Mechanism Of Aldosterone Induced Autophagy In HUVECs And The Role Of Autophagy In Ald-induced HUVEC’ Dysfunction

Objective:To investigate the mechanism of aldosterone（Ald）-induced autophagy in the human umbilical vascular endothelial cells（HUVECs）.To investigate the role of autophagy in Ald-induced HUVECs’dysfunction.Methods:HUVECs were treated by Ald at different times or with gradient concentrations.Autophagy related protein expressions[microtubule-associated protein 1 light chain 3（MAP1-LC3,LC3）,Beclin-1,and Sequestosome-1（SQSTM1/p62）]were detected by western blot.HUVECs were stimulated with optimal time and concentration,cell viability was examined by MTT method.Cells were respectively pretreated with a mineralcoticoid receptor（MR）antagonist（Eplerenone）,a mitochondrial K_ATP channel inhibitor（5-HD）,an antioxidant（Tempol）,an inhibitor of autophagy（3-MA）and an enhancer of autophagy（Rapamycin）under optimal Ald stimulation.Expressions of LC3,Beclin-1 and SQSTM1/p62 were observed by western blot.Productions of nitric oxide in HUVECs were detected by Nitrate/Nitrite Assay Kit.Expressions of cell factors（IL-6,IL-8,IL-10,TGF-β,TNF-α,and ET-1）were detected by quantitative real-time PCR（Q-PCR）and enzyme linked immunosorbent assay（Elisa）kit.Interaction of MR with autophagy protein Beclin-1 was confirmed by co-immunoprecipitation and immune co-localization technology.Effects of overexpression of Beclin-1 on Ald-induced changes of cell factors were also examined by Q-PCR and Elisa kit.Results:Ald increased the ratio of LC3-II to LC3-I,Beclin-1 expression and decreased SQSTM1/p62 expression dose-dependently and time-dependently in the HUVECs without affecting cell viability.Both of Eplerenone and Tempol inhibited the increase of autophagy levels induced by Ald.5-HD had no effect on the Ald-induced increase of autophagy.3-MA and Rapamycin inhibited or enhanced the levels of autophagy induced by Ald.Production of nitric oxide was decreased under Ald stimulation.Both of 3-MA and Rapamycin pretreatments had no effect on decrease of nitric oxide production induced by Ald.In addition,Ald increased the expressions of IL-6、IL-8、IL-10、TGF-β.3-MA had no effect on Ald-induced increases of expression of IL-6、IL-8、IL-10、TGF-β.Rapamycin and overexpression of Beclin-1 inhibited the increases of expression of IL-6、IL-8、TGF-β.Conclusion:Ald induced increase of autophagy by binding to the mineralocorticoid receptor（MR）and subsequently increasing the level of intracellular reactive oxygen species in the HUVECs.Autophagy had no effect on the reduction of nitric oxide production induced by Ald.However,Autophagy may play a protective role in Ald-induced endothelial cell inflammatory response through the interaction of Beclin-1 and MR,and prevented the translocation of MR into the nucleus to increase the expressions of IL-6,IL-8 and TGF-β.