利伐沙班对内毒素干扰人脐静脉内皮细胞损伤的影响

Effect of rivaroxaban on the injury during endotoxin-induced damage to human umbilical vein endothelial cells

目的探讨凝血因子Ⅹa(FⅩa)抑制剂利伐沙班对内毒素诱导的人脐静脉内皮细胞(HUVEC)损伤的作用及其机制。方法体外培养HUVEC,待细胞生长至80%融合时,按随机数字表法分为4组:空白对照组(DMEM培养基)、脂多糖(LPS)组(100μg/LLPS培养16h)、FⅩa+LPS组(100nmol/LFⅩa预处理24h后加入LPS)、FⅩa+RIV+LPS组(100nmol/LFⅩa+1μmol/L利伐沙班预处理24h后加入LPS)。各组细胞处理后用CCK-8细胞增殖及细胞毒性检测试剂盒检测细胞活性,用细胞划痕实验检测细胞迁移能力,用流式细胞仪检测细胞凋亡情况,用Transwell小室法及伊文思蓝检测单层内皮细胞屏障通透性,用酶联免疫吸附试验(ELISA)检测细胞肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-1β、IL-6)水平,用蛋白质免疫印迹试验(WesternBlot)检测细胞核转录因子-?κB(NF-κB)和丝裂素活化蛋白激酶(MAPK)炎症信号通路关键蛋白的表达。结果与空白对照组比较,LPS组细胞活性降低,细胞迁移能力增加,凋亡细胞增多,单层内皮细胞屏障通透性增加,TNF-α、IL-1β和IL-6等促炎因子分泌增加,炎症信号通路关键蛋白磷酸化c-Jun氨基末端激酶(p-JNK)、磷酸化p38MAPK(p-p38MAPK)、磷酸化转化生长因子激酶1(p-TAK1)和磷酸化NF-κBp65(p-NF-κBp65)表达增加,说明LPS可以刺激血管内皮细胞的炎症反应,从而对细胞活性、凋亡及功能有明显影响。FⅩa+LPS组除IL-6水平显著高于LPS组外,其他指标与LPS组比较差异均无统计学意义。与FⅩa+LPS组比较,FⅩa+RIV+LPS组细胞活性明显提高(A值:0.42±0.02比0.33±0.02),细胞迁移能力明显下降(倍:1.78±0.17比2.24±0.20),凋亡细胞明显减少〔(11.30±0.70)%比(21.03±0.19)%〕,单层内皮细胞通透性明显降低〔(149±12)%比(253±15)%〕,炎性因子水平明显降低〔IL-1β(ng/L):163.2±20.7比477.8±20.2,IL-6(ng/L):69.3±0.5比238.0±24.1,TNF-α(ng/L):117.0±13.1比196.2±4.5),炎症信号通路中p-TAK1和p-NF-κBp65蛋白表达水平明显降低(p-TAK1/TAK1:0.74±0.09比1.85±0.15,p-NF-κBp65/NF-κBp65:1.15±0.17比2.36±0.20),差异均有统计学意义(均P<0.05);而p-JNK和p-p38MAPK蛋白表达水平差异无统计学意义(p-JNK/JNK:1.64±0.12比1.65±0.15,p-p38MAPK/p38MAPK:2.31±0.32比2.35±0.20,均P>0.05)。结论利伐沙班可以有效缓解内毒素刺激HUVEC的炎症反应,其作用机制可能与抑制NF-κB信号通路激活而非MAPK信号通路有关。

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.