Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai 200233, China.
Mol Biol Rep. 2011 Jun;38(5):3471-80. doi: 10.1007/s11033-010-0457-8. Epub 2010 Nov 18.
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.
植物乳杆菌 (LP) 以前曾被用于治疗和预防肠道疾病。然而，LP 表面层黏附蛋白 (SLAP) 抑制上皮细胞破裂的作用尚不完全清楚。本研究旨在探讨纯化的 SLAP 对感染肠致病性大肠杆菌 (EPEC) 的 Caco-2 细胞的保护作用。还评估了 ERK 在 LP 介导的紧密连接 (TJ) 损伤抑制中的作用，以确定 LP 在上皮细胞中发挥保护作用的分子机制。使用猪胃粘蛋白-Sepharose 4B 柱从 LP 细胞中提取和纯化 SLAP。使用基于竞争的黏附测定法测量 SLAP 介导的细菌黏附抑制作用。通过实时 PCR、western blot 和免疫荧光显微镜组合评估 SLAP 处理细胞中 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 磷酸化水平。通过测量跨上皮电阻 (TER) 和葡聚糖通透性来分析细胞通透性。通过流式细胞术评估 SLAP 对上皮细胞凋亡水平的影响。这些实验的结果表明，SLAP 处理降低了 EPEC 与 Caco-2 细胞的黏附水平。SLAP 处理还增强了 TJ 蛋白在 mRNA 和蛋白水平的表达，并影响了 F-肌动蛋白的分布。尽管 ERK 水平保持不变，但 SLAP 处理增加了 ERK 磷酸化。用 SLAP 处理的 Caco-2 细胞表现出增加的 TER 和减少的大分子通透性，伴随着凋亡水平的降低。综上所述，这些结果表明 LP 产生的 SLAP 可保护肠道上皮细胞免受 EPEC 诱导的损伤，其机制可能涉及 ERK 激活。
