Rho Kinase Is Involved In The Protection Effect Of Molecular Hydrogen Against LPS-induced Human Intestinal Epithelial Barrier Dysfunction

Objective: Sepsis is infection-induced life-threatening systemic inflammation reaction syndrome(SIRS). Sepsis is usually consequent to heavy trauma, major operation or other serious stress events. Patients at different age groups can all suffer from sepsis. The incidence rate and fatality rate of sepsis are extremely high, and the survivors are often unavoidably in trouble with perpetual complications and sequelae thus get lower quality of life. The destruction of intestinal barrier has always being considered to be one of the "motor" of septic infections and multiple organ dysfunction syndrome(MODS). The composition of intestinal barrier is fairly complicated, which participate in immune system of human body and increasingly become the hotspot of sepsis-related research. Molecular hydrogen exist in atmospheric layer naturally; some species of bacteria within human GI tract can also generate molecular hydrogen as gas, and also, there are even also some molecular hydrogen exist in human bodies. It is reported that molecular hydrogen is medicative to multiple diseases at a certain concentration, and has been proved by several models. The therapeutic mechanisms of molecular hydrogen is not clear yet. Rho kinase(ROCK) is a serine/threonine kinase, which widely exist within mammal cells, participating in various intra-/inter cellular activities mainly through the regulation of cell skeleton. The research aimed at investigating the effects of molecular hydrogen to human intestinal epithelial cell barrier functions using in-vitro sepsis model and whether Rho kinase is involved in the mechanism of hydrogen effects.Methods: Human intestinal epithelial cells Caco-2 were cultured in high glucose DMEM medium with 20% fetal bovine serum. Cell passage was conducted until cells in log phase were collected for experiments. Part 1: Establishing the Caco-2 cellular monolayer barrier with Transwell chamber, or seeded cells onto 96-/6-well plates. The experiments were divided into 6 groups: control group(C group), 0.1mg/L Escherichia coli lipopolysaccharide(LPS) treatment group(L1 group), 1mg/L LPS treatment group(L2 group), 10mg/L LPStreatment group(L3 group), 50mg/L LPS treatment group(L4 group), 100mg/L LPS treatment group(L5 group). 24 h after treatments, the changes of integrity and permeability of Caco-2 cellular monolayer barrier in each group were tested with tranepithelial electrical resistance(TEER) values and FITC-dextran permeability. MTT method was used to observe cell apoptosis levels. Western Blot experiments were used to examine the expression levels of intercellular tight junction protein ZO-1. The proper concentration of LPS was chosen for the treatment. Part 2: Establishing the cellular models as part 1. The experiments were divided into 5 groups: control group(C group), hydrogen-rich medium group(H group), LPS-treatment group(L group), hydrogen-rich medium + LPS treatment(HL group), ROCK inhibitor(Y-27632) + LPS treatment group(YL group). Equivalent volume of high glucose DMEM medium or 0.6mmol/L hydrogen-rich medium was administered to cells in control group and hydrogen-rich medium treatment group separately. The treatment concentration of LPS was 50mg/L, and the treatment concentration of Y-27632 is 25μmol/L. TEER values were tested at 0h, 6h, 12 h, 24 h, and FITC-dextran permeability was tested at 24 h separately. MTT method was used to test cell apoptosis levels. Western Blot experiment was conducted to examine the expression levels of intercellular tight junction protein ZO-1 and ROCK protein at 6h, 12 h and 24 h. Real time-polymerase chain reaction(RT-PCR) was conducted to examine the expression levels of ZO-1 m RNA and ROCK m RNA at 6h, 12 h, 24 h. Cell immunofluorescence experiment was performed to observe the change of ZO-1 distribution at 24 h after treatments.Results: Part 1: Compared to control group, 0.1mg/L LPS failed to change the transepithelial electrical resistance(TEER) value and FITC-dextran permeability significantly(P>0.05), or increase cell apoptosis(P>0.05), or decrease the expression levels of intercellular tight junction protein ZO-1 of Caco-2 significantly(P>0.05); 1mg/L LPS and 10mg/L LPS could change the TEER value and FITC-dextran permeability significantly(P<0.05), and increased Caco-2 cell apoptosis(P<0.05), but failed to change the expression level of ZO-1 protein(P>0.05). 50mg/L LPS and 100mg/L LPS could both decrease TEER values and increase FICT-dextran permeability(P<0.05), which means destructing the integrity of Caco-2 cellular monolayer barrier and increasing its permeability. These two groups also showed increased Caco-2 cells apoptosis and decreased ZO-1 protein expression of Caco-2(P<0.05). Therefore, this research chose 50mg/L as LPS treatment concentration. Part 2: Compared to control group, LPS decreased TEER value and increased FITC-dextran permeability, while decreasing the expression of ZO-1 m RNA and protein and inducing ROCK m RNA and protein overexpression, the changes are statistically significant(P<0.05). LPS induced the intercellular protein ZO-1 move into Caco-2 cells, and the continuity of ZO-1 distribution on cell membrane was disturbed. Compared to LPS group, the addition of hydrogen-rich medium or ROCK inhibitor Y-27632 weakened the influence of LPS to Caco-2 cell monolayer TEER and FITC-dextran permeability(P<0.05); hydrogen-rich medium alleviated the LPS-induced increase of cell apoptosis(P<0.05), while ROCK inhibitor showed no effect on LPS-induced increase of cell apoptosis(P > 0.05); the addition of hydrogen-rich medium or ROCK inhibitor attenuated the LPS-induced decreased of ZO-1 m RNA and ZO-1 protein expression while inhibiting LPS-induced ROCK m RNA and ROCK protein overexpression, the changes were statistically significant(P < 0.05). Hydrogen-rich medium or ROCK inhibitor also ameliated the redistribution of ZO-1 protein induced by LPS. The results revealed hydrogen-rich medium and ROCK inhibitor can both alleviate the destructive effects of LPS to intestinal epithelial cell barrier. In hydrogen-rich medium group, ROCK m RNA expression levels and ROCK protein expression levels were lower than LPS group(P<0.05), which suggest ROCK protein may be involved in the protective effects of hydrogen to intestinal epithelial cell barrier.Conclusion: LPS can injure intestinal barrier directly in vitro; hydrogen-rich medium can alleviate the destruction, and Rho kinase may be involved in this protective effects of molecular hydrogen.