【摘要】 为研究环孢霉素 A(cyclosporin A,CsA)对缺氧/复氧诱导人脐静脉内皮细胞(ECV-304)与中性粒细胞粘附的影响,本工作以缺氧/复氧诱导粘附为模型,采用β-N-乙酰氨基己糖苷酶比色法检测粘附率,流式细胞术检测ECV-304细胞表面粘附分子E-选择素(E-selectin)、细胞间粘附分子-1(ICAM-1)的表达,Fenton反应测定活性氧(reactive oxygen species,ROS)的含量,West-ern-blot法检测ECV-304细胞亲环素A(cyclophilin A,CyPA)、磷酸化及总细胞外信号调节激酶(ERK1/2)蛋白的表达。结果发现,ECV-304细胞经缺氧/复氧处理后,ROS释放增多,E-selectin、ICAM-1的表达上调,其表面中性粒细胞的粘附增加,CsA能显著抑制缺氧/复氧的上述作用。缺氧/复氧后,CyPA蛋白表达明显上调,ERK1/2显著活化,细胞总ERK1/2蛋白表达无明显改变。CyPA抑制剂CsA以及CyPA反义寡核苷酸均明显减轻缺氧/复氧诱导的ERK1/2激活,显著减少ECV-304细胞与中性粒细胞粘附。ERK1/2信号通路特异性阻断剂PD98059亦显著抑制ECV-304细胞与中性粒细胞的粘附。上述结果提示,CsA抑制缺氧/复氧诱导的ECV-304细胞与中性粒细胞粘附,并可能通过抑制ROS-Cyclophilin A-ERK1/2的信号转导途径实现。更多还原

【Abstract】 To investigate the inhibition of cyclosporin A (CsA) on neutrophil adhesion to human umbilical vein endothelial cells (HUVECs,ECV-304) induced by hypoxia/reoxygenation and further explore its mechanism, a 1 h hypoxia/4 h reoxygenation model was reproducedusing ECV-304. The adhesion rate of neutrophils to ECV-304 was determined by measuring the activity of endogenous hexosaminidase. Theexpression of endothelial cell adhesion molecules of E-selectin and ICAM-1 was measured by flow cytometry. The expression of cyclophilinA (CyPA) and the activation of ERK1/2 was compared among experimental groups by Western blot. The content of reactive oxygen species(ROS) was measurd by Fenton reaction. After being stimulated with 1 h hypoxia/4 h reoxygenation, ECV-304 showed an enhanced neutrophiladhensiveness in association with an increased surface expression of E-selectin and ICAM-1. In parallel, the content of ROS was alsoincreased. These effects were significantly suppressed by the addition of CsA. Most importantly, the expression of CyPA was significantlyincreased following 1 h hypoxia/4 h reoxygenation, which was accompanied with an increased activation of ERK1/2. Treatment with CyPAinhibitor CsA and CyPA antisense oligonucleotides significantly inhibited the activation of ERK1/2 and decreased the adhesion of neutrophilsto ECV-304. The specific ERK1/2 inhibitor PD98059 caused an inhibition of neutrophil adhesion to hypoxia/reoxygenation-stimulated ECV-304. Our data confirm that CsA inhibits neutrophil adhesion to hypoxia/reoxygenation stimulated ECV-304 by a mechanism involvinginhibition of the signal transduction of ROS, CyPA and ERK1/2.更多还原