褪黑素对脂多糖诱导血管内皮细胞损伤的保护机制研究

Protective Effects of Melatonin on Human Umbilical Vein Endothelial Cell Injury Induced by LPS

目的:研究褪黑素(melatonin,MLT)对人脐静脉内皮细胞(HUVECs)损伤的保护作用及其机制探讨。方法:不同浓度的褪黑素作用于体外培养的内皮细胞脂质过氧化损伤模型,实验分为5组,即正常对照组(Ctrl),脂多糖(LPS)氧化损伤组:在培养基中加入2mmol/L的LPS诱导损伤4 h;LPS加MLT低剂量(200μmol/L)组、中剂量(400μmol/L)组、高剂量(600μmol/L)组。采用MTT法观察MLT对HUVECs活性的影响;用双波长荧光分光光度法测定HUVECs细胞内游离钙离子浓度;检测各组内皮细胞匀浆中丙二醛(MDA)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性;用ELISA法测定培养的细胞上清液中白细胞介素6(IL-6)表达的变化;并测定细胞凋亡率。结果:①LPS作用后血管内皮细胞损伤明显,细胞增殖减少,细胞培养上清液和细胞匀浆中MDA含量、细胞内钙离子浓度和IL-6均升高,SOD、GSH-Px活性下降,凋亡率可达38.9±1.1%,均与正常对照组有统计学差异(P<0.01);②加入MLT可明显减轻LPS对抗氧化酶SOD、GSH-Px的影响,同时MDA含量、细胞内钙离子浓度和IL-6均明显下降,并显著减少凋亡细胞数量,各指标差异有统计学意义(P<0.01)。结论:褪黑素可保护和修复LPS引起的血管内皮细胞损伤,其作用途径可能与保护细胞的线粒体,提高了该细胞的抗氧化酶活性,降低细胞内钙离子浓度作用有关。

Objective： To investigate the protective effect ofmelatonin （MLT） on human umbilical vein endothelial cells （HUVECs） injury and their preliminary mechanism. Methods： Lipopolysaccharide （LPS） was used to injure HUVECs in vitro. The cultured HUVECs were treated with MLT （200 ixmol/L, 400 p, mol/L, 600 ixmol/L） for 8 h, and the survival ratio of HUVECs was detected with MTT as- say. The cultured cells were loaded by Fura-2/AM and the change of [Ca2~] in HUVECs was measured by fluorospeetrophotometry. The contents ofmalondialdehyde （MDA）, superoxide dismutase （SOD）, reduced glutathione （GSH-Px） were measured by the commercial kits. The levels of tissue plasminogen activator IL-6 in the supematant were measured by enzyme linked immunosorbent assay （ELISA） kits. Apoptosis rate of the HUVECs was analyzed by flow cytometry. Results： LPS significantly increased the contents of MDA, IL-6 while decreased HUVECs viability, the contents of SOD and GSH-Px, and meantime increased the apoptosis rate about 38.9＋ 1.1% ex- pression in the injured groups. MLT at different dosages could depress the content of MDA and IL-6, improve the activity of SOD and GSH-Px compared with that in injury control group, both of which were depended on the concentration of MLT. Conclusions： The find- ings demonstrate that MLT displayed a protective action on LPS induced injury in human umbilical vein endothelial cells. Its effect may be related with the protection to cell chondriosome improvement in the antioxygenic activity of cells, and with suppressing the mobiliza- tion of cytosolic calcium.