An engineering probiotic producing defensin-5 ameliorating dextran sodium sulfate-induced mice colitis via Inhibiting NF-kB pathway
An engineering probiotic producing defensin-5 ameliorating dextran sodium sulfate-induced mice colitis via Inhibiting NF-kB pathway
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产生防御素 5 的工程益生菌通过抑制 NF-kB 通路改善右旋糖酐硫酸钠诱导的小鼠结肠炎
DOI：
10.1186/s12967-020-02272-5
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发表时间：
2020-03
期刊：
影响因子：
--
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Zeng L;Tan J;Xue M;Liu L;Wang M;Liang L;Deng J;Chen W;Chen Y
人防御素-5 （HD-5）是一种关键的抗菌肽，在宿主免疫防御中起着重要作用，但半衰期短极大地限制了其临床应用。方法构建pN8148-SHD-5载体，将质粒转染至乳酸乳球菌（lactococcus lactis， L.）。构建重组菌株NZ9000SHD-5，该菌株连续产生成熟的HD-5。NZ9000SHD-5给予葡聚糖硫酸钠（DSS）诱导的结肠炎模型。苏木精-伊红染色分析损伤肠的变化。采用fitc -葡聚糖渗透性试验检测肠通透性的变化，western blotting或酶联免疫吸附法检测紧密连接蛋白ZO-1、occludin和细胞因子的变化。在Caco-2细胞单层中，采用经上皮电阻法分析其通透性，采用western blotting和免疫荧光法检测TJ蛋白。此外，通过研究NF-κB信号通路，进一步分析NZ9000SHD-5处理对体外诱导肠道保护的分子机制。结果口服NZ9000SHD-5可显著减少结肠腺结构破坏和炎症细胞浸润，下调几种炎症相关分子的表达，并保持上皮屏障的完整性。在体外实验中也观察到同样的保护作用，LPS应用前用NZ9000SHD-5培养上清预处理巨噬细胞可显著降低磷酸化核转录因子-κB (NF-κB) p65及其抑制剂i -κB α的表达。结论NZ9000SHD-5可通过抑制NF-κB信号通路减轻dss诱导的粘膜损伤，可能成为治疗溃疡性结肠炎的新手段。
BackgroundHuman 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 mechanismMethodsWe constructed the pN8148-SHD-5 vector, and transfected this plasmid intoLactococcus 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.ResultsWe 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.ConclusionsThese 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.
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