The effects of Kv1.5 on sepsis-induced endothelial cell injury

Abstract

Objective To investigate the correlation between Kv1.5 protein and lipopolysaccharide (LPS)-induced endothelial cell injury. Methods LPS effected on human umbilical vein endothelial cells (HUVECs) in a concentration gradient (0.5, 1.0, 5.0, 10.0 μg/ml)and time gradient (0, 6, 12, 24, 48 h), then measure the cell apoptosis rate by flow cytometry to establish the model of endothelial cell injury, and detect the level of endothelial cells Kv1.5 protein by Western blotting; different concentrations of mephetyl tetrazole (MT, Kv1.5-specific channel blockers) (75, 125, 250, 500 mmol/L) was used to pretreatment 30 min to block Kv1.5 channels, then used flow cytometry to evaluate endothelial cell apoptosis rate, Enzyme linked immunosorbent assay (ELISA) assay to detect the concentrations of endothelial cell injury markers endocan and VCAM-1. Results Incubated HUVECs with 0.5, 1, 5, 10 μg/mlfor 24 h, the rates of Endothelial cells apoptosis were (33.530±1.266)%, (35.530±0.551)%, (48.270±0.901)%, (51.600±2.179)%, it is statistical different compared with control group (the values of P were 0.025, 0.027, 0.011, 0.004). Then treated HUVECs for 6, 12, 24, 48 h, LPS could induce HUVECs endothelial cell injury, the rates of Endothelial cells apoptosis were (30.200±1.113)%, (32.470±0.814)%, (46.630±0.513)%, (50.870±1.498)% (the values of P were 0.027, 0.025, 0.012, 0.006). Respectively, the rate of Endothelial cells apoptosis was obvious when treated with 5 μg/ml LPS for 24 h. Endothelial cells apoptosis was induced by LPS in a concentration and time-dependent, endothelial cell injury model was 5 μg/ml LPS treatment for 24 h. Kv1.5 protein expression in all groups treated with LPS of different concentration was significantly increased (P=0.036, 0.027, 0.008, 0.005) in LPS group(12, 24、48 h) (the values of P were 0.004, 0.036, 0.007), the expression of Kv1.5 protein increased in a dose and time-dependent partly. Compared with LPS group, endothelial cell apoptosis was significantly lower in MT groups (75, 125, 250, 500 mmol/L, P=0.000). When MT concentration was increased to 125 mmol/L, HUVECs apoptosis rate was significantly reduced, and the concentration of endocan (P=0.001, 0.000, 0.001, 0.006) and VCAM-1 (P=0.006, 0.000, 0.000, 0.000) secretion was decreased. Conclusion LPS-induced injured HUVECs express more Kv1.5 protein; Kv1.5 channel blockers can reduce the LPS-induced injury of HUVECs. Key words: Kv1.5; Human umbilical vein endothelial cells; Mephetyl tetrazole; Endothelial cell injury