氢对内毒素诱导人结肠上皮细胞闭锁小带蛋白1表达的影响

Effect of hydrogen on endotoxin-induced expression of zonula occludens-1 in human colon epithelial cells

目的探讨氢对内毒素诱导人结肠上皮细胞（Caco-2细胞）闭锁小带蛋白1（ZO-1）表达的影响。方法常规培养Caco-2细胞，接种于Transwell小室或培养孔，采用随机数字表法分为4组（n=45）：对照组（C组）采用高糖DMEM培养基培养；富氢培养基组（H组）采用含0．6mmol／L氢气的富氢培养基孵育；内毒素组（E组）采用含50μg／ml脂多糖的高糖DMEM培养基孵育；富氢培养基＋内毒素组（HE组）采用含50μg／ml脂多糖＋0．6mmol／L氢气的富氢培养基孵育。于孵育或培养前、孵育或培养6、12、24h时测定细胞跨膜电阻（TEER），于孵育或培养24h时采用MTT法检测Caco-2细胞活力，于孵育或培养6、12和24h时采用RT-PCR法检测Caco-2细胞ZO-1mRNA的表达水平，于孵育或培养24h时细胞免疫荧光技术检测Caco-2细胞ZO-1的分布情况。结果与c组比较，E组和HE组孵育或培养6、12和24h时TEER值降低，ZO-1mRNA表达下调（P〈0．05），H组上述指标差异无统计学意义（P〉0．05）；与E组比较，HE组孵育或培养6、12和24h时TEER升高，ZO-1mRNA表达上调（P〈0．05）。E组细胞膜ZO-1分布不连续，胞浆ZO-1分布增多，HE组细胞膜ZO-1分布比E组增多，渐趋连续，胞浆ZO-1分布减少。结论氢减轻内毒素诱导人结肠上皮细胞屏障损伤的机制与上调ZO-1表达，改善ZO-1重分布有关。

Objective To investigate the effect of hydrogen on endotoxin-induced expression of zonula occludens-1 （ZO-1） in human colon epithelial cells （Caco-2 cells）. Methods Caco-2 cells were cuhured routinely, seeded in Transwell chambers or wells, and randomly divided into 4 groups （n = 45 each） using a random number table： control group （group C ）; hydrogen-rich culture medium group （ group H） ; endotoxin group （group E） ; hydrogen-rich culture medium ＋ endotoxin group （ group HE）. The cells were cultured in high-glucose DMEM culture medium in group C. The cells were incubated in hydrogen-rich culture medium containing hydrogen 0.6 mmol/L in group H. The cells were incubated in high- glucose DMEM culture medium containing 50 μg/ml lipopolysaccharide in group E. The cells were incuba- ted in hydrogen-rich culture medium containing 50 μg/ml lipopolysaccharide and 0.6 mmol/L hydrogen in group HE. Transepithelial electrical resistance （TEER） was measured before incubation or culture, and at 6, 12 and 24 h of incubation or culture. The viability of Caco-2 ceils was measured by methyl thiazolyl tet- razolium assay at 24 h of incubation or culture. The expression of ZO-1 mRNA in Caco-2 cells was determined using real-time reverse transcriptase polymerase chain reaction at 6, 12 and 24 h of incubation or culture. The distribution of ZO-1 in Caco-2 cells was observed by immunofluorescenee at 24 of incubation or culture. Results Compared with group C, TEER was significantly decreased at 6, 12 and 24 h of incubation or cuhure, and the expression of ZO-1 mRNA was significantly down-regulated in E and HE groups （P〈0.05） , and no significant change was found in the parameters mentioned above in group H （P〉0.05）. Compared with group E, TEER was significantly increased at 6, 12 and 24 h of incubation or culture, and the expression of ZO-1 mRNA was significantly up-regulated in group HE （P〈0.05）. The distribution of ZO-1 protein in cell membrane became discontinuous, and the distribution of ZO-1 protein in cytoplasm was significantly increased in group E. Compared with group E, the distribution of ZO-1 protein in cell mem- brane was significantly increased and gradually became continuous, and the distribution of ZO-1 protein in cytoplasm was significantly decreased in group HE. Conclusion The mechanism by which hydrogen reduces the damage to human colon epithelial cell barrier is related to up-regulation of ZO-1 expression and improvement in the redistribution of ZO-1 protein.