Lactobacillus plantarum surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic Escherichia coli
Lactobacillus plantarum surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic Escherichia coli
复制标题
植物乳杆菌表面层粘附蛋白保护肠上皮细胞免受致病性大肠杆菌引起的紧密连接损伤
DOI：
10.1007/s11033-010-0457-8
复制
发表时间：
2011-06-01
影响因子：
2.8
通讯作者：
Qin, Huanlong
中科院分区：
文献类型：
--
作者：
Liu, Zhihua;Shen, Tongyi;Qin, Huanlong
植物乳杆菌（LP）先前已用于治疗和预防肠道病症和疾病。然而，LP表面层粘附蛋白（SLP）在抑制上皮细胞破坏中的作用尚未完全了解。本研究旨在探讨纯化的Sodium对肠致病性大肠杆菌（EPEC）感染的Caco-2细胞的保护作用。ERK在LP介导的紧密连接（TJ）损伤抑制中的作用也进行了评价，以确定LP在上皮细胞中保护作用的分子机制。用猪胃粘蛋白-Sepharose 4 B柱从LP细胞中提取并纯化血清。使用基于竞争的粘附测定来测量SLAP介导的细菌粘附抑制。TJ相关蛋白的表达，TJ结构的维持，以及细胞外信号调节激酶（ERK）和ERK磷酸化的水平进行了评估，在SLAP处理的细胞通过实时PCR，蛋白质印迹和免疫荧光显微镜的组合。通过测量跨上皮电阻（TER）和葡聚糖渗透性来分析细胞渗透性。通过流式细胞术评估了Sodium对上皮细胞凋亡水平的影响。这些实验的结果表明，用Sodium处理降低了EPEC对Caco-2细胞的粘附水平。Sodium处理还增强了TJ蛋白在mRNA和蛋白水平的表达，并影响F-肌动蛋白的分布。虽然ERK水平保持不变，但ERK磷酸化水平通过Sodium处理增加。用Sodium处理的Caco-2细胞表现出TER增加和大分子渗透性降低，这伴随着凋亡水平的降低。总之，这些结果表明，脂蛋白产生的SLEP保护肠上皮细胞免受EPEC诱导的损伤，可能是通过涉及ERK激活的机制。
Lactobacillus plantarum (LP) has previously been used for the treatment and prevention of intestinal disorders and disease. However, the role of the LP surface layer adhesive protein (SLAP) in inhibition of epithelial cell disruption is not fully understood. The aim of the present study was to investigate the protective effects of purified SLAP on Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). The role of ERK in LP-mediated inhibition of tight junction (TJ) injury was also evaluated in order to determine the molecular mechanisms underlying the protective effects of LP in epithelial cells. SLAP was extracted and purified from LP cells using a porcine stomach mucin-Sepharose 4B column. SLAP-mediated inhibition of bacterial adhesion was measured using a competition-based adhesion assay. Expression of TJ-associated proteins, maintenance of TJ structure, and levels of extracellular signal regulated kinase (ERK) and ERK phosphorylation were assessed in SLAP-treated cells by a combination of real-time PCR, western blotting, and immunofluorescence microscopy. Cell permeability was analyzed by measurement of trans-epithelial electrical resistance (TER) and dextran permeability. The effect of SLAP on levels of apoptosis in epithelial cells was assessed by flow cytometry. Results from these experiments revealed that treatment with SLAP decreased the level of adhesion of EPEC to Caco-2 cells. SLAP treatment also enhanced expression of TJ proteins at both the mRNA and protein levels and affected F-actin distribution. Although ERK levels remained unchanged, ERK phosphorylation was increased by SLAP treatment. Caco-2 cells treated with SLAP exhibited increased TER and decreased macromolecular permeability, which was accompanied by a decrease in the level of apoptosis. Together, these results suggest that LP-produced SLAP protects intestinal epithelial cells from EPEC-induced injury, likely through a mechanism involving ERK activation.