ABSTRACT
Objective To evaluate the effect and mechanism of rivaroxaban, an inhibitor of coagulation factor Ⅹa (FⅩa), on endotoxin-induced injury to human umbilical vein endothelial cells (HUVEC). Methods When cultured HUVEC grow to 80% fusion, they were divided into four groups according to the random number method: blank control group (DMEM medium), lipopolysaccharide (LPS) group (cells were challenged by 100 μg/L LPS for 16 hours), FⅩa+LPS group (cells were challenged by LPS for 16 hours after they were cultured with 100 nmol/L FⅩa for 24 hours), and FⅩa+RIV+LPS group (cells were challenged by LPS for 16 hours after they were cultured with 100 nmol/L FXa and 1 μmol/L rivaroxaban for 24 hours). After each group of cells were challenged with LPS, the cell activity was detected by the cell proliferation and toxicity kit (CCK-8); the cell migration ability was detected by cell scratch experiments;the abilities of cells migration were measured by scratch-wound-healing assay; the apoptosis of cells were evaluated using flow cytometry; the endothelial barrier of cells was assessed by Transwell and Evans blue; the levels of tumor necrosis factor-α(TNF-α), interleukin (IL-1β, IL-6) were detected by the enzyme linked immunosorbent assay (ELISA); the expressions of nuclear factor-κB (NF-κB) and mitogen activated protein kinase (MAPK) signaling pathway were detected by Western Blot. Results Compared with blank control group, the cell viability in LPS group was significantly decreased, and the migration ability, number of apoptotic cells, and barrier permeability of endothelial cells was significantly increased, the levels of TNF-α, IL-1β and IL-6 were significantly increased, and the expressions of phosphorylation of c-Jun N-terminal kinase (p-JNK), phosphorylation of p38MAPK (p-p38MAPK), phosphorylation of transforming growth factor kinase 1 (p-TAK1) and phosphorylation of NF-κBp65 (p-NF-κBp65) were significantly increased. It indicated that LPS could stimulate the inflammatory response of vascular endothelial cells, and had a significant impact on cell activity, apoptosis and function. There was no significant difference in above indexes between FⅩa+LPS group and LPS group, except for the level of IL-6 being higher in FⅩa+LPS group. Compared with FⅩa+LPS group, in FⅩa+RIV+LPS group, the cell activity was significantly increased (A value: 0.42±0.02 vs. 0.33±0.02), and migration ability was significantly decreased (folds: 1.78±0.17 vs. 2.24±0.20), the number of apoptotic cells was significantly decreased [(11.30±0.70)% vs. (21.03±0.19)%], and permeability of monolayers endothelial cells was significantly decreased [(149±12)% vs. (253±15)%], the levels of inflammatory cytokines were significantly decreased [IL-1β(ng/L): 163.2±20.7 vs. 477.8±20.2, IL-6 (ng/L): 69.3±0.5 vs. 238.0±24.1, TNF-α(ng/L): 117.0±13.1 vs. 196.2±4.5], the expressions of p-TAK1 and p-NF-κBp65 were significantly decreased (p-TAK1/TAK1: 0.74±0.09 vs. 1.85±0.15, p-NF-κBp65/NF-κBp65: 1.15±0.17 vs. 2.36±0.20), with statistically significant differences (all P <0.05). There was no significant difference in the p-JNK, p-p38MAPK expressions between FⅩa+RIV+LPS group and FⅩa+LPS group (p-JNK/JNK: 1.64±0.12 vs. 1.65±0.15, p-p38MAPK/p38MAPK: 2.31±0.32 vs. 2.35±0.20, both P > 0.05). Conclusion Rivaroxaban can effectively relieve the inflammatory response of HUVEC stimulated by LPS, which may be related to the inhibition of NF-κB signaling pathway activation rather than MAPK signaling pathway.