Background Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2’-fucosyllactose (2’-FL) and 6’-sialyllactose (6’-SL) can reduce NEC through inhibition of TLR4 signaling. Methods NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2’-FL, 6’-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling. Results Supplementation of infant formula with either 2’-FL and/or 6’-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2’-FL and 6’-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2’-FL and 6’-SL, but not lactose, to dock into the binding pocket of the TLR4–MD2 complex, explaining their ability to inhibit TLR4 signaling. Conclusions 2’-FL and 6’-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition. Impact Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2’-FL and 6’-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2’-FL and 6’-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2’-FL and 6’-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

中文翻译：

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人乳寡糖 2'-岩藻糖基乳糖和 6'-唾液酸乳糖通过抑制 Toll 样受体 4 信号传导防止坏死性小肠结肠炎的发展

背景 坏死性小肠结肠炎 (NEC) 是通过肠上皮细胞中过度的 Toll 样受体 4 (TLR4) 信号传导发展而来的。母乳富含不易消化的低聚糖，可通过不明机制预防 NEC。我们现在假设人乳寡糖 2'-岩藻糖基乳糖 (2'-FL) 和 6'-唾液酸乳糖 (6'-SL) 可以通过抑制 TLR4 信号来减少 NEC。方法在新生小鼠和早产仔猪中诱导NEC，并在婴儿配方奶粉中添加2'-FL、6'-SL或乳糖。在手术切除时获得肠组织。HMO 对 TLR4 的抑制在 IEC-6 肠上皮细胞、小鼠和人体组织外植体中以及通过计算机模拟进行了评估。结果 添加了 2'-FL 和/或 6'-SL 的婴儿配方奶粉，但不添加母体乳糖，通过减少细胞凋亡、炎症、体重减轻和组织学外观减少小鼠和仔猪的 NEC。从机制上讲，2'-FL 和 6'-SL（但不是乳糖）减少了 TLR4 介导的核因子 kappa 轻链增强剂，在小鼠和人类肠道中激活 B 细胞 (NF-kB) 炎症信号。引人注目的是，计算机模拟显示 2'-FL 和 6'-SL，但不是乳糖，停靠在 TLR4-MD2 复合物的结合口袋中，这解释了它们抑制 TLR4 信号传导的能力。结论 2'-FL 和 6'-SL，但不是乳糖，可预防小鼠和仔猪模型中的 NEC，并减轻人类回肠中的 NEC 炎症，部分通过抑制 TLR4。影响 坏死性小肠结肠炎 (NEC) 是早产儿发病率和死亡率的主要原因，发生在肠道细菌定植和配方喂养以及先天免疫受体 Toll 样受体 4 (TLR4) 激活的情况下。母乳通过不清楚的机制预防 NEC。我们现在表明，源自乳糖的富含母乳的人乳寡糖 (HMO) 通过抑制 TLR4 来预防 NEC。人乳寡糖 2'-FL 和 6'-SL，但不是骨架糖乳糖，可防止小鼠和仔猪发生 NEC。2'-FL 和 6'-SL 但不抑制乳糖在培养的肠细胞、来自小鼠肠道的类肠细胞和 NEC 患者手术切除时获得的人类肠道外植体中的 TLR4 信号传导。在寻找所涉及的机制时，发现 2'-FL 和 6'-SL 但不是乳糖直接结合 TLR4，解释了对 NEC 的抑制和保护。这些发现可能会影响临床实践，因为表明使用 HMO 可以作为有 NEC 发展风险的早产儿的预防策略。