目的:探讨二十碳五烯酸(EPA)对热打击后肠黏膜上皮细胞通透性改变的影响.方法:使用Caco-2细胞株建立肠上皮细胞紧密连接模型,加入25、50、100、150 μmol/L EPA培养96 h,进行43 ℃ 1 h的热打击.分别使用CCK-8法检测细胞增殖,transwell测定单层跨膜电阻抗(TEER)值和对大分子物质辣根过氧化物酶(HRP)的通透性,Western blot法检测occludin蛋白的表达水平,显微镜下观察考马斯亮蓝染色细胞骨架的变化.结果:50 μmol/L的EPA促进细胞增殖的效果最强,升高TEER和防止HRP通透性升高的作用最大(与其他各浓度组比较,P均〈0.01),对occludin表达的增加作用最明显,并有益于维持细胞骨架的正常结构.结论:50 μmol/L的EPA能够保护肠上皮紧密连接,对肠黏膜屏障有一定的保护作用.
Objective:To discuss the effect of eicosapentaenoic acid (EPA) on tight junction (T J) dysfunction of intestinal epithelium after heat stress. Methods: Human intestinal epithelial Caco-2 cells were pre-incubated with various concentration of EPA (25,50,100,150 μmol/L) for 96 hours, and then they were exposed to a temperature of 43 ℃ for 1 hour. Cell counting kit-8 (CCK-8) assay was used to detect the cellular proliferation. The barrier integrity of TJ was measured by detecting transepithelial electrical resistance (TEER) and horseradish peroxi- dase (HRP) permeability with transwell. Level of TJ protein occludin was assayed by Western blot. Change in cytoskeleton was observed by microscopy after Coomassie blue staining. Results: EPA in 50 μmol/L concentration showed the most effective effects on promoting cell proliferation, and a decrease in TEER and impairment of intestinal permeability induced by heat exposure (all P ~ 0.01) were attenuated as compared to the other concentration groups. The function of occludin expression was most significantly elevated by 50 μmol/L EPA. The distortion of cytoskeleton was also effectively prevented by pretreatment with 50μmol/L EPA. Conclusions. This study indicates that 50 μmol/L EPA is potent in protecting intestinal epithelial barrier function against heat-induced permeability dysfunction and damage of tight junction.