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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 effect of eicosapentaenoic acid (EPA) on the expression of tight junction protein (ZO-1) in intestinal epithelial cells (Caco-2) after adhesion-invasive E. coli (AIEC) infection. Methods Caco-2 cell lines were used to establish the close model of intestinal epithelial cells in vitro. The models were divided into four groups: EPA group (0, 25, 50, 100, 200μmol / L) 200μmol / L) + E.coli LF82 combined treatment group, with different concentrations of EPA intervention on the basis of 96 h, E.coli LF82 intervention were 0 h, 6 h, 12 h. The Caco-2 cell model was evaluated by cell morphology observation, MTT assay of cell growth curve, and alkaline phosphatase (ALP) activity assay on both sides of cell membrane. Flow cytometry was used to detect the effects of different concentrations of EPA on the apoptosis of Caco-2 cells. RT-q PCR was used to detect the expression of ZO-1 m RNA in Caco-2 cells treated with EPA and / or E.coli LF82. The changes of TNF-α in the supernatant of Caco-2 cells were detected by enzyme-linked immunosorbent assay. Results After treatment with EPA25 and 50 μmol / L, the survival rate of Caco-2 cells was higher than that of 0 group (P <0.05). The cell viability of the cells treated with EPA100 and 200 μmol / L decreased in a concentration-dependent manner , All lower than 0 concentration group (P <0.05). When the EPA concentration was 100 and 200 μmol / L, the apoptosis rate was increased compared with the 0 concentration group (P <0.05). The expression of ZO-1 mRNA in Caco-2 cells treated with LF82 alone for 6 h and 12 h decreased with the prolongation of treatment time, both of which were lower than those of untreated group (P <0.05). The expression of ZO-1 m RNA in Caco-2 cells treated with EPA 25,50 μmol / L and E.coli LF82 for 6 h or 12 h increased with the increase of EPA concentration, which was higher than that of E.coli LF82 alone group (P < 0.05). The secretion of TNF-α in 6 h and 12 h groups treated with E. coli LF82 alone increased with the prolongation of intervention time, both of which were significantly higher than those of untreated group (P <0.05). EPA25, 50μmol / L and LF82 treatment for 6 h or 12 h, TNF-α secretion in the supernatant decreased with the increase of EPA concentration, which were less than that of LF82 alone treatment group (P <0.05). Conclusions EPA can effectively prevent the destruction of tight junctions of intestinal epithelial cells induced by E. coli LF82, inhibit the secretion of inflammatory cytokines and have some protective effects on intestinal mucosal barrier.