Expression of NADPH Oxidase and Production of Reactive Oxygen Species in Aorta in an Active Immunization Mouse Model with AT1-EC2 Peptide

摘要

The antibody against AT1-EC2 plays a role in some kinds of inflammatory vascular diseases including malignant hypertension,preeclampsia,and renal-allograft rejection,but the detailed mechanisms remain unclear.In order to investigate the changes of NADPH oxidase and reactive oxygen species in the aorta in a mouse model which can produce AT1-EC2 antibody by active immunization with AT1-EC2 peptide,15 mice were divided into three groups:control group,AT1-EC2-immunized group,and AT1-EC2-immunized and valsartan-treated group.In AT1-EC2-immunized group and AT1-EC2-immunized and valsartan-treated group,the mice were immunized by 50 μg peptide subcutaneously at multiple points for 4 times:0,5,10,and 15 days after the experiment.In AT1-EC2-immunized and valsartan-treated group,valsartan was given at a dose of 100 mg/kg every day for 20 days.After the experiment,the mice were sacrificed under anesthesia and the aortas were obtained and frozen in liquid nitrogen for the preparation of frozen section slides and other experiments.The titer of AT1-EC2 was assayed by using ELISA.The level of NOX1 mRNA in the aorta was determined by using RT-PCR.The expression of NOX1 was detected by using Western blotting.Confocal scanning microscopy was used to assay the α-actin and NOX1 expression in the aortic tissue.The O 2.production was detected in situ after DHE staining.The mice produced high level antibody against AT1-EC2 in AT1-EC2-immunized group and AT1-EC2-immunized and valsartan-treated group,and the level of NOX1 mRNA in the aortic tissues was 1.6±0.4 times higher and the NOX1 protein expression was higher in AT1-EC2-immunized group than in control group.There were no significant differences in the level of NOX1 mRNA and protein expression between control group and AT1-EC2-immunized and valsartan-treated group.The expression and co-localization of α-actin and NOX1 in AT1-EC2-immunized group increased significantly as compared with those in control group,and the O 2.production increased about 2.7 times as compared with control group.There were no significant differences between control group and AT1-EC2-immunized and valsartan-treated group.It is concluded that active immunization with AT1-EC2 can activate NOX1-ROS,and increase vascular inflammation,which can be inhibited by AT1 receptor blocker valsartan.This may partially explain the mechanism of the pathogenesis of inflammatory vascular diseases related to antibody against AT1-EC2.