Hao Li-Jun, Lin Yan, Zhang Wei, Tian Jiao, Wang Ya, Chen Peng-De, Hu Chong-Kang, Zeng Ling-Chao, Yang Jie, Wang Bao-Xi, Jiang Xun
Department of Pediatrics, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2017 Jun;19(6):693-698. doi: 10.7499/j.issn.1008-8830.2017.06.016.
To investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection.
The Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant.
After EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05).
EPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.
探讨紧密连接蛋白ZO-1在肠上皮细胞（Caco-2细胞）中的表达变化以及二十碳五烯酸（EPA）对侵袭性大肠杆菌（E.coli）LF82感染后的保护作用。
采用Caco-2细胞系建立肠上皮细胞紧密连接的体外模型。将Caco-2细胞分为EPA处理组（0、25、50、100和200 μmol/L EPA）以及EPA（0、25、50、100和200 μmol/L EPA）+E.coli LF82处理（0、6和12小时）组。用显微镜观察细胞的形态特征。采用MTT法测定细胞生长曲线。比较细胞膜两侧碱性磷酸酶（ALP）的活性以评估Caco-2细胞模型。采用MTT法和流式细胞术研究不同浓度EPA对Caco-2细胞存活率和凋亡率的影响。采用RT-qPCR检测EPA和/或E.coli LF82处理后Caco-2细胞中ZO-1的mRNA表达。采用ELISA检测培养上清液中肿瘤坏死因子-α（TNF-α）水平的变化。
EPA处理（25和50 μmol/L）后，Caco-2细胞的增殖呈剂量依赖性诱导。细胞存活率显著高于对照组（P<0.05）。EPA处理（100和200 μmol/L）组对Caco-2细胞的增殖具有显著的剂量依赖性抑制作用。细胞存活率显著低于对照组（P<0.05）。EPA处理（100和200 μmol/L）组与对照组相比，细胞凋亡率显著增加（P<0.05）。E.coli LF82处理6小时和12小时组，随着处理时间的延长，Caco-2细胞中ZO-1的mRNA表达降低，且显著低于未处理组（P<0.05）。用E.coli LF82和25或50 μmol/L EPA处理6或12小时的Caco-2细胞，随着EPA浓度的增加，ZO-1的mRNA表达增加，且显著高于单独用E.coli LF82处理的Caco-2细胞（P<0.05）。单独用E.coli LF82处理6或12小时的Caco-2细胞，随着处理时间的延长，TNF-α的分泌增加，且显著高于未处理的Caco-2细胞（P<0.05）。用E.coli LF82和25或50 μmol/L EPA处理6或12小时的Caco-2细胞，随着EPA浓度的增加，TNF-α的分泌减少，且显著低于单独用E.coli LF82处理的Caco-2细胞（P<0.05）。
EPA可有效预防E.coli LF82感染诱导的肠上皮细胞紧密连接破坏，并抑制炎症因子的分泌。因此，它对肠黏膜屏障具有一定的保护作用。
