蝮蛇毒血小板抑制因子减轻LPS诱导的内皮细胞损伤

AHV-PI attenuates endothelial cell injury induced by LPS

目的:探讨蝮蛇毒血小板抑制因子(AHV-PI)对体外脂多糖(LPS)诱导的人脐静脉血管内皮细胞(HUVECs)损伤的影响及其作用机制。方法:体外培养HUVECs,运用LPS(1 mg/L)诱导HUVECs炎症损伤模型,实验分为空白对照组、LPS组、AHV-PI组和AHV-PI+LPS组。MTT比色法检测HUVECs的活力,倒置显微镜观察HUVECs的形态变化,筛选出AHV-PI最适浓度为5 mg/L。流式细胞术检测细胞凋亡;免疫组化法观察胞内组织型纤溶酶原激活物(T-PA)以及纤溶酶原激活物抑制剂-1(PAI-1)表达情况;ELISA法检测HUVECs上清液中细胞间黏附分子-1(ICAM-1)和组织因子(TF)含量;免疫荧光染色检测胞核内NF-κB亚基p65激活转位情况。结果:LPS组细胞明显梭形化,长宽比增大,呈成纤维细胞状,胞浆出现颗粒样物质。AHV-PI浓度低于5 mg/L时对HUVECs的活性和形态没有明显影响,但可减轻LPS引起的HUVECs的活力抑制和形态改变。与对照组比较,LPS组上清液中TF和ICAM-1含量升高,胞内T-PA和PAI-1表达减少;与LPS组相比,AHV-PI+LPS组上清液中TF和ICAM-1的含量显著降低,细胞内T-PA和PAI-1表达增多,细胞核内NF-κB p65表达减少。结论:AHV-PI能减轻HUVECs损伤,其保护机制与抑制细胞因子分泌及NF-κB活化有关。

AIM:To investigate the effect of platelet inhibitor from Agkistrodon halys venom(AHV-PI)on lipopolysaccharide(LPS)induced human umbilical vein endothelial cells(HUVECs)injury in vitro,and to explore its mechanism.METHODS:Cultured HUVECs were induced inflammatory injury by LPS(1 mg/L).The experiment was divided into blank control group,LPS group,AHV-PI group and AHV-PI+LPS group.The viability of HUVECs was measured by MTT assay.The morphological changes of HUVECs were observed under inverted microscope.The optimum concentration of AHV-PI at 5 mg/L was selected.Flow cytometry was used to detect apoptosis of HUVECs.Immunohistochemical method was used to observe the expression of tissue type plasminogen activator(t-PA)and plasminogen activator indhibitor-1(PAI-1)of HUVECs.ELISA was used to detect the concentrations of intercellular adhesion molecule-1(ICAM-1)and tissue factor(TF)in the supernatant.The activation and translocation of NF-κB subunit p65 were observed by immunofluorescence staining.RESULTS:The HUVECs were spindle shaped,the ratio of length to width was increased,the cells were fibroblast-like,and granular substance appeared in the cytoplasm in LPS group.The viability and morphological changes of HUVECs were not significantly affected as treated with AHV-PI at concentration of 0~5 mg/L,but the viability of HUVECs induced by LPS was inhibited and the morphological changes were alleviated.Compared with the blank control group,the levels of TF and ICAM-1 in the supernatant increased,and the expression of t-PA and PAI-1 in the HUVECs was decreased in LPS group.Compared with the LPS group,the contents of TF and ICAM-1 in the supernatant were significantly decreased,the expression of t-PA and PAI-1 in the HUVECs was increased and the expression of nucleus NF-κB p65 was decreased in AHV-PI+LPS group(P<0.05).CONCLUSION:AHV-PI reduces HUVECs damage.The protective mechanism is related to the inhibition of cytokine secretion and NF-κB activation.