Abstract: [Abstract] Objective To investigate the role of Rho kinase in the protection effects of hydrogen to intestinal epithelial barrier function against sepsis. Methods Caco-2 cells were cultured routinely, and divided into 6 groups randomly: control group, hydrogen-rich medium group, LPS-treatment group, hydrogen+ LPS-treament group, Y-27632 (ROCK inhibitor) treatment group and Y-27632+ LPS-treatment group. Hydrogen-rich medium group were treated with the 0.6mmol/L hydrogen-rich media. The concentration of LPS and Y-27632 were 100μg/ml and 25μmol/L separately. After the Caco-2 monolayer models were established, the transepithelial electrical resistance (TEER) values were measured regularly. When the TEER values reached 800Ω?cm2, the treatments were administered. Then TEER values were measured at 6h, 12h and 24h, and FITC-dextran permeability were detected at 24h. Cells were seeded on 6-well cell culture clusters. After cell density reached 80%~90%, treatments were given randomly. The real time-polymerase chain reaction (RT-PCR) was conducted to assess mRNA levels of ZO-1 and Rho kinase; ZO-1 and Rho kinase protein levels were detected with Western Blot. Results Compared with control group, TEER values of hydrogen-rich medium group elevated in 12h and 24h (P<0.05), no statistical significance in FITC-dextran permeability, neither in mRNA or protein levels of ZO-1 and ROCK (P>0.05); TEER values of Y-27632 treatment group at 6h, 12h and 24h elevated (P<0.05), FITC-dextran permeability did not change obviously (P>0.05), mRNA of ZO-1 increased and mRNA of ROCK decreased (P<0.05); the TEER values of LPS-treatment group reduced at 6h, 12h and 24h, FITC-dextran permeability increased markedly, mRNA and protein s of ZO-1 decreased, mRNA and protein s of ROCK increased (all P<0.05); compared with LPS-treatment group, TEER values in Y-27632+ LPS-treatment group rose significantly, FITC-dextran permeability decreased, mRNA s of ZO-1 increased, mRNA s of ROCK decreased (P<0.05); TEER values in hydrogen+ LPS-treament group increased, FITC-dextran permeability reduced markedly, protein s of ZO-1 increased, protein s of ROCK decreased (P<0.05). Conclusion Hydrogen can protect intestinal barrier function against sepsis, ameliorating the integrity of intestinal epithelium, reducing its permeability and increasing the s of tight junction proteins. The suppression of Rho kinase over reduced by LPS maybe involved in these protective effects of hydrogen.

Key words: Rho kinase, Hydrogen, LPS, epithelial barrier