Effects Of 17?-estradiol Protecting Vascular Smooth Muscle Cells From Oxidative Stress-Induced Senescence Via G Protein-coupled Estrogen Receptor And Its Mechanism

Background:Atherosclerosis(AS)is a common disease in the aged,which pathological characteristics is the deposition of cholesterol and other lipids and thickening arterial wall.Vascular smooth muscle cells(VSMCs)is the main cells in the arterial wall,and migration,proliferation and apoptosis of VSMCs play a vital role during the course of AS.Recent studies find that homeostasis change in aging arteries may promote the development of AS by aging patients and animal models.And it is reported that the senescent VSMCs is separated from the AS plaque.It is well known that estrogen can prohibit the course of AS through the classic estrogen receptor a and?.However,with the development of research,the third receptor related to estrogen has been found,the G protein-coupled estrogen receptor(GPER).Estrogen can regulate cell proliferation,apoptosis and inflammation though rapid non-genomic action mediated by GPER.Studies have shown that vascular endothelial cells and VSMCs could express endogenous GPER,and estrogen has anti-AS effect caused by inhibiting inflammatory response in endothelial cell through GPER.Other studies have demonstrated that estrogen could protect VSMCs from oxidative stress induced senescence.However,whether GPER plays an important role for estrogen protecting VSMCs from oxidative stress-induced senescence is still no report,this research project tries to explore the role of GPER and its related mechanisms,and supply the experimental evidence for treatment of eatrogen-related diseases such as atherosclerosis.Part 1:Effects of 17?-estradiol protecting vascular smooth muscle cells from oxidative stress-induced senescence via G protein-coupled estrogen receptorObjective:To explore whether estrogen protects vascular smooth muscle cells(VSMCs)from oxidative stress-induced senescence via G protein-coupled estrogen receptor(GPER).Method:Cultured VSMCs of 3-4 passages were pre-treated with 150 ?M hydrogen peroxide(H2O2)for 2 hours,then treated with 10-8mol/L 17?-estradiol(E2),10-6M GPER agonist and antagonist(G1 and G15)separately.In each group,the VSMCs senescence was detected by senescence-associated beta-galactosidase(SA-?-Gal)staining,cell cycle was detected by Flow cytometry,and GPER mRNA and protein expression were detected by RT-qPCR and western blot separately.Results:After treated with H2O2,the rate of positive SA-?-Gal staining VSMCs increased obviously,cell cycle in VSMCs was arrested in G0/G1 phase,GPER mRNA and protein expression in VSMCs decreased significantly.E2 and G1 could inhibit these effects of H2O2 on VSMCs,while G15 could block the inhibitory effects of G1 completely and E2 partly.Conclusion:Estrogen could protect vascular smooth muscle cells from oxidative stress-induced senescence via GPER.Part 2:The mechanism of 17p-estradiol protecting vascular smooth muscle cells from oxidative stress-induced senescence via G protein-coupled estrogen receptorObjective:To explore the mechanism of 17?-estradiol protecting vascular smooth muscle cells(VSMCs)from oxidative stress-induced senescence via G protein-coupled estrogen receptor(GPER).Method:VSMCs treatment in each experimental group was the same as that of the first part.In each group silent information regulator 1(SIRT1)mRNA was detected by RT-qPCR,and the expression of SIRT1,Acetyl-p53 and p21 protein were measured by western blot.Results:After treated with H2O2,the expression of SIRT1 mRNA and protein in VSMCs decreased significantly,and E2 and G1 could inhibit these effects of H2O2 on VSMCs,while G15 could block the inhibitory effects of E2 and G1.At the same time,western blot results showed that Actyl-p53 and p21 protein expression in VSMCs obviously increased after treatment with H2O2,while E2 or G1 could decrease Actyl-p53 and p21 protein expression significantly.G15 pretreatment could inhibit the effect of E2 and G1 on VSMCs.Conclusion:17?-estradiol could protect against oxidative stress-induced senescence in VSMCs mediated by GPER through SIRTl/p53/p21 signaling pathway.