Roseburia hominis对肝硬化大鼠肠道黏膜保护作用及机制研究

Alterations in the gut microbiota are important in complications of end-stage liver cirrhosis. Mutations in the NOD2 gene are strongly associated with liver cirrhosis , which disturbed the right reconization of pathogen assocaited molecular patterns from the intestine. Previous studies have shown that In liver cirrhosis the numbers of R. hominis are significantly lower compared to control. Our previous preliminary study also shows that R. hominis has the potential protective effects for the intestinal barrier and liver, 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 CCL4 –induced LC animal models. And we also try to understand the role of R. hominis 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 R. hominis in the intestinal mucosal protection in a deeper level, which will reveal the role of R. hominis in the development of LC and provide a scientific basis for R. hominis to be developed into probiotics.

肝硬化的发生发展与肠道菌群变化密切相关。NOD2 基因突变会造成肠黏膜屏障功能紊乱，促进肝硬化发生发展。本团队前期研究显示R. hominis在肝硬化病人肠道的含量显著下降，具有益生菌潜质。本课题组预实验也发现R. hominis具有潜在的保护肝硬化大鼠肝脏和肠黏膜的作用，但其机制仍不明确，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R. hominis干预肝硬化大鼠动物模型，从肠道菌群、肝脏损伤、肠道黏膜机械屏障和免疫屏障等方面观察其对肝脏和肠道黏膜的保护作用，并采用正向和反向调控Caco-2细胞上NOD2的表达，探明R. hominis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R. hominis对肠道黏膜保护作用的分子机制，为开发R. hominis为肠道益生菌提供科学依据。

本项目圆满完成了既定任务。动物实验结果显示：R.hominis具有减少肝损伤，保护肝功能、降低血液内毒素的作用；可以显著降低促炎因子IL-1和TNF-a水平，增加抑炎因子IL-13水平，增加炎症调节因子IL-18水平；改善肝细胞损伤及肠道机械屏障结构受损程度；可以升高肠组织ZO-1和NOD-2基因mRNA表达水平；可以显著升高肝硬化大鼠肠道菌群的多样性和丰富度。.细胞实验结果显示：IFN-γ和TNF-α显著升高NOD2的表达量，而siNOD2处理降低NOD2的表达；RH剂量依赖性的增加NOD2在上皮细胞的表达，在1×109具有显著促进的效果；RH显著促进TRAF4，SOCS-3，TNF-α，IFN-γ的分泌，但显著降低IL-10的分泌；RH显著激活Caco-2细胞的NF-kB信号通路；特异性抑制剂PDTC可降低RH升高的NOD2表达量，升高RH降低的炎症因子TRAF4和SOCS-3表达量。本课题资助的成果发表于中国微生态杂志、中国临床药理学杂志、Front Microbiol、J Glob Antimicrob Resist（均标注项目编号），培养博士研究生1名，硕士研究生2名。