血管生成素4对脂多糖诱导的人脐静脉内皮细胞损伤的影响

Effects of angiopoietin 4 on lipopolysaccharide-induced injury of human umbilical vein endothelial cells

目的:观察血管生成素4(Ang-4)对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)损伤的影响,并探讨其可能的作用机制。方法:EnVision法免疫组化鉴定HUVECs。LPS诱导细胞损伤,不同剂量的Ang-4干预。显微镜下观察细胞形态,MTT法检测细胞活力,ELISA法检测von Willebrand因子(vWF)和肿瘤坏死因子(TNF-α)含量,real-time PCR检测Toll样受体4(TLR4)、核因子κB(NF-κB)p65和TNF-αmRNA含量变化。结果:免疫组化示胞浆内人Ⅷ因子相关抗原呈阳性;与正常组比,LPS组细胞增殖活力降低(P<0.01),vWF及TNF-α分泌增多(P<0.01),且TLR4、NF-κB p65和TNF-αmRNA表达升高(P<0.01);Ang-4(100μg/L)组可恢复细胞活力,降低vWF及TNF-α含量(P<0.01),抑制LPS所致TLR4、NF-κB p65和TNF-αmRNA的表达(P<0.01)。结论:Ang-4可拮抗LPS诱导的HUVECs损伤,这可能与抑制TLR4-NF-κB p65-TNF-α信号通路的活化有关。

AIM： To observe the effects of angiopoietin 4 （Ang-4） on lipopolysaccharide （LPS） -induced injury of human umbilical vein endothelial cells （HUVECs）. METHODS ： The EnVision immunohistochemical method was used to identify the HUVECs. After pre-treated with different doses of Ang-4 for 0.5 h, HUVECs was exposed to LPS at concentration of 10 mg/L for 24 h. The cell viability was evaluated by MTF assay. The content of tumor necrosis factor-al- pha （TNF-α） in the supernatant and the concentrations of intracellular and supernatant von Willebrand factor （vWF） were detected by ELISA. The mRNA levels of Toll-like receptor 4 （TLR4）, NF-κB p65 and TNF-α were determined by realtime PCR. RESULTS： Factor Ⅷ in the cytoplasm was positive in the HUVECs. Compared with normal group, LPS reduced the cell viability （P 〈 0.01 ）, and significantly increased the secretion of TNF-α and vWF （P 〈 0.01 ）. The mRNA expression of TLR4, NF-κB p65 and TNF-a also increased （P 〈0.01 ）. Ang-4 at concentration of 100 μg/L enhanced the cell viability （P 〈 0.01 ）, reduced the content of vWF and TNF-α, and inhibited the LPS-induced increases in the mRNA levels of TLR4, NF-κB p65 and TNF-α （P 〈0.01 ）. CONCLUSION： Ang-4 antagonizes LPS-induced damage in HUVECs by inhibiting TLR4-NF-κB p65-TNF-ot signaling pathways.