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目的:探讨Apelin-13在脂多糖(LPS)诱导的血管内皮细胞凋亡中的作用及其可能的机制。方法:体外培养原代人脐静脉内皮细胞株(HUVECs),给予LPS(1μg/ml)作用24h建立细胞凋亡模型,采用酶联免疫法测定Apelin-13的含量;RT-PCR法检测血管紧张肽受体样蛋白质J(APJ)受体、凋亡因子Fas mRNA表达的变化;荧光探针DCFH-DA检测细胞内活性氧(ROS)水平;Caspase-3荧光测定试剂盒检测细胞内Caspase-3的活性。结果:与对照组相比,LPS使细胞内Apelin-13的含量降低(P<0.05),而APJ受体的mRNA表达无明显变化;使Caspase-3的活性、Fas的mRNA表达及ROS的生成明显增加(P<0.05)。Apelin-13预处理能显著降低LPS诱导的细胞凋亡,使细胞内ROS产生明显减少(P<0.05);APJ siRNA转染则进一步促进了细胞凋亡及ROS生成(P<0.05)。此外,与对照组相比,H2O2孵育后能降低细胞内Apelin-13含量(P<0.05),而抑制NADPH酶或消除ROS可逆转LPS诱导的Apelin-13含量的降低(P<0.05)。结论:Apelin/APJ系统参与了LPS诱导的HUVECs凋亡,可能通过拮抗LPS诱导的细胞内ROS水平升高抑制HUVECs凋亡,发挥其保护作用。
AIM: To investigate the role of Apelin-13 in lipopolysaccharide (LPS) -induced apoptosis of vascular endothelial cells and its possible mechanism. Methods: Primary cultured human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Apoptosis model was induced by LPS (1μg / ml) for 24h. The content of Apelin-13 was determined by enzyme-linked immunosorbent assay (ELISA) (APJ) receptor and Fas mRNA expression. The level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. Caspase-3 was detected by Caspase-3 fluorescence assay kit Activity. Results: Compared with the control group, LPS decreased the content of Apelin-13 (P <0.05), while the mRNA expression of APJ did not change significantly. The activity of Caspase-3, the expression of Fas mRNA and the production of ROS Significantly increased (P <0.05). Apelin-13 preconditioning could significantly decrease the apoptosis induced by LPS and significantly reduce the intracellular ROS production (P <0.05). APJ siRNA transfection further enhanced the apoptosis and ROS production (P <0.05). In addition, compared with the control group, H2O2 increased the intracellular Apelin-13 level (P <0.05), whereas the inhibition of NADPH or ROS abolished the decrease of Apelin-13 level induced by LPS (P <0.05). CONCLUSION: Apelin / APJ system participates in LPS-induced apoptosis of HUVECs and may play a protective role by antagonizing the increase of intracellular ROS induced by LPS.