The Role And Mechanism Of Hydrogen-rich Medium Regulating Monocyte Adhesion And Vascular Endothelial Permeability

Objective:Sepsis is a very common clinical complication of systemic inflammatory response caused by a variety of infections, while due to its complex pathogenesis, there is no effective treatment. The increased endothelial cell permeability plays an important role in pathological process of death in patients with sepsis. Various adhesion molecules and chemokines regulate the migration, adhesion, accumulation, ultimately destroy the endothelial cells with high permeability. Vascular endothelial-cadherin participates in junction between normal cells, regulating vascular endothelial permeability. In recent years, hydrogen as an effective free radical scavenger, has shown selective antioxidant and anti-inflammatory effect, and it can be used in treatment of many diseases. In this study, we use LPS to stimulate vascular endothelial cells, and explore the role and mechanism of hydrogen-rich medium in the regulation of monocyte adhesion and vascular endothelial permeability.Methods:Human umbilical vein endothelial cells (HUVECs) and monocytes U937were cultured for experimental study after cell recovery.Part1:HUVECs were randomly divided into four groups:control group, hydrogen-rich medium group (0.6mmol/L), LPS group and LPS+H2group.1μg/mL LPS was added into the last two groups. When forming a monolayer, monocytes were added into each group after6,12and24h respectively. After coculture of90mins, adhesion was detected using Wright-Giemsa. Supernatants were collected to detect concentrations of vascular cell adhesion molecule-1(VCAM-1) and E-selectin using ELISA. The barrier function was detected by Transendothelial/endothelial Electrical Resistance(TEER). The expression of VE-cadherin was measured by Western blot. Cells were dyeing with immuno fluorescence to show distribution of VE-cadherin after incubation of24h.Part2:There were five groups:control group, LPS group, LPS+H2group, LPS+Y-27632(10μmol/L) group and LPS+Y-27632group (20μmol/L). After the incubation, endothelial barrier permeability were tested by TEER, and the concentration of VCAM-1and E-selectin were detected using ELISA. The expression of VE-cadherin and ROCK were measured by Western blot.Results:Part1:LPS could irritate monocytes adhesion, and increase the levels of adhesion molecules. After LPS stimulation, there was a significantly decreased values of TEER indicating a high barrier permeability, a low level of VE-cadherin, And it is not complete in cell-cell connects. While in LPS+H2group, monocyte adhesion and adhesion molecules decreased, while the levels of TEER values and the VE-cadherin increased, VE-cadherin is also complete and well-distributed.Part2:There showed the same trend of indicator in control group, LPS group and LPS+H2group, as the first part. As the inhibitor of ROCK, Y-27632could inhibit the effect of LPS on the barrier permeability, including TEER values and levels of VE-cadherin, similar to LPS+H2group. LPS could increase the levels of ROCK, while its level was decreased in LPS+H2group and groups with ROCK inhibitor Y-27632. The concentrations of Y-27632(20μmol/L) showed a better effect of permeability protection compared with Y-27632(10μmol/L).Conclusion:Hydrogen-rich medium could reduce the release of adhesion molecules induced by LPS then lessen monocytes adhesion to HUVEC. Meanwhile hydrogen shown identical function with ROCK inhibitor Y-27632in the effect on the TEER values and the levels of VE-cadherin. Possibly hydrogen regulated vascular endothelial cell permeability by participating in Rho/ROCK pathway.