Mechanism Of Resveratrol Attenuating Vascular Endothelial Cell Senescence By Inhibiting Ferroptosis

Background and Objective:Atherosclerosis(AS)is a chronic inflammatory process characterized by lipid accumulation and inflammatory cell infiltration.It is the pathological basis for many acute cardiovascular and cerebrovascular events such as coronary artery disease,myocardial infarction,and stroke.The pathogenesis of AS is complex and closely related to multiple factors.Several studies have confirmed that vascular endothelial cell senescence is the initial change and important part in the development of AS.Ferroptosis,a novel iron-dependent type of cell death,is involved in the senescence of various cells,including vascular smooth muscle cells and endothelial cells,and plays an important role in the progression of AS.Resveratrol,a kind of plant polyphenolic compound,can delay cellular senescence and exert anti-AS effects through mechanisms such as reduce oxidative stress,alleviating inflammation and improving mitochondrial function.But its effects on ferroptosis in vascular endothelial cells have not been reported so far.Therefore,the aim of this study was to investigate whether resveratrol could play a protective role against AS by antagonizing ferroptosis in vascular endothelial cells and thereby delaying cellular senescence.Methods:1.Construction of endothelial cell senescence model: Human umbilical vein endothelial cells(HUVECs)were treated with angiotensin II human(AngⅡ)for 48 h to establish senescence model.The cell viability of HUVECs was measured by the CCK-8 kit to select the optimal treatment concentration of AngⅡ;β-galactosidase staining was used to detect the senescence of HUVECs;flow cytometry was used to detect the cell cycle and Western Blot(WB)was used to detect senescence-associated protein expression levels of P53.P21 and P16.2.Ferroptosis-related assay in AngⅡ-induced senescent HUVECs: According to whether ferroptosis inhibitor(Fer-1)was added,cells were divided into control group(normal culture medium),AngⅡ group(10-5 mol/L AngⅡ),AngⅡ + Fer-1 group(10-5mol/L AngⅡ + 10 μmol/L Fer-1),and Fer-1 group(10 μmol/L Fer-1).The levels of intracellular Glutathione(GSH)and Malonaldehyde(MDA)were measured by GSH and MDA assay kits,the levels of intracellular Reactive oxygen species(ROS)and lipid ROS in HUVECs were detected by 2’,7’-dichlorofluorescein diacetate(DCFHDA)and C11-BODIPY581/591 fluorescent probe method and the protein expression levels of Ferritin heavy chain1(FTH1).Glutathione peroxidase 4(GPX4)and Solute carrier protein 7 family member 11(SLC7A11)were measured by WB;β-galactosidase staining to detect cellular senescence;Senescence-associated protein expression levels of P53.P21 and P16 were measured by WB.3.To detect the effect and mechanism of resveratrol on ferroptosis in senescent HUVECs.HUVECs were intervened with 5,10 and 20 μmol/L resveratrol,and the CCK-8 was used to detect the cell viability of HUVECs to select the optimal drug concentration of resveratrol;according to the treatment,the cells were divided into control group(normal culture medium),AngⅡ group(10-5 mol/L AngⅡ),resveratrol group(10-5 mol/L AngⅡ + 20 μmol/L resveratrol),Erastin group(10-5 mol/L AngⅡ +20 μmol/L resveratrol + 30 μmol/L SLC7A11 inhibitor Erastin),and RSL3 group(10-5mol/L AngⅡ + 20 μmol/L resveratrol + 3 μmol/L GPX4 inhibitor RSL3).The CCK-8kit detected cell viability of HUVECs in each group,GSH and MDA assay kits detected intracellular GSH and MDA levels,DCFH-DA and C11-BODIPY581/591 probe detected intracellular ROS and lipid ROS levels in HUVECs and FTH1.GPX4 and SLC7A11 were detected by WB;β-galactosidase staining detected cell senescence and WB detected senescence-associated protein expression levels of P53.P21 and P16.Results:1.AngⅡ induced endothelial cell senescence model: Based on the results of CCK-8 experiment,10-5 mol/L AngⅡ concentration was selected as the optimal modeling concentration to induce cell senescence in this study.After HUVECs are treated with AngⅡ,positive cells increased in β-galactosidase staining;flow cytometry results showed that cells underwent cycle arrest and G0/G1 phase cells increased;WB results showed that the senescence-associated protein expression levels of P53.P21 and P16 were elevated2.Ferroptosis-related assays in aging HUVECs: Compared with the control group,MDA.ROS and lipid ROS levels were increased,GSH levels were decreased and the protein expression levels of anti-ferroptosis factor FTH1,GPX4 and SLC7A11 were decreased in the AngⅡ group of HUVECs.In the AngⅡ + Fer-1 group,cell proliferation was enhanced,MDA.ROS and lipid ROS levels were decreased,GSH levels were increased,and the protein expression levels of anti-ferroptosis factor FTH1,GPX4 and SLC7A11 were increased;the number of β-galactosidase-positive stained cells was decreased and the senescence-associated protein expression levels of P53.P21 and P16 were decreased compared with the AngⅡ group.3.The effect and mechanism of resveratrol on ferroptosis in senescent HUVECs:compared with the AngⅡ group,HUVECs in the resveratrol group enhanced cell viability,decreased MDA.ROS and lipid ROS levels,increased GSH levels,increased the protein expression levels of anti-ferroptosis factor FTH1,GPX4 and SLC7A11 in cells,decreased number of β-galactosidase-positive stained cells and the senescenceassociated protein expression levels of P53.P21 and P16 were reduced.And compared with the resveratrol group,the Erastin and RSL3 groups attenuated the inhibitory effect of resveratrol on AngⅡ-induced senescence and ferroptosis in HUVECs.Conclusion:Ferroptosis is involved in AngⅡ-induced HUVECs senescence.Inhibition of ferroptosis can attenuate AngⅡ-induced HUVECs senescence.Resveratrol constricts ferroptosis by activating the SLC7A11/GPX4 pathway to postpone cell senescence.