Activation Of TACE/EGFR Signaling Pathways In Vascular Endothelial Cells By Astragaloside Ⅲ

Background and Objective: Cardiovascular disease(CVD)has become one of the most harmful diseases around the world.Vascular endothelial cells(VECs)covering the surface of vascular lumen can synthesize and release a variety of vasoactive substances and cytokines,which are not only of importance for the maintenance of vascular homeostasis and vascular function,but also play important roles in the development of CVD.Astragaloside Ⅲ(AS-Ⅲ)is one of the bioactive triterpenoid saponins contained in Astragali Radix.Previous studies have found that AS-Ⅲ has potent anti-inflammatory effects on VECs,however underlying mechanisms are unclear.This work is aimed to study the effect of AS-Ⅲ on the activation of tumor necrosis factor-α converting enzyme(TACE)and TACE-dependent regulation of epithelial growth factor receptor(EGFR)-mediated signaling pathways in VECs,and further to investigate the molecular mechanisms involved in activation of TACE,so as to provide a new laboratory basis for the cardiovascular protective effect of Chinese herbal medicine Astragali Radix.Methods: Mouse endothelial b End.3 cells were used in this study.(1)b End.3 cells growing in 96-well cell culture plates were treated with different concentrations of AS-Ⅲ,and then cell viability was evaluated by conducting the WST-1 assay.(2)b End.3 cells growing in 6-well cell culture plates were treated with different concentrations of AS-Ⅲand TACE inhibitor(TAPI-0),and then TACE activity assay kit was used to measure the sheddase activity.(3)b End.3 cells were treated with AS-Ⅲ and various signaling pathways inhibitors,and the phosphorylation of TACE,ERK1/2,AKT,EGFR and p38 were investigated by Western blotting analysis.Results:(1)Compared with the control group,in the range from 10 to 1000 n M,AS-Ⅲdid not reduce the cell viability(P>0.05);however,10 μM AS-Ⅲ significantly reduced the cell viability of b End.3 cells(P<0.01);(2)Compared with the control group,AS-Ⅲinduced phosphorylation of TACE at Thr735 and enhanced its sheddase activity,both of which were significantly inhibited by TAPI-0 pretreatment(P<0.01);(3)Compared with the control group,AS-Ⅲ induced a rapid TACE-dependent EGFR transactivation and activation of ERK1/2 and AKT pathways(P<0.01).AS-Ⅲ induced phosphorylation of p38.Both TACE phosphorylation and EGFR transactivation induced by AS-III were significantly inhibited by the use of p38 inhibitor(SB203580)(P<0.01).Conclusion: AS-Ⅲ activates TACE-dependent EGFR signaling in VECs in a p38-dependent fashion,which may contribute to its cardiovascular protective effects and shed light on the studying of other astragalosides.