红毛藻多糖抑制大肠杆菌黏附Caco-2细胞的机制

Inhibitory Mechanism of Escherichia coli Adhesion to Caco-2 Monolayers by Polysaccharides from Bangia fusco-purpurea

基于体外人结肠腺癌细胞(Caco-2)单层模型,研究红毛藻多糖(Bangia fusco-purpurea polysaccharide,BFP)对大肠杆菌(Escherichia coli)黏附Caco-2细胞的影响以及潜在作用机制。采用CFDA-SE荧光标记技术分析BFP对E.coli黏附Caco-2细胞单层的影响;利用实时聚合酶链式反应分析BFP对Caco-2细胞表面整合素β1和E.coli黏附素fimH及E.coli黏附Caco-2细胞导致细胞产生的炎症因子(白细胞介素(interleukin,IL)-1β、IL-8、肿瘤坏死因子(tumor necrosis factor,TNF)-α)和细胞紧密连接蛋白(闭锁小带蛋白-1(zonula occludens-1,ZO-1)与Occludin)的基因表达情况,最终基于Western Blot分析验证ZO-1、Occludin蛋白表达。结果表明,BFP在质量浓度为400~800μg/mL时能够显著抑制E.coli黏附Caco-2细胞单层;BFP主要通过下调Caco-2细胞表面整合素β1和E.coli黏附素基因fimH的表达抑制细菌的黏附。此外,BFP可显著抑制E.coli及其培养上清液诱导Caco-2细胞产生的炎症细胞因子基因表达的上调、紧密连接蛋白(ZO-1与Occludin)及其基因表达的下调。综上所述,BFP能够抑制E.coli对Caco-2细胞单层的黏附,实验结果可为其开发新型的抗菌产品以及BFP的高值利用和精深加工提供参考。

In this study,the effect of Bangia fusco-purpurea polysaccharide(BFP)on Escherichia coli adhesion to Caco-2 monolayers and its potential mechanism was studied using an in vitro human colon adenocarcinoma cell(Caco-2)monolayer model.The effect of BFP on the adhesion of E.coli to Caco-2 monolayers was analyzed by fluorescence labeling with carboxyfluorescein diacetate and succinimidyl ester(CFDA-SE),and its effect on the gene expression of integrinβ1 in Caco-2 cells,the adhesin FimH in E.coli,and inflammatory factors(IL-1β,IL-8 and TNF-α)induced by E.coli adhesion to Caco-2 cells and the tight junction proteins zonula occludens 1(ZO-1)and occludin were analyzed using real-time polymerase chain reaction(real-time PCR).Also,the protein expression of ZO-1 and occludin in Caco-2 monolayers was detected using Western Blot analysis.Our results showed that BFP at concentrations of 400–800μg/mL significantly inhibited the adhesion of E.coli to Caco-2 monolayers mainly by down-regulating the gene expression of integrinβ1 in Caco-2 cells and FimH in E.coli.In addition,BFP significantly inhibited the up-regulation of inflammatory cytokine gene expression and the down-regulation of ZO-1 and occludin protein and gene expression induced by in Caco-2 cells induced by E.coli and its culture supernatant.In conclusion,BFP inhibited the adhesion of E.coli to Caco-2 cell monolayers,which will lay a scientific basis for the development of novel antimicrobial products and the high-value utilization and deep processing of BFP.