Necrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates and has very limited therapeutic options. Sialylated human milk oligosaccharides (SHMOs) improve pathological changes in experimental NEC models. The objectives of this study were to investigate the involvement of NLRP3 inflammasome in NEC pathology and to explore the effects of SHMOs on toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/NLRP3 inflammatory pathway in experimental NEC. The intestinal-tissue segments were collected from NEC infants, NLRP3 and caspase-1 positive cell were examined by immunohistochemistry. Newborn rats were hand-fed with formula containing or non-containing SHMOs (1500 mg/L) and exposed to hypoxia/cold stress to induce experimental NEC. The NEC pathological scores were evaluated; ileum protein expression of membrane TLR4 (mTLR4), inhibitor κB-α (IκB-α), NF-κB p65 subunit and phospho-NF-κB p65, as well as NLRP3 and caspase-1 were analyzed; ileum concentrations of interleukin-1β, interleukin-6, tumor necrosis factor-α (TNF-α) were also measured. Human colon epithelial Caco-2 cells were pre-treated with or without SHMOs and stimulated with TLR4 activator, lipopolysaccharide. Cell viabilities, mitochondrial membrane potential and supernatant matrix metalloprotease 2 (MMP-2) activities were analyzed. Increased frequencies of NLRP3 and caspase-1 positive cells were found in the lamina propria of damaged intestinal area of NEC neonates. SHMOs supplementation reduced NEC incidence and pathological damage scores of rats challenged with hypoxia/cold stress. Accumulation of interleukin-1β, interleukin-6 and TNF-α in NEC group were attenuated in SHMOs + NEC group. Protein expression of mTLR4, NLRP3 and caspase-1 were elevated, cytoplasmic IκB-α were reduced, nuclear phospho-NF-κB p65 were increased in the ileum of NEC rats. SHMOs supplementation ameliorated the elevation of mTLR4, NLRP3 and caspase-1, restored IκB-α in the cytoplasmic fraction and reduced phospho-NF-κB p65 in the nuclear fraction in the ileum of NEC rats. SHMOs pre-treatment improved Caco-2 cell viability, mitigated loss of mitochondrial membrane potential and modulated MMP-2 activities in the presence of lipopolysaccharide in-vitro. This study provided clinical evidence of involvement of NLRP3 inflammasome in NEC pathology, and demonstrated the protective actions of SHMOs might be owing to the suppression of TLR4/NF-κB/NLRP3-mediated inflammation in NEC.

中文翻译：

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唾液酸化的人乳低聚糖通过抑制坏死性小肠结肠炎大鼠中的Toll样受体4 / NLRP3炎性体途径来预防肠道炎症

坏死性小肠结肠炎（NEC）在新生儿中仍然是致命的胃肠道疾病，治疗选择非常有限。唾液酸化的人乳寡糖（SHMO）可改善NEC实验模型的病理变化。这项研究的目的是调查NLRP3炎性小体在NEC病理学中的参与，并探讨SHMOs在实验性NEC中对Toll样受体4（TLR4）/核因子κB（NF-κB）/ NLRP3炎症途径的影响。收集NEC婴儿的肠组织片段，通过免疫组织化学检查NLRP3和caspase-1阳性细胞。新生大鼠用含或不含SHMOs（1500 mg / L）的配方奶喂养，并暴露于低氧/冷应激下以诱导实验性NEC。评估NEC病理评分；膜TLR4（mTLR4）的回肠蛋白表达，分析了抑制剂κB-α（IκB-α），NF-κBp65亚基和磷酸化NF-κBp65以及NLRP3和caspase-1。还测量了回肠的白介素-1β，白介素-6，肿瘤坏死因子-α（TNF-α）浓度。用或不用SHMO预处理人结肠上皮Caco-2细胞，并用TLR4激活剂脂多糖刺激。分析细胞活力，线粒体膜电位和上清基质金属蛋白酶2（MMP-2）活性。在NEC新生儿肠道受损区域的固有层中发现NLRP3和caspase-1阳性细胞的频率增加。补充SHMOs可以降低缺氧/冷应激所致大鼠的NEC发生率和病理损伤评分。SHMOs + NEC组NEC组白细胞介素-1β，白细胞介素-6和TNF-α的积累减弱。NEC大鼠回肠中mTLR4，NLRP3和caspase-1的蛋白表达升高，胞质IκB-α降低，核磷酸-NF-κBp65升高。补充SHMOs改善了NEC大鼠回肠中mTLR4，NLRP3和caspase-1的表达，恢复了细胞质部分的IκB-α，减少了核部分的磷酸化NF-κBp65。SHMO的预处理改善了Caco-2细胞的活力，减轻了线粒体膜电位的损失，并在存在脂多糖的情况下调节了MMP-2的活性。这项研究提供了NLRP3炎性体参与NEC病理的临床证据，并证明SHMOs的保护作用可能是由于抑制NEC中的TLR4 /NF-κB/ NLRP3介导的炎症。NEC大鼠回肠中核磷酸-NF-κBp65水平升高。补充SHMOs改善了NEC大鼠回肠中mTLR4，NLRP3和caspase-1的表达，恢复了细胞质部分的IκB-α，减少了核部分的磷酸化NF-κBp65。SHMO的预处理改善了Caco-2细胞的活力，减轻了线粒体膜电位的损失，并在存在脂多糖的情况下调节了MMP-2的活性。这项研究提供了NLRP3炎性体参与NEC病理的临床证据，并证明SHMOs的保护作用可能是由于抑制NEC中的TLR4 /NF-κB/ NLRP3介导的炎症。NEC大鼠回肠中核磷酸-NF-κBp65水平升高。补充SHMOs改善了NEC大鼠回肠中mTLR4，NLRP3和caspase-1的表达，恢复了细胞质部分的IκB-α，减少了核部分的磷酸化NF-κBp65。SHMO的预处理改善了Caco-2细胞的活力，减轻了线粒体膜电位的损失，并在存在脂多糖的情况下调节了MMP-2的活性。这项研究提供了NLRP3炎性体参与NEC病理的临床证据，并证明SHMOs的保护作用可能是由于抑制NEC中的TLR4 /NF-κB/ NLRP3介导的炎症。NEC大鼠回肠细胞质部分恢复IκB-α，核部分磷酸-NF-κBp65降低。SHMO的预处理改善了Caco-2细胞的活力，减轻了线粒体膜电位的损失，并在存在脂多糖的情况下调节了MMP-2的活性。这项研究提供了NLRP3炎性体参与NEC病理的临床证据，并证明SHMOs的保护作用可能是由于抑制NEC中的TLR4 /NF-κB/ NLRP3介导的炎症。NEC大鼠回肠细胞质部分恢复IκB-α，核部分磷酸-NF-κBp65降低。SHMO的预处理改善了Caco-2细胞的活力，减轻了线粒体膜电位的损失，并在存在脂多糖的情况下调节了MMP-2的活性。这项研究提供了NLRP3炎性体参与NEC病理的临床证据，并证明SHMOs的保护作用可能是由于抑制NEC中的TLR4 /NF-κB/ NLRP3介导的炎症。