Zeng Lishan, Tan Jiasheng, Xue Meng, Liu Le, Wang Mingming, Liang Liping, Deng Jun, Chen Wei, Chen Ye
Department of Gastroenterology, State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, People's Republic of China.
Department of Gastroenterology, Dongguan Third People's Hospital, Affiliated Dongguan Shilong People's Hospital of Southern Medical University, Dongguan, Guangdong, People's Republic of China.
J Transl Med. 2020 Mar 2;18(1):107. doi: 10.1186/s12967-020-02272-5.
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.
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.
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.
人防御素5（HD - 5）是一种关键的抗菌肽，在宿主免疫防御中发挥重要作用，但其短半衰期极大地限制了其临床应用。本研究旨在探讨产生HD - 5的工程益生菌对肠道屏障的影响，并探究其潜在机制。方法：构建pN8148 - SHD - 5载体，并将该质粒转染至乳酸乳球菌（L. lactis）中，构建出能持续产生成熟HD - 5的重组NZ9000SHD - 5菌株。将NZ9000SHD - 5适当给予葡聚糖硫酸钠（DSS）诱导的结肠炎模型。通过苏木精 - 伊红染色分析受损肠道的变化。通过FITC - 葡聚糖通透性试验检测肠道通透性变化，通过蛋白质免疫印迹法或酶联免疫吸附测定法分析紧密连接（TJ）蛋白ZO - 1和闭合蛋白以及细胞因子。在Caco - 2细胞单层中，通过跨上皮电阻分析通透性，并通过蛋白质免疫印迹法和免疫荧光检测TJ蛋白。此外，研究NF - κB信号通路以进一步分析NZ9000SHD - 5处理在体外诱导肠道保护的分子机制。
我们发现口服NZ9000SHD - 5可显著减少结肠腺结构破坏和炎性细胞浸润，下调几种炎症相关分子的表达，并保持上皮屏障完整性。在体外实验中也观察到了相同的保护作用，并且在应用脂多糖（LPS）之前用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IκBα的表达。
这些结果表明NZ9000SHD - 5可通过抑制NF - κB信号通路减轻DSS诱导的黏膜损伤，并且NZ9000SHD - 5可能是溃疡性结肠炎的一种新型治疗手段。
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