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

Abstract

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 cultured 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 incubated 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 cells was measured by methyl thiazolyl tetrazolium 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 immunofluorescence at 24 of incubation or culture. Results Compared with group C, TEER was significantly decreased at 6, 12 and 24 h of incubation or culture, and the expression of ZO-1 mRNA was significantly down-regulated in E and HE groups (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 membrane 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. Key words: Hydrogen; Endotoxemia; Tight junctions; Intestines