亚砷酸钠对人脐静脉内皮细胞损伤及鞘氨醇激酶1/1-磷酸鞘氨醇信号轴的影响

Effects of sodium arsenite on human umbilical vein endothelial cell injury and sphingosine kinases 1/sphingosine 1-phosphate signaling axis

背景:血管内皮细胞是砷毒作用的关键靶点,砷致内皮细胞损伤的分子机制有待进一步研究。目的:研究亚砷酸钠对人脐静脉内皮细胞的损伤及鞘氨醇激酶1/1-磷酸鞘氨醇(SPHK1/S1P)信号轴的影响。方法:分离培养并鉴定人脐静脉内皮细胞,将人脐静脉内皮细胞暴露于含0,5,10,15,20,25μmol/L亚砷酸钠的培养液24 h,CCK-8法检测细胞存活率;倒置显微镜观察细胞形态;荧光探针DCFH-DA染色检测细胞内活性氧含量;Annexin V-FITC/PI双标记结合流式细胞术及TUNEL法检测细胞凋亡;ELISA检测细胞内1-磷酸鞘氨醇含量;实时荧光定量PCR、Western blot分别检测血管细胞黏附分子1、鞘氨醇激酶1、1-磷酸鞘氨醇受体1的mRNA和蛋白表达。结果与结论:①与对照组(0μmol/L亚砷酸钠)相比,随着亚砷酸钠浓度的增加:细胞存活率逐渐降低,且呈剂量-效应关系;细胞融合率逐渐降低,细胞间隙逐渐增宽,脱落漂浮的细胞逐渐增多;细胞内活性氧含量逐渐升高;细胞凋亡率逐渐升高;细胞内1-磷酸鞘氨醇含量逐渐升高;血管细胞黏附分子1、鞘氨醇激酶1 mRNA和蛋白的相对表达量逐渐升高,1-磷酸鞘氨醇受体1 mRNA和蛋白相对表达量逐渐降低。②结果提示,亚砷酸钠诱导的人脐静脉内皮细胞损伤可能与SPHK1/S1P信号轴激活、1-磷酸鞘氨醇受体1下调有关。SPHK1/S1P信号轴可能成为砷致心血管损伤的新靶点。

BACKGROUND:Vascular endothelial cells are the main target of arsenic toxicity and the molecular mechanism of arsenic-induced endothelial cell injury needs to be further studied.OBJECTIVE:To study the effects of sodium arsenite on human umbilical vein endothelial cell injury and sphingosine kinase 1/sphingosine 1-phosphate signaling axis of human umbilical vein endothelial cells.METHODS:Human umbilical vein endothelial cells were isolated,cultured,and identified.The cells were treated with 0,5,10,15,20,25μmol/L sodium arsenite for 24 hours.Cell viability was detected by cell counting kit-8.Cell morphology was observed by inverted phase contrast microscope.The content of intracellular reactive oxygen species was detected by fluorescent probe DCFH-DA.Annexin V-FITC/PI double labeling combined with flow cytometry and TUNEL were used to detect apoptosis.The content of sphingosine 1-phosphate in cells was detected by ELISA.The mRNA and protein expressions of vascular cell adhesion molecule-1,sphingosine kinase 1,and sphingosine 1-phosphate were detected by real-time fluorescence quantitative PCR and western blot respectively.RESULTS AND CONCLUSION:Compared with the control group(0μmol/L sodium arsenite),the cell viability decreased gradually along with the increased concentration of sodium arsenite in a dose-dependent manner;the cell fusion rate decreased gradually,the cell gap widened gradually,and the number of exfoliated and floating cells increased gradually;the reactive oxygen species content and apoptosis rate increased gradually;the sphingosine 1-phosphate content in cells increased gradually;the relative mRNA and protein expressions of vascular cell adhesion molecule-1 and sphingosine kinase 1 increased gradually,but the relative mRNA and protein expression of sphingosine kinase 1 decreased gradually.To conclude,sodium arsenite-injured human umbilical vein endothelial cell injury may be related to the activation of sphingosine kinase 1/sphingosine 1-phosphate signaling axis and the downregulation of sphingosine 1-phosphate receptor 1.Sphingosine kinase 1/sphingosine 1-phosphate signaling axis may become a new target of arsenic-induced cardiovascular injury.