Adrenomedullin2Antagonizing Oxidative Stress Ameliorated Endothelial Dysfunction Induced By Angiotensin â…¡

Objective:To investigate the effect of adrenomedullin2on blood pressure in angiotensin Ⅱ-induced hypertension rat, the protective effect of adrenomedullin2against oxidative stress in vascular endothelium and its possible mechanism.Methods:(1)18male,6-week-old SD rats were randomly divided into three groups:control group (n=6), Ang Ⅱ group (n=6) and AngⅡ+ADM2group (n=6). Mini-osmotic pumps were implanted in the back subcutaneously. The control group was infused with saline and the Ang Ⅱ was infused at a dose of150ng/kg/min with or without ADM2(500ng/kg/h). After two weeks, blood pressure was measured by the way of carotid intubation. The plasma was collected for the detection of MDA content, NO content and the activity of SOD, CAT and NOS. The in situ oxidation of fluorescent dye DHE was used for detecting superoxide in rat arterys. The isometric myograph was used for examing the effect of ADM2on the change of tension (%) in rat arterial rings, with or without endothelium, which were preconstricted by Ang Ⅱ.(2) HUVEC and EA.hy926cell lines were cultured and treated with different concentration of Ang Ⅱ and ADM2. Intracellular ROS were evaluated by2’,7’-dichlorodihydro-fluorescein diacetate (DCFH-DA) and western blot was used to detect the protein expression. Membrane protein and cytosolic protein were separated to identify the translocation of p47phox from cytosol to membrane. Co-immunoprecipitation was performed for the p47phox phosphorylation assessment. NADPH oxidase activity was measured by a luminescence assay and NADPH/NADP+ratio was calculated according to the reduction of MTT. Two pairs of shRNA targeting homo ADM2were designed and synthesized. Then the complementary single stranded oligonucleotides (ss oligo) were annealed to create a double stranded oligonucleotide (ds oligo).The ds oligoes were ligated to plasmid vector and retroviral packaging was finished in293FT cells. ADM2-shRNA retroviruses were transfected into endothelial cells and the expression of ADM2was analyzed by western blot.Results:(1) ADM2could dramatically decrease the blood pressure in angiotensin Ⅱ-induced hypertension rat model. The activities of plasma SOD and CAT were significantly upregulated and the MDA content was reduced. ADM2decreased superoxide formation in vessel wall and enhanced NO content and the activities of eNOS.(2) ADM2induced relaxation of the vascular rings preconstricted by AngⅡ in a concentration-dependent and endothelium-dependent manner.(3) ADM2could ameliorate the ROS generation induced by Ang Ⅱ in a concentration-dependent manner in vascular endothelium, decreased the subunits expressions of NADPH oxidase and reduced the NADPH oxidase activity by inhibiting the membrane translocation and phosphorylation of p47phox.(4) After the ADM2protein expression was effectively down-regulated, the protein expressions of p47phox and Nox4were increased and eNOS was reduced in HUVEC.Conclusions:Adrenomedullin2antagonized oxidative stress by reducing the subunits expressions of NADPH oxidase and down-regulating the NADPH oxidase activity, subsequently ameliorated endothelial dysfunction and decreased the blood pressure of angiotensin Ⅱ-induced hypertension.