Mechanism Of 17β-estradiol Inhibiting Hydrogen Peroxide Induced Senescence Of Human Umbilical Vein Endothelial Cells By Up Regulating SIRT3 Expression And Promoting Autophagy

Purpose:17β-estradiol(17β-E2)can inhibit the aging of vascular endothelial cells(VEC)and delay the progression of atherosclerosis.However,the underlying mechanism still a mystery.In this study,we investigated the role of SIRT3 in 17 βE2 induced autophagy and 17 β-E2 mediated hydrogen peroxide(H2O2)-induced senescence in human umbilical vein endothelial cells(HUVECs).Materials and methods1.Human umbilical vein endothelial cells were intervened with 0,0.1,0.3 and 0.4 μ mol/L 17 β-E2.The SIRT3 mRNAwere detected by reverse transcription polymerase chain reaction(RT-PCR)The SIRT3 protein expression were detected by Western blot(wb).2.Construct SIRT3 plasmid and amplify the promoter fragment.The luciferase based SIRT3 reporter plasmid and the negative control psmd14 promoter were transfected into HUVEC cells respectively,and the cells were exposed to different concentrations of 17 β-E2.Then the cells were harvested.Then Dual liciferase determind the lucifercase activity.3.Human umbilical vein endothelial cells were transfected with SIRT3 siRNA to understand the expression of SIRT3 gene.The cells treated with 17 β-E2 were collected and analyzed by Western blotting with anti-LC3 or p62 antibodies.4.HUVEC cells were transfected with SIRT3 siRNA and treated with vehicle or 0.1μmol/L 17β-E2.For the co-treatment group,cells were pretreated with 0.1μmol/L 17β-E2 for 1 h and then stimulated with 200μmol/L H2O2 in the presence of 0.1μmol/L 17β-E2 for 24 h.The cells were harvested and subjected to immunoblot analysis using antibodies against p-Rb or Rb.Senescence associated b-galactosidase staining was also performed to measure senescence.5.HUVEC cells transfected with SIRT3 siRNA were pre-treated with 0.1μmol/L 17β-E2 for 1 h and then cotreated with 0.1 μmol/L 17β-E2 and 200M H2O2 for 24 h.The cells were then subjected to immunostaining using antibodies against SIRT3,VDAC1 and LC3.The percentage of cells with colocalization of SIRT3,VDAC1 and LC3 was quantified.Results:1.17β-E2 increases the expression levels of SIRT3 mRNA in a dose-dependent manner in HUVEC cells.Consistently,immunot blot analysis shows that the protein expression levels of SIRT3 are elevated,when HUVEC cells are exposed to different concentrations of 17β-E2.2.17β-E2 increases SIRT3 promoter activity and exhibits the maximum effect at the concentration of 0.1 μmol/L.As mentioned in the Materials and Methods,when the fragment is reverse oriented,it serves as a promoter for PSMD13 gene.In contrast,17β-E2 does not display any effect on the PSMD13 promoter.3.In control siRNA-transfected cells,17β-E2 dramatically increases the ratio of LC3II to LC3-I,whereas siRNA-mediated silencing of SIRT3 gene expression significantly inhibits17β-E2-induced autophagy.wealsomeasured autophagic flux using immunoblot analysis with antip62.Consistently,17β-E2 induces a dramatic degradation of p62 in control siRNA-transfected cells,whereas SIRT3 knockdown signficanlty inhibits 17β-E2-induced decrease in p62 protein levels.4.In control siRNA-transfected HUVEC cells,H2O2 induces a remarkable increase in the phosphoryaltion of Rb and 17β-E2 significantlyinhibits H2O2’s effect.In contrast,when SIRT3 expression is depleted in HUVEC cells using SIRT3 siRNA,17β-E2 fails to block H2O2-induced increase in Rb phosphorylation(Fig.4a and b).Similarly,17β-E2 significantly inhibits H2O2-induced increase in the number of SA-b-gal-positive cells(Fig.4c and d).However,when we knocked down SIRT3 gene expression,17β-E2 loses the capacity to thwart H2O2-induced increase in the number of SA-b-galpositive cells(Fig.4c and d).5.When control siRNA-transfected HUVEC cells are exposed to H2O2 and 17β-E2,35%of the cells display the co-localization of SIRT3,VDAC1 and LC3.By comparison,SIRT3 knockdown impairs the co-localization of these proteins following the treatments with H2O2 and 17β-E2.Conclusion1.Estrogen upregulated SIRT3 expression by activating SIRT3 promoter.2.The expression of SIRT3 gene is requried for 17β-E2-induced autophagy3.The expression of SIRT3 gene is requried for17β-E2-induced autophagy4.SIRT3 mediates the fusion of autophagosomes and dysfunctional mitochondria induced by H2O2.