Apelin-13通过调节活性氧水平抑制脂多糖诱导的内皮细胞凋亡的机制研究

Apelin-13 inhibits lipopolysaccharide-induced endothelial cells apoptosis by regulating reactive oxygen species production

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。

Objective：To investigate the effect of Apelin-13 on apoptosis of human umbilical vein endothelial cells（HUVECs）induced by lipopolysaccharide（LPS）.Method：HUVECs were cultured in vitro and treated by LPS of 1μg/ml for 24 hours to induce apoptosis model.Enzyme immunoassay（EIA）was carried out using an enzyme immunoassay kit for Apelin-13.The mRNA expression of APJ and Fas was detected by RT-PCR.Intracellular reactive oxygen species（ROS）levels were measured by fluorescence method.The caspase-3activity in HUVECs was determined by using a caspase-3fluorescent assay kit.Result：Compared with control group,LPS significantly decreased Apelin-13 production,but enhanced apoptotic cascades,such as Fas mRNA expression and Caspase-3activation,and increased intracellular ROS production（both P〈0.05）,with no influence on APJ mRNA expression.These effects of LPS were abrogated by Apelin-13 pretreatment but further romoted by knockdown of APJ using specific siRNA（both P〈0.05）.In addition,treatment with H2O2 remarkably reduced Apelin-13 production in HUVECs.Inhibition of NADPH oxidase or scavenging ROS significantly increased Apelin-13 production in LPS-stimulated HUVECs.Conclusion：The Apelin/APJ system is involved in LPS-induced HUVECs apoptosis.Apelin-13 can protect HUVECs from LPS-induced apoptosis via down-regulating intracellular ROS production.