| Objective:Sepsis is a systemic inflammatory response syndrome induced by infection, which is the leading cause of deaths in intensive care patients, there is no effective treatment. In sepsis,as the target cells and the effect cells of the inflammatory response, vascular endothelial cells were activated continuousely,and were severe damaged, eventually leading to organs damage and failure. Therefore, vascular endothelial cells as a targets for sepsis are of great significance. Hydrogen has the anti-oxidation,anti-inflammatory and anti-apoptosis effectives, and additionnaly hydrogen can ajust signal translation. So hydrogen can cure many diseases, such as brain,heart,liver,lung and kidney damage caused by ischemia-reperfusion, neurodegenerative diseases, diabetes, etc. Previous studies have found that hydrogen inhalation in sepsis mice has obvious protective effects,but its specific mechanism is unclear. Based on the previous studies, in this research we use LPS to induce endothelial cell damage, explore whether hydrogen has protective effects on vascular endothelial cell and the possible mechanisms, in order to provide experimental and theoretical basis for its clinical application in the future.Methods:Human umbilical vein endothelial cells were inoculated respectively with densities of1×104/mL and1×106/mL at96and6well Plates,200μL/well or3mL/well.Part I:Used the concentrations of0μg/mL,0.25μg/mL,0.50μg/mL,1.0μg/mL,2.0μg/mL and4.0μg/mL LPS to stimulate the cells. Cell proliferation activity was detected by MTT,24after LPS stimulation. Choosed the suitable concentration of LPS according to the results to establishcell damage model. Given the concentration of0.15mmol/L,0.3mmol/L and0.6mmol/L hydrogen-rich medium and measure cell activity and LDH release after24h, and then choosed the best hydrogen concentration.Part II:According to the first Part of the experiment, choosed1μg/mL LPS and hydrogen-saturated medium for this Part. Human umbilical vein endothelial cells were randomly divided into four groups (N=5):Normally cultured cells group (group C), Normally cultured cells with hydrogen treatment group (group H2), Normally cultured cells with LPS group (group LPS), Normally cultured cells with LPS and hydrogen treatment group (group LPS+H2). Groups C and LPS were given with normal medium; H2and LPS+H2groups were given with hydrogen-saturated medium, joined the1μg/mL concentration of LPS in Group LPS and group LPS+H2, but joined the same amount of physiological saline in C group and H2group at the same time. Cell apoptosis,ICAM-land VCAM-1expression were detected by flow cytometry at24h after LPS stimulation. The MDA levels in supernatants were measured at3h,6h,12h and24h after LPS stimulation.Part Ⅲ:The activity of SOD and CAT in supernatants were measured at3h,6h,12h and24h after LPS stimulation by application kits. Nrf2nuclear translocation and expression were detected respectively by immunofluorescence and Western Blot assay after incubation for24h.Results:Part Ⅰ:MTT results showed that cell growth was restrained after1μg/mL LPS stimulation Compared with normal cell significant (P＜0.05). Selected the1μg/mL LPS for the next experiments. Compared Group H2with Group C, there is no significant influence on cell poliferation activity and LDH release (P＞0.05).But hydrogen could significantly improve the activity and LDH release in the vascular endothelial cells stimulated by LPS (P＜0.05). And the0.6mmol/L hydrogen-rich medium was the most effective.Part Ⅱ:Compared with group C and H2, the number of apoptosis, the expressions of VCAM-1and ICAM-1of group LPS were significantly increased (P＜0.05); Compared with group LPS, the number of apoptosis, the expression of VCAM-1and ICAM-1of group LPS+H2were significantly reduced (P＜0.05); Compared with group C and H2, MDA levels in cell supernatant were significantly increased at3h,6h,12h and24h (P＜0.05); Compared with group LPS, MDA levels in cell supernatant of group LPS+H2were significantly reduced (P＜0.05).Part Ⅲ:At3h,6h,12h and24h after LPS stimulation, compared with group C and H2,the SOD and CAT activity in cell supernatant of group LPS were significantly reduced (P＜0.05); Compared with group LPS, SOD and CAT activity in cell supernatant of group LPS+H2were significantly increased (P＜0.05). Compared with group C and H2,the leve in the nucleus and expression of Nrf2of groups LPS and LPS+H2were significantly increased (P＜0.05),and compared with group LPS,the leve in the nucleus and expression of Nrf2of LPS+H2were significantly increased (P＜0.05).Conclusion The hydrogen-rich medium can effectively improve LPS-induced endothelial cell apoptosis, inhibit LPS-induced adhesion molecules expression and injury,which may be associated with promoting the Nrf2translateing from the cytoplasm to the nucleus and expression. |
PDF Full Text Request