Effects Of Astragaloside Ⅳ On Isoproterenol-induced Aortic Endothelial Function Damage And Its Underlying Mechanism

ObjectiveTo eveluate the effect of isoproterenol（ISO）on vascular function in cardiac hypertrophy model,and to assess the effect of Astragaloside IV（AsⅣ）on Iso-induced vascular dysfunction and the underlying mechanisms were investigated.Otherwise,we go further study to explore the effect of AsⅣ on the endothelial cells dysfunction induced by oxidative stress and its underlying mechanisms.MethodsSprague-Dawley（SD）rats were treated with Iso（10mg/kg/d）alone or in combination with AsⅣ（50mg/kg/d）.We divided the rats into four groups（n = 10）:the control group,Iso group,Iso plus Propranolol（Pro,40 mg/kg/day,i.g）,Iso plus AsⅣ（AsⅣ,50 mg/kg/day,i.g）.The ratios of heart weight/body weight and left ventricular weight/body weight were measured,the vascular reactivity of aortas was determined.The production of superoxide anion in situ was evaluated by hydroethidine（DHE）.The nitric oxide（NO）level in rat serum was determined with Griess method,the peroxynitrite（ONOO-）content of plasm was measured by ELISA.The ratio of eNOS dimer/ monomer,eNOS p65 and IκB-αprotein expression of the aortas were quantified by Western blot.RT-PCR was used to quantify m RNA expression of IL^-1β,IL-6 and TNF-α.Human umbilical vein endothelial cells（HUVECs）were divided into control,H₂O₂ 200μmol·L^-1,H₂O₂+AsⅣ(25 μmol·L^-1),H₂O₂+AsⅣ(50 μmol·L^-1),H₂O₂+AsⅣ(100 μmol·L^-1).We used MTT assay to evaluated the cell viability;intracellular superoxide anion was determined by DHE;NO production in the culture supernatants was determined by NO assay kit;BH₄ level was measured by HPLC;We measured the concentration of IL^-1β,IL-6 and TNF-α in the culture supernatants by ELISA;eNOS,eNOS dimer/monomer,nuclear-cytoplasmic NF-κB p65,the cytoplasmic IκB-α protein expression were quantified by Western blot.ResultsAnimal experiments showed,compared with the Control group,ISO obviously increased the ratios of heart weight/body weight（HMI）and left ventricular weight/body weight（LVMI）,increased the vasoconstriction response to phenylephrine,enhanced superoxide anion generation in rat aortas,increased eNOS protein expression while decreased its dimmer/monomer ratio and tetrahydrobiopterin（BH₄）level in aortas,reduced the NO production in the serum while increased the plasmatic peroxynitrite（ONOO-）.Moreover,ISO increased the ratio of nuclear-to-cytosolic protein expression of the NF-κB p65 subunit while decreased its inhibitor protein expression of IκB-α,increased m RNA expression of IL^-1β,IL-6 and TNF-α of the aortas.However,pre-treatment with AsⅣ could reverse these changes.Cell experiments show,compared with control group,H₂O₂-treated alone could decrease the cell viability,the NO production was also decreased,the BH₄ level was decreased,the intracellular superoxide anion generation were increased significantly,the total protein expression of eNOS was increased while its dimer/monomer ratio was obviously decreased,the protein expression of p65 in nucleus was increased while the cytoplasmic p65 protein expression was decreased,its inhibitor IκB-α protein expression was decreased,the production of IL^-1β,IL-6 and TNF-α in the culture supernatants were increased.Compared with the H₂O₂ group,pre-treatment with AsⅣ of various concentrations could reverse these conditions,and the differences had dose-dependent.ConclusionAsⅣ can protects against the vascular dysfunction in ISO-induced myocardial hypertrophy rat model,its mechanism may be via attenuating eNOS uncoupling-mediated oxidative stress,inhibiting the activation of ROS-NF-κB signaling pathways induced by oxidative stress so as to regulate the downstream inflammatory genes,thus alleviating the inflammatory response.