R. intestinalis 对艰难梭菌感染小鼠肠道黏膜的保护作用及机制研究

Clostridium difficile infections (CDI) is considered to be the main cause of bacterial infectious diarrhea in nosocomial setting and the dysbiosis of intestinal microbiota is strongly associated with CDI. We found some alterations of species and metabolisms of the human intestinal flora early in CDI patients. Butyrate-producing anaerobic bacteria, especially R.intestinalis from Lachnospiraceae family are significantly depleted, which may play vital roles in the developemt of CDI. Our previous preliminary study also shows that R.intestinalis has the potential protective effects for the intestinal barrier, while the mechanisms remain unclear. In this project, we aim to explore the protective effects from the intestinal microbial composition, intestinal inflammation, intestinal physical barrier and immune barrier in a mouse model of CDI. And we also try to understand the role of R.intestinalis on the expression of the NOD1 key regulatory factor by up-regulating and down-regulating the expression of NOD1 in Caco-2 cells. Meanwhile, we also explore the changes of key protein in the NOD1 related signaling transduction pathways, and the expression of inflammatory cytokines. According to our present study, we will understand the specifc mechanisms of R.intestinalis in the intestinal mucosal protection in a deeper level, which will reveal the role of R.intestinalis in the development of CDI and provide a theoretical basis for R.intestinalis to be developed into probiotics.

艰难梭菌感染(CDI)是院内感染性腹泻及肠炎的重要病因之一，肠道菌群紊乱与其发生密切相关。我们前期发现CDI患者肠道菌群种类和代谢改变，产丁酸厌氧菌比例明显降低，其中以毛螺菌科R.intestinalis 降低最显著，其可能为CDI发生发展中的潜在关键功能菌。课题组预实验也发现R.intestinalis具有潜在肠道黏膜保护功能，但作用机制不明。课题组拟构建R.intestinalis 干预小鼠艰难梭菌感染动物模型，从肠道菌群、炎症指标、肠道黏膜屏障等方面观察其肠道黏膜保护作用，并正、反向调控Caco-2细胞上NOD1的表达，探明R.intestinalis对NOD1表达关键调控因子的影响，观察相关信号转导通路蛋白活化情况及下游炎症因子表达差异，明确其对肠道黏膜保护作用的分子机制，进而揭示在CDI发生发展中的作用，为今后开发R.intestinalis为肠道益生菌防治CDI提供理论依据。

抗生素相关的菌群失调是艰难梭菌感染(CDI)发病和进展的关键因素。课题组前期研究发现CDI患者中与产丁酸相关的Roseburia intestinalis显著降低，其可能是CDI相关的关键功能菌。我们构建了R.intestinalis干预小鼠艰难梭菌感染动物模型，结果发现其对艰难梭菌感染小鼠的肠道黏膜具有显著的保护性作用，R.intestinalis干预可以防止体重减轻，减少结肠的组织学损伤，并抑制炎症细胞因子IL-4、IL-9、IL-10和TNF-α等。R.intestinalis干预小鼠肠道微生物群组成发生改变，包括Akkermansia在内的有益共生菌显著富集。更重要的是，R.intestinalis可以增加乙酸、丙酸、丁酸等短链脂肪酸的生成。肠道组织转录组分析发现R.intestinalis干预可显著降低CDI诱导的炎症因子相关通路中肠转录基因的上调。.同时，本研究通过调控HT-29细胞NOD1的表达和对关键信号蛋白阻断，观察R.intestinalis发酵产物对相关信号转导通路蛋白表达及下游炎症因子的影响。结果发现，应用NOD1 siRNA处理可显著降低HT-29细胞中NOD1的表达，NOD1 siRNA干扰后HT-29细胞的炎症因子变化趋势和正常组相似，但IL-10和IFN-r等炎症因子的水平显著下降，提示R.intestinalis发酵产物可能通过NOD1介导的通路发挥抗炎因子的调控作用。对NF-KB和MAPKs信号转导通路关键蛋白进行特异性阻断，发现p65、JAK在NOD1 siRNA干扰HT-29细胞中表达显著降低，提示R.intestinalis可能通过该通路发挥作用。R.intestinalis归属于产丁酸毛螺菌科，且R.intestinalis干预可显著逆转CDI 诱导的丁酸减少，提示其免疫调控作用可能是通过其代谢产物丁酸，我们研究发现丁酸钠可在体外抑制IL-6/JAK2/STAT3信号传导，后续需进一步深入探究R.intestinalis是否亦通过阻断IL-6/JAK2/STAT3轴的活化来发挥抗炎作用。.综上所述，肠道产丁酸菌R.intestinalis通过维持肠道上皮完整性和调节免疫反应、肠道菌群和代谢物组成来发挥对艰难梭菌感染小鼠肠道黏膜的保护作用。本研究为今后开发R.intestinalis用作改善CDI的潜在制剂提供理论依据。