坏死性小肠结肠炎(NEC)是早产儿最常见的胃肠道急症。导致回肠和结肠炎性坏死的推测机制包括病原体识别受体Toll样受体4(TLR4)的活化和转化生长因子β(TGFβ)水平的降低。细胞外烟酰胺磷酸核糖基转移酶(eNAMPT),一种新的损伤相关分子模式(DAMP),是TLR4配体,在许多炎性疾病过程中发挥作用。为了检验ENAMPT参与NEC的假设,一种eNAMPT中和单克隆抗体,ALT-100用于建立良好的NEC动物模型中。从定时怀孕的水坝中过早分娩的早产Sprague-Dawley幼崽暴露于缺氧/体温过低,并随机分配给对照寄养的母鼠,分娩后48小时注射生理盐水(媒介物);对照+mAB-fosterdam-fed大鼠,分娩后48小时IP注射10µgALT-100;NEC-口服管,注射生理盐水的配方喂养大鼠;和NEC+mAb配方喂养大鼠,在48h时IP注射10µgALT-100。在剖腹产后96h处理远端回肠以进行组织学检查,生物化学,分子,和RNA测序研究。与对照组相比,盐水处理的NEC幼崽表现出显著增加的粪便血液和组织学回肠损伤(q<0.0001),在ALT-100mAb处理的NEC幼崽中发现显著降低(q<0.01)。回肠组织中的实时PCR显示,与接受ALT-100mAb的幼犬相比,NEC幼犬中的NAMPT增加(p<0.01)。血清肿瘤坏死因子α(TNFα)水平升高,白细胞介素6(IL-6),白细胞介素-8(IL-8),与NEC+mAb幼崽相比,在NEC幼崽中观察到NAMPT(p<0.01)。最后,RNA-Seq证实NEC幼仔中TGFβ和TLR4信号通路失调,其通过ALT-100mAb处理减弱。这些数据强烈支持eNAMPT参与NEC病理生物学和eNAMPT中和作为解决对NEC疗法的未满足需求的策略。
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of prematurity. Postulated mechanisms leading to inflammatory necrosis of the ileum and colon include activation of the pathogen recognition receptor Toll-like receptor 4 (TLR4) and decreased levels of transforming growth factor beta (TGFβ). Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a novel damage-associated molecular pattern (
DAMP), is a TLR4 ligand and plays a role in a number of inflammatory disease processes. To test the hypothesis that eNAMPT is involved in NEC, an eNAMPT-neutralizing monoclonal antibody, ALT-100, was used in a well-established animal model of NEC. Preterm Sprague-Dawley pups delivered prematurely from timed-pregnant dams were exposed to hypoxia/hypothermia and randomized to control-foster mother dam-fed rats, injected IP with saline (vehicle) 48 h after delivery; control + mAB-foster dam-fed rats, injected IP with 10 µg of ALT-100 at 48 h post-delivery; NEC-orally gavaged, formula-fed rats injected with saline; and NEC + mAb-formula-fed rats, injected IP with 10 µg of ALT-100 at 48 h. The distal ileum was processed 96 h after C-section delivery for histological, biochemical, molecular, and RNA sequencing studies. Saline-treated NEC pups exhibited markedly increased fecal blood and histologic ileal damage compared to controls (q < 0.0001), and findings significantly reduced in ALT-100 mAb-treated NEC pups (q < 0.01). Real-time PCR in ileal tissues revealed increased NAMPT in NEC pups compared to pups that received the ALT-100 mAb (p < 0.01). Elevated serum levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), interleukin-8 (IL-8), and NAMPT were observed in NEC pups compared to NEC + mAb pups (p < 0.01). Finally, RNA-Seq confirmed dysregulated TGFβ and TLR4 signaling pathways in NEC pups that were attenuated by ALT-100 mAb treatment. These data strongly support the involvement of eNAMPT in NEC pathobiology and eNAMPT neutralization as a strategy to address the unmet need for NEC therapeutics.