Protective Effect Of The Total Flavonoids In Carya Cathayensis Sarg.Leaves On HUVECs Senescence Induced By Ang Ⅱ Via MiR-34a/SIRT1 Activation

Objective:Studies have shown that cellular senescence of endothelial cells is closely related to atherosclerosis(AS).Our preliminary study found that the total flavonoids in Carya cathayensis Sarg.leaves(TFs)had antioxidant activity,regulating cell proliferation and so on,and had potential application value in regulating cell maturation and senescence.This study was aimed to explore the anti-aging and the preliminary mechanism of TFs by studying its effects on the aging of human umbilical vein endothelial cells(HUVECs)induced by angiotensin II(Ang II)and the role of miR-34a/SIRTl in TFs regulating cell senescence.This experiment provide therapeutic targets for AS,at the same time providing a new approach and avenues for the prevention and treatment of AS.Method:Culturing HUVEC cells in vitro to build cellular senescence model by the per-treatment AngⅡ.Cell viability was measured by the modified MTs method,β-galactosidase activity and cell proliferation activity were detected,mRNAs and miR-34a that related to senescence were detected by qPCR,and the expression of proteins in HUVECs cells were evaluated by western blotting(WB).In order to detect the roles of the miR-34a and SIRT1in the mechanism of TFs inhibit the cells senescence,miR-34a mimic,miR-34a inhibitor and SIRT1 inhibitor NAM were transferred into HUVEC respectively,SYBR qPCR and WB were used to detect the expression of miR-34a and SIRT1.Result:(1)Compared to the control group,the Ang Ⅱ group induced by the per-treatment 48h of the 1×10-6mol/L Ang Ⅱ,the cells activity was decreased obviously(P<0.05),and β-gal staining cells were significantly increased(P<0.01),cell cycle arrest at G0/G1 phase with decreasing S phase cell percentage(P<0.05).(2)While compared to Ang Ⅱ group,the cells tread with TFs could increase cell viability(P<0.05),decrease β-gal activity(all P<0.05)and G0/G1 phase percentage(P<0.05),increase S phage cell percentage(P<0.05)and NO contents(all P<0.05),moreover,the expression of P53 and P21 were down-regulated and the level of SIRT1、CDK2 and E2F-1 were up-regulated.(3)qPCR results indicated that miR-34a was changed obviously after transfected by miR-34a mimic and inhibitor,and the expression of SIRT1 respectively decreased and increased after transfection.TFs could inhibit the expression of miR-34a,increase the expression of SIRT1 and P-gal staining cells were significantly decreased.(4)Furthermore,SIRT1 inhibitor NAM also abolished the decrease in expression of acetylation P53 and p21 and the increase in expression of CDK2 and E2F-1 inferred by TFs alone.Conclusion:(1)TFs could protect HUVECs against Ang Ⅱ-induced senescence,and delay senescence.(2)The mechanism of TFs prevents Ang Ⅱ-induced HUVEC senescence by inhibited the expression of miR-34a via activation the signal path of SIRT1/P53/P21/E2F-1.