研究其对小鼠结肠炎动物模型肠道黏膜免疫的保护作用。结果：研究发现，NOD2 siRNA可有效干扰HT-29细胞的表达，F. prausnitzii活菌及其发酵滤液对抗炎因子IL-10和IFN-gamma的表达有促进作用，对炎症因子IL-12的表达有抑制作用，但对TNF-α无显著调控作用。NOD2 siRNA后，IL-10和IFN-gamma蛋白表达水平显著下降，提示肠道共生F. prausnitzii及其发酵产物可能通过NOD2介导的信号转导通路发挥作用。对NF-κB和MAPKs信号转导通路关键蛋白进行特异性阻断，未发现其对下游产物表达有显著的影响，提示肠道共生F. prausnitzii可能通过其他通路发挥作用。研究发现，肠道共生F. prausnitzii对小鼠结肠炎动物模型小鼠具有显著的肠道黏膜保护作用。结论：肠道共生F. prausnitzii具有显著的抗炎作用，可通过NOD2进行识别，其发酵产物可能是其主要的活性成分，是一株潜在的益生菌株。