Department of Pediatrics, Brody School of Medicine, East Carolina University, Greenville, NC, 27834, USA.
Present Address: Clinical Neonatologist, Mercy Medical Center, Cedar Rapids, IA, 52403, USA.
BMC Pediatr. 2018 Nov 27;18(1):372. doi: 10.1186/s12887-018-1346-x.
Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Neonates weighing <1500 grams are at the highest risk for acquiring NEC, with a prevalence of nearly 7-10%, mortality up to 30%, and several long-term complications among survivors. Despite advancements in neonatal medicine, this disease remains a challenge to treat. The aim of this study is to investigate the effect of NEC on gut epithelial tight junctions and its barrier function using a NEC mouse model.
Three-day old C57BL/6 mouse pups were fed with Esbilac formula every 3 hours and then subjected to hypoxia twice daily followed by cold stress. Dam fed pups from the same litters served as controls. Pups were observed and sacrificed 96 hours after the treatments and intestines were removed for experiments. The successful induction of NEC was confirmed by histopathology. Changes in tight junction proteins in NEC intestines were studied by western blotting and immunofluorescent microscopy using specific protein markers. The gut leakage in NEC was visualized using biotin tracer molecules.
Our study results demonstrate that we induced NEC in >50% of experimental pups, pups lost nearly 40% of weight and their intestines showed gross changes and microscopic changes associated with NEC. There were inflammatory changes with loss of tight junction barrier function and disruption of tight junction claudin proteins in the intestines of NEC mouse model. We have demonstrated for the first time that NEC intestines develop increased leakiness as visualized by biotin tracer leakage.
NEC leads to breakdown of epithelial barrier due to changes in tight junction proteins with increased leakiness which may explain the transmigration of microbes and microbial products from the gut lumen into the blood stream leading to sepsis like signs clinically witnessed.
坏死性小肠结肠炎（NEC）是早产儿死亡的主要原因。体重<1500 克的新生儿罹患 NEC 的风险最高，患病率接近 7-10%，死亡率高达 30%，幸存者存在多种长期并发症。尽管新生儿医学取得了进步，但这种疾病的治疗仍然具有挑战性。本研究旨在使用 NEC 小鼠模型研究 NEC 对肠道上皮紧密连接及其屏障功能的影响。
3 天大的 C57BL/6 幼鼠每 3 小时喂食 Esbilac 配方，然后每天接受两次缺氧处理，随后进行冷应激。来自同一窝的母鼠喂养的幼鼠作为对照。处理后 96 小时观察并处死幼鼠，取出肠道进行实验。通过组织病理学确认 NEC 的成功诱导。使用特定的蛋白质标记物通过 Western 印迹和免疫荧光显微镜研究 NEC 肠道中紧密连接蛋白的变化。使用生物素示踪分子可视化 NEC 中的肠道渗漏。
我们的研究结果表明，我们在>50%的实验幼鼠中诱导了 NEC，幼鼠体重减轻了近 40%，其肠道出现了与 NEC 相关的大体和微观变化。NEC 小鼠模型的肠道存在炎症变化，紧密连接屏障功能丧失，紧密连接蛋白 claudin 破坏。我们首次证明，NEC 肠道通透性增加，可通过生物素示踪渗漏可视化。
NEC 导致上皮屏障的破坏，这是由于紧密连接蛋白的变化导致通透性增加，这可能解释了微生物和微生物产物从肠道腔转移到血液中，导致临床上观察到类似败血症的迹象。
