二十碳五烯酸对E.coli LF82感染后肠上皮细胞紧密连接蛋白ZO-1 mRNA的影响

Effect of eicosapentaenoic acid on m RNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection

目的探讨二十碳五烯酸(EPA)对黏附侵袭性大肠杆菌(AIEC)LF82感染后肠上皮细胞(Caco-2)紧密连接蛋白(ZO-1)表达的影响。方法用Caco-2细胞株建立体外肠上皮细胞紧密连接模型,分为EPA处理组,EPA 0、25、50、100、200μmol/L干预96 h;以及EPA(EPA 0、25、50、100、200μmol/L)+E.coli LF82联合处理组,在不同浓度EPA干预96 h的基础上,予E.coli LF82分别干预0 h、6 h、12 h。通过细胞形态学观察,MTT法测定细胞生长曲线,以及细胞膜两侧碱性磷酸酶(ALP)活性检测对Caco-2细胞模型进行评价。流式细胞术检测不同浓度EPA对Caco-2细胞凋亡的影响。RT-q PCR检测EPA和/或E.coli LF82作用于Caco-2细胞后ZO-1 m RNA的表达情况。酶联免疫吸附法检测Caco-2细胞上清中TNF-α的变化。结果 EPA25、50μmol/L处理后,Caco-2细胞存活率均高于0浓度组,且增高呈浓度依赖性(P<0.05);EPA100、200μmol/L处理组的细胞存活率下降呈浓度依赖性,均低于0浓度组(P<0.05)。EPA浓度为100、200μmol/L时,细胞凋亡率较0浓度组增加(P<0.05)。单独E.coli LF82干预Caco-2细胞6 h、12 h后,ZO-1 m RNA表达随处理时间延长而减少,均低于未处理组(P<0.05)。EPA 25、50μmol/L干预联合E.coli LF82处理6 h或12 h,Caco-2细胞的ZO-1 m RNA表达随EPA浓度增加而增加,均高于E.coli LF82单独处理组(P<0.05)。单独E.coli LF82处理6 h、12 h组的TNF-α分泌随干预时间延长而增加,均高于未处理组(P<0.05)。EPA25、50μmol/L联合LF82处理6 h或12 h,细胞上清液中TNF-α分泌量随EPA浓度的增加而减少,均少于单独LF82处理组(P<0.05)。结论 EPA能有效预防E.coli LF82感染后肠上皮细胞紧密连接的破坏,抑制炎症因子的分泌,对肠黏膜屏障有一定的保护作用。

Objective 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. Methods 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 inCaco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-or （TNF-α） in culture supernatant. Results 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-I 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）. Conclusions 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.