人乳寡糖单体DSLNT对新生儿坏死性小肠结肠炎防治的应用基础研究

Necrotizing enterocolitis (NEC) is the leading cause of mortality and mobility in preterm infants. The limitation of approach to prevent and control the development of NEC results in the emergency of research for new pharmaceutical therapy against NEC. DSLNT, a human breast milk derived oligosaccharide with unclear biological function, was found to show anti-NEC effects. However, the mechanism of how DSLNT exerts protection against NEC was largely unknown. TLR4/NF-κB signaling pathway was critically associated with cell proliferation, apoptosis as well as inflammatory cascade. Overactive TLR4/NF-κB signaling transduction in the surface of intestinal epithelial cell plays an initial role in and underlies the pathology of NEC. Based on our previous search, we have hypothesized that the anti-NEC effects of DSLNT could be attributed to its inhibitory effects on TLR4/NF-κB signaling pathway and subsequent inflammatory responses. Thereby, we plan to investigate the secretory pattern of DSLNT in human breast milk, the stability, toxic and side effects of DSLNT in order to search for the feasibility of DSLNT to show Intestinal protective effects. Next, we intend to investigate the effects of DSLNT on TLR4/NF-κB signaling pathway and proliferation, apoptosis as well as inflammatory cytokine release in fetal ileum epithelial cells and in neonatal rats, to illustrate the possibility as well as the molecular mechanism of DSLNT in protection against NEC. Our study will provide a new therapeutic strategy and possibility to prevent and control NEC.

新生儿坏死性小肠结肠炎(NEC)是早产儿致死致残的重要原因，防治手段有限，探索NEC的药物防治策略迫在眉睫。DSLNT是母乳来源、功能不明的单体寡糖，初步探索发现其具备抗NEC作用，但作用机制不明。TLR4/NF-κB信号通路与细胞增殖/凋亡以及炎症因子分泌密切相关，早产儿肠细胞表面特征性TLR4/NF-κB信号转导异常活跃是NEC的关键病理基础和始动因素。我们在本项目组前期研究基础上提出立项假设：DSLNT的抗NEC作用与其对TLR4/NF-κB信号通路和下游炎症级联反应的抑制有关。为此本项目拟通过考察DSLNT在乳汁中的分泌规律、稳定性和毒副作用，探讨DSLNT发挥肠道保护作用的可行性；通过动物和细胞实验，研究DSLNT对TLR4/NF-κB信号通路、胎儿回肠IEC细胞的增殖/凋亡和炎症反应的影响，探索DSLNT防治NEC的可能性和分子机制，以期为防治NEC提供新的治疗方法和可能性。

新生儿坏死性小肠结肠炎(NEC)时至今日依然是严重威胁新生儿生命的肠道急症；母乳喂养是降低NEC发生率的重要手段，而人乳中一种唾液酸化寡糖单体双唾液酸-乳糖-N四糖(DSLNT)在动物研究中被发现是重要的抗NEC保护成分。本项目致力于考察DLSNT对肠道的保护作用机制，主要研究内容和发现为：(1)DSLNT对新生肠道肥大细胞(MC)具有调节作用：对NEC新生儿肠道病变部位的病理组织切片进行免疫组化(IHC)染色，发现NEC患儿肠道固有层中存在大量的MC蛋白酶风暴，继而运用新生儿巨结肠手术中获取的废弃肠道原位组织，对其进行MC-chymase刺激，发现chymase可使肠上皮从基底膜上脱落，因而首次发现MC的异常激活是NEC的重要致病基础；用缺氧/冷刺激构建新生大鼠NEC模型，并在该模型中验证DSLNT在人乳生理浓度范围之内具有降低NEC大鼠肠道病理评分、减少MC聚集、维持肠道代谢稳态的作用；最后在环切皮MC体外激活模型中，发现DSLNT可显著抑制MC在外源性刺激脂多糖(LPS)和内源性刺激(C5a)条件下的激活和脱颗粒释放产物，证实DSLNT具有MC膜稳定性；(2)富含DSLNT的人乳酸性糖(SHMOs)抑制肠道异常激活的TLR4/NLRP3炎症小体通路保护肠道上皮：IHC和western blots结果表明，NEC新生儿和新生大鼠肠道中NLRP3炎症小体相关蛋白表达增加，SHMOs显著降低NEC新生大鼠肠道中TLR4的表达，同时增加NF-κB p65的抑制性蛋白IκB-的表达并减少phospho-NF-κB p65的穿核，从而减少下游NLRP3/caspase1/IL-1β炎症通路的激活；用LPS构建人肠道细胞Caco-2炎症模型，并在此基础上运用MTT细胞活力检测、流式细胞技术等方法考察了SHMOs对上皮细胞的作用，结果表明SHMOs对LPS刺激引起的上皮细胞增殖抑制、细胞周期阻滞、线粒体功能损伤具有逆转作用，表明SHMOs具有独立于调节肠道菌群之外的上皮保护作用。综上，本项目发现MC作为基本的先天免疫细胞类型，参与了NEC的病理进程，同时发现DSLNT是具有抗NEC作用的天然的先天免疫调节剂；SHMOs对肠道上皮层具有直接和独立的保护作用，是抗NEC药学防御策略研发的天然结构蓝本。