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.
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。