Abstract：
BACKGROUND: Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation.
OBJECTIVE: This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells.
METHODS: Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence.
RESULTS: AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05).
CONCLUSIONS: AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.
OBJECTIVE: This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells.
METHODS: Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence.
RESULTS: AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05).
CONCLUSIONS: AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.
摘要：
背景：肠道微生物群失衡和肠道屏障受损导致坏死性小肠结肠炎（NEC）的发展。自体诱导因子-2(AI-2)在修复肠道损伤和减轻炎症中起着至关重要的作用。
目的：本研究旨在研究AI-2对NEC肠闭塞带1（ZO-1）和闭塞蛋白表达的影响。我们使用NEC小鼠在体内和体外使用脂多糖（LPS）刺激的肠细胞评估了其作用。
方法：使用组织学染色和评分评估新生小鼠肠道的病理变化。使用细胞计数试剂盒-8(CCK-8)测定法测量细胞增殖,以确定LPS和AI-2干预的最佳条件。采用实时定量聚合酶链反应(RT-qPCR)分析基质金属蛋白酶-3(MMP3)mRNA水平,蛋白酶激活受体2（PAR2），白细胞介素-1β（IL-1β），IL-6使用蛋白质印迹评估MMP3，PAR2，ZO-1和闭塞蛋白的蛋白质水平，免疫组织化学，或免疫荧光。
结果：AI-2减轻了NEC诱导的肠损伤（P<0.05），增强了损伤的IEC-6细胞的增殖（P<0.05）。AI-2干预降低了肠组织和细胞中MMP3和PAR2的mRNA和蛋白表达(P<0.05)。此外,它增加了ZO-1和occludin的蛋白质水平（P<0.05），同时降低IL-1β和IL-6mRNA的表达(P<0.05)。
结论：AI-2干预增强了紧密连接蛋白（ZO-1和occludin）的表达，减轻NEC新生小鼠和IEC-6细胞的肠道损伤，可能通过调节PAR2和MMP3信号传导。AI-2有望成为NEC的保护性干预措施。AI-2在修复肠道损伤和减轻炎症中起着至关重要的作用。
目的：本研究旨在研究AI-2对NEC肠闭塞带1（ZO-1）和闭塞蛋白表达的影响。我们使用NEC小鼠在体内和体外使用脂多糖（LPS）刺激的肠细胞评估了其作用。
方法：使用组织学染色和评分评估新生小鼠肠道的病理变化。使用细胞计数试剂盒-8(CCK-8)测定法测量细胞增殖,以确定LPS和AI-2干预的最佳条件。采用实时定量聚合酶链反应(RT-qPCR)分析基质金属蛋白酶-3(MMP3)mRNA水平,蛋白酶激活受体2（PAR2），白细胞介素-1β（IL-1β），IL-6使用蛋白质印迹评估MMP3，PAR2，ZO-1和闭塞蛋白的蛋白质水平，免疫组织化学，或免疫荧光。
结果：AI-2减轻了NEC诱导的肠损伤（P<0.05），增强了损伤的IEC-6细胞的增殖（P<0.05）。AI-2干预降低了肠组织和细胞中MMP3和PAR2的mRNA和蛋白表达(P<0.05)。此外,它增加了ZO-1和occludin的蛋白质水平（P<0.05），同时降低IL-1β和IL-6mRNA的表达(P<0.05)。
结论：AI-2干预增强了紧密连接蛋白（ZO-1和occludin）的表达，减轻NEC新生小鼠和IEC-6细胞的肠道损伤，可能通过调节PAR2和MMP3信号传导。AI-2有望成为NEC的保护性干预措施。AI-2在修复肠道损伤和减轻炎症中起着至关重要的作用。
