Role of RhoA in amelioration of lipopolysaccharide-induced human intestinal barrier dysfunction by hydrogen-rich saline: in vitro experiment

Objective To evaluate the role of RhoA in amelioration of lipopolysaccharide (LPS)-induced human intestinal barrier dysfunction by hydrogen-rich saline. Methods Caco-2 cells were plated on transwells at a density of 2×105/ml (400 μl/well), and randomly divided into 5 groups (n=50 each) using a random number table: control group (group C), hydrogen-rich saline group (HRS group), LPS group, LPS + hydrogen-rich saline group (group LPS+ HRS), and LPS + RhoA inhibitor C3 exoenzyme group (group LPS+ C3). Cells were cultured in the medium containing 0.6 mmol/L hydrogen-rich saline in HRS and LPS+ HRS groups, or in the medium containing 100 μg/ml LPS in LPS, LPS+ HRS and LPS+ C3 groups.In group LPS+ C3, C3 exoenzyme 2.5 μg/ml was added at 1 h before addition of LPS.Five wells were selected at 0, 3, 6, 12, 24, 36 and 48 h of incubation with LPS, and the transepithelial electrical resistance (TEER) and permeability coefficient (PE) were measured.Five wells were selected at 24 h of incubation with LPS for determination of occludin and E-cadherin expression (by Western blot and immunofluorescence) and RhoA activity (using GST pull-down assay). Results Compared with group C, TEER was significantly decreased, PE and RhoA activity were increased, and the expression of occludin and E-cadherin was down-regulated at 6-48 h of incubation with LPS in LPS, LPS+ HRS and LPS+ C3 groups (P 0.05). Compared with group LPS, TEER was significantly increased, PE and RhoA activity were decreased, and the expression of occludin and E-cadherin was up-regulated at 6-48 h of incubation with LPS in LPS+ HRS and LPS+ C3 groups (P<0.05). Compared with group LPS, the distribution of occluding and E-cadherin in the cell membrane was significantly increased, the distribution in the cytoplasm was decreased, and the structures of occludin and E-cadherin partly recovered in LPS+ HRS and LPS+ C3 groups. Conclusion Hydrogen-rich saline can alleviate LPS-induced intestinal barrier dysfunction via inhibiting RhoA activation, thus up-regulating occludin and E-cadherin expression and inhibiting the disturbance of the distribution. Key words: RhoA GTP-binding protein; Hydrogen; Endotoxemia; Intestinal mucosa