新生儿坏死性小肠结肠炎中肠道菌群失调介导肠道粘膜屏障损伤的病理机制研究

Currently, the pathogenesis of NEC is believed to be multifactorial, including dysbiosis of the gut and impairment of the intestinal mucosal barrier. Tight junctions serve to form tight seals between intestinal epithelial cells and are thought to be extremely important to the intergrity of the mucosal barrier. In our pervious study, we have demonstrated that preterm newborns with NEC had less bacterial diversity, an increase in abundance of Proteobacteria, and a decrease in other bacteria phyla. In addition, the structure and function of tight junction was impaired in both NEC newborns and rat pups. Lipopolysaccharides (LPS) treatment could reduce the expression of tight junction- and cytoskeleton-associated molecules such as claudin-1 and F-actin. However, the underlying mechanism is still unclear. It has been reported that HIF-1α could bind the hypoxia response elements in the claudin-1 promoter region. We hypothesize that dysbiosis-mediated regulation of the intestinal mucosal barrier in NEC via the TLRs/Myd88/HIF-1α pathway. In supporting this hypothesis, we first assess the diversity and microbial succession in human and rat NEC, using 16S rRNA gene-base sequencing method. Next, the effect of dysbiosis on the mucosal barrier is detected in rat NEC model and intestinal epithelial cells by immunostaining, western blot, transmission electron microscopy. Intestinal permeability is also measured by detecting transepithelial electrical resistance (TER) and FITC-dextran concentrations. To determine whether the TLRs/Myd88/HIF-1α signaling pathway is involved, Myd88-deficient and Myd88-overexpressed enterocytes will be generated by transduction of adeno-associated virus particles. HIF-1α siRNA is also generated. The expression and nuclear translocation of HIF-1α were measured in vitro and in vivo. The expression of tight junction proteins (claudin-1 and its phosphorylation, and other molecules such as occludin, claudin-2, ZO-1, etc.), Rho activation, MLCK activity, and cytoskeleton-associated proteins will also be measured. At last, the effect of probiotics on the incidence of NEC and regulation of the structure and function of tight junction are detected. Current work will support the manipulation of the gut microbiota and regulation of the intestinal mucosal barrier in the prevention and treatment of NEC in the future.

肠道粘膜屏障损伤是导致新生儿坏死性小肠结肠炎（NEC）的重要因素。紧密连接（TJ）是维持肠道粘膜屏障的重要结构。本项目关注的科学问题是：NEC中肠道菌群失调影响肠道粘膜屏障的致病机制。我们预实验发现：NEC早产儿肠道菌群中变形菌门数量显著增加；细菌成分LPS可减少TJ关键蛋白claudin-1的表达，而且TJ结构和功能受损。鉴此，本项目拟研究和明确NEC中肠道菌群动态分布特征；明确LPS处理对肠上皮细胞株和NEC疾病模型大鼠肠上皮细胞中HIF-1α核转位活性及其靶基因claudin-1的表达和亚细胞分布、细胞骨架重组相关蛋白表达和TJ超微结构的影响；明确微生态制剂对NEC模型的干预效果以及对各组大鼠肠壁中TJ结构和功能的影响。籍此，揭示肠道菌群失调通过TLRs/Myd88/HIF-1α通路介导肠道粘膜屏障损伤的分子机制，并为优化NEC肠道粘膜屏障损伤的治疗策略提供新的理论视野和实验依据。

坏死性小肠结肠炎（necrotizing enterocolitis，NEC）是新生儿期特有的肠道炎症性疾病，是导致新生儿尤其是早产儿和低出生体重儿死亡的病因之一。研究认为，益生菌可通过调节肠道菌群结构，减少病原菌定植，降低NEC的发病率和死亡率，但其中的具体作用机制尚不明确。在本研究中，通过建立NEC小鼠模型，验证目前临床常用的益生菌双歧杆菌四联活菌复合物对NEC的作用，并通过16SrDNA测序技术检测肠道菌群结构变化，通过分离肠道固有层免疫细胞，应用流式细胞术分析免疫细胞功能变化，同时通过检测肠道上皮细胞紧密连接相关蛋白变化、肠道渗透性变化等指标，评估肠道黏膜屏障功能改变，并深入探讨其中的作用机制。同时，深入探究了低氧诱导因子（hypoxia-induced factor-1a, HIF-1α）在新生儿坏死性小肠结肠炎 （necrotizing enterocolitis, NEC）发病过程中对肠道屏障的调节作用及其分子机制。本研究认为，双歧杆菌四联活菌复合物可通过调节肠道菌群结构，抑制RORγt+细胞炎症因子表达，减轻肠道炎症。并进一步证实肠道屏障损伤在NEC发生中的作用，并且揭示双歧杆菌四联活菌复合物可能通过促进肠道上皮细胞PXR表达，同时抑制JNK的磷酸化，促进紧密连接相关蛋白表达，增强肠黏膜屏障功能，从而减轻NEC。本研究结果将对阐明益生菌减轻NEC的作用机制提供理论依据。而HIF-1α在NEC发病过程中可能起到适应性保护的作用，其作用机制可能是通过激活HIF-1α/Claudin-1通路对肠道屏障发挥保护作用。