[Elafin-expressing probiotic Escherichia coli Nissle 1917 protects against experimental colitis].

Objective: To construct the gene modified probiotic Escherichia coli nissle1917 (EcN) which can express human Elafin protein and to explore its protective effect on the acute colitis in mice. Methods: The recombinant plasmid with human Elafin gene was constructed and then transferred to EcN. Western blot results confirmed that the engineered probiotic expressed Elafin successfully in vitro. C57/BL6J mouse was used in this study and were randomly divided into 4 groups according to different treatment: PBS gavage (PBS group); DSS administrated (DSS group); DSS administrated with wild-type EcN (EcN-WT) gavage (EcN-WT group); DSS administrated with EcN-Elafin gavage (EcN-Elafin group). Body weight and disease activity index (DAI) were measured every day. The length of mice colons in each group were measured after euthanasia. The degree of inflammation of intestinal mucosa in each group was measured through histopathological scoring. The proportion of neutrophils and macrophages infiltrated into colon lamina propria was detected by flow cytometry. The protein expression levels of pro-inflammatory cytokines TNF-α, IL-6 and chemokine CXCL-1 in colonic tissue were quantified by enzyme-linked immunosorbent assay. Results: Elafin protein could be detected in the supernatant of EcN-Elafin culture medium and EcN-Elafin homogenates. Compared with DSS group, the weight loss and DAI score of EcN-Elafin group and EcN-WT group were both significantly improved. The colon length of EcN-Elafin group was significantly longer than that of DSS group. The histological score of colitis in EcN-Elafin group was significantly lower than that in DSS group (5.3±2.3 vs 9.3±1.4, P<0.05). In EcN-Elafin group, the proportion of neutrophils[(8.65±1.49)% vs (17.60±2.16)%, P<0.01]and macrophages[(3.79±0.26)% vs (5.73±0.45)%, P<0.01]infiltrated into the colon lamina propria was significantly decreased compared with DSS group. The protein expression levels of TNF-α, IL-6 and CXCL-1 in EcN-Elafin group and EcN-WT group were significantly lower than those in DSS group. Conclusion: Elafin-expressing EcN can protect against DSS-induced acute colitis in mice and may have provided an effective and cost-efficient method for the treatment of IBD.目的： 构建能够表达人重组蛋白Elafin的益生菌大肠杆菌Nissle 1917（EcN），并探讨其对葡聚糖硫酸钠（DSS）诱导的急性小鼠结肠炎的保护作用。 方法： 构建带有Elafin基因的重组质粒，将其转入感受态EcN，构建能够表达Elafin蛋白的益生菌EcN-Elafin。通过Western印迹证实该益生菌在体外成功表达Elafin。C57/BL6J小鼠随机分为4组：健康对照组（PBS组）、结肠炎组（DSS组）、野生型EcN（EcN-WT）治疗结肠炎组（EcN-WT组）、EcN-Elafin治疗结肠炎组（EcN-Elafin组）。每天同一时间测量小鼠体重、观察小鼠排便情况及计算疾病活动度指数（DAI）。小鼠处以安乐死后测量结肠长度，苏木精-伊红（HE）染色及组织病理学评分比较各组肠黏膜炎症程度，流式细胞术检测结肠固有层浸润中性粒细胞和巨噬细胞的比例，酶联免疫吸附法（ELISA）定量结肠组织中的肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6及趋化因子CXCL-1的蛋白表达水平。 结果： 在EcN-Elafin的培养基上清和菌体沉淀中均能检测到Elafin蛋白。与DSS组相比，EcN-Elafin组和EcN-WT组小鼠体重减轻状况和DAI评分均明显改善。EcN-Elafin组小鼠结肠长度显著长于DSS组。EcN-Elafin组结肠炎组织学评分显著低于DSS组（5.3±2.3比9.3±1.4，P<0.05）。与DSS组小鼠相比，EcN-Elafin组小鼠结肠固有层浸润的中性粒细胞［（8.65±1.49）% 比（17.60±2.16）%，P<0.01］和巨噬细胞［（3.79±0.26）% 比（5.73±0.45）%，P<0.01］比例均显著降低。EcN-Elafin组和EcN-WT组结肠中TNF-α、IL-6、和CXCL-1蛋白质表达水平均显著低于DSS组。 结论： 表达Elafin的益生菌EcN-Elafin能够显著减轻DSS诱导的急性小鼠结肠炎，对肠黏膜炎症具有保护作用，为临床炎症性肠病的治疗提供一种新的经济有效的方法。.