BACKGROUND Human defensin-5 (HD-5) is a key antimicrobial peptide which plays an important role in host immune defense, while the short half-life greatly limits its clinical application. The purpose of this study was to investigate the effects of an engineering probiotic producing HD-5 on intestinal barrier and explore its underlying mechanism METHODS: We constructed the pN8148-SHD-5 vector, and transfected this plasmid into Lactococcus lactis (L. lactis) to create the recombinant NZ9000SHD-5 strain, which continuously produces mature HD-5. NZ9000SHD-5 was administrated appropriately in a dextran sodium sulfate (DSS)-induced colitis model. Alterations in the wounded intestine were analyzed by hematoxylin-eosin staining. The changes of intestinal permeability were detected by FITC-dextran permeability test, the tight junction (TJ) proteins ZO-1 and occludin and cytokines were analyzed by western blotting or enzyme linked immunosorbent assay. In Caco-2 cell monolayers, the permeability were analyzed by transepithelial electrical resistance, and the TJ proteins were detected by western blotting and immunofluorescence. In addition, NF-κB signaling pathway was investigated to further analyze the molecular mechanism of NZ9000SHD-5 treatment on inducing intestinal protection in vitro. RESULTS We found oral administration with NZ9000SHD-5 significantly reduced colonic glandular structure destruction and inflammatory cell infiltration, downregulated expression of several inflammation-related molecules and preserved epithelial barrier integrity. The same protective effects were observed in in vitro experiments, and pretreatment of macrophages with NZ9000SHD-5 culture supernatants prior to LPS application significantly reduced the expression of phosphorylated nuclear transcription factor-kappa B (NF-κB) p65 and its inhibitor IκBα. CONCLUSIONS These results indicate the NZ9000SHD-5 can alleviate DSS-induced mucosal damage by suppressing NF-κB signaling pathway, and NZ9000SHD-5 may be a novel therapeutic means for ulcerative colitis.

中文翻译：

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通过抑制NF-kB途径，生产一种能产生益生菌的工程防御素5来改善右旋糖酐硫酸钠诱导的小鼠结肠炎。

背景技术人防御素5（HD-5）是一种关键的抗菌肽，在宿主的免疫防御中起着重要的作用，而半衰期短则极大地限制了其临床应用。这项研究的目的是研究产生工程性益生菌的HD-5对肠屏障的作用，并探讨其潜在机制。方法：我们构建了pN8148-SHD-5载体，并将该质粒转染到乳酸乳球菌（L. lactis）中创建重组NZ9000SHD-5菌株，该菌株可连续产生成熟的HD-5。在右旋糖酐硫酸钠（DSS）诱导的结肠炎模型中适当施用NZ9000SHD-5。用苏木精-曙红染色分析受伤肠的变化。通过FITC-葡聚糖通透性试验检测肠道通透性的变化，通过蛋白质印迹或酶联免疫吸附分析法分析紧密连接（TJ）蛋白ZO-1和闭合蛋白和细胞因子。在Caco-2细胞单层中，通过跨上皮电阻分析通透性，并通过Western印迹和免疫荧光检测TJ蛋白。此外，还研究了NF-κB信号通路，以进一步分析NZ9000SHD-5治疗诱导肠道保护的分子机制。结果我们发现口服NZ9000SHD-5可以显着减少结肠腺结构破坏和炎性细胞浸润，下调一些炎症相关分子的表达并保留上皮屏障完整性。在体外实验中观察到了相同的保护作用，在应用LPS之前，使用NZ9000SHD-5培养上清液对巨噬细胞进行预处理和预处理可以显着降低磷酸化核转录因子-κB（NF-κB）p65及其抑制剂IκBα的表达。结论这些结果表明NZ9000SHD-5可以通过抑制NF-κB信号通路减轻DSS引起的粘膜损伤，而NZ9000SHD-5可能是溃疡性结肠炎的一种新型治疗手段。