Effects And Study Of Atorvastatin On The Autophagy Of Seneium Vaseular Endothelial Cells

Part Ⅰ Construction and identification of human umbilical vein endothelial cells senescence modelObjective:This experiment establish the endothelial cell senescence model by angiotensin-Ⅱ (AngⅡ)indueing human umbilical vein endothelial cells (HUVECs) aging, and explore the influence of Atorvastatin on autophagy of vascular endothelial cell.Methods:We culture HUVECs in vitro and the3to5generation cells were used in our study. Experiment was divided into:(1) control group:no intervention of factors;(2) add1×10-5mol/L of AngⅡ;(3) add1×10-6mol/L of AngⅡ:(4) add1×10-7mol/L of AngⅡ.Cells were collected48hours after the intervention. Senescence β—gal staining and cell cycle analysis were used to identify cell aging status.Results:1. Flow cytometry results of cell cycle analysis:compared to the control group, with the concentration of Angll increased, the G0/G1phase cell percentage gradually increased the percentage of S phase cells gradually reduced, no significant difference in the G2phase. And compared to the control group, the10-1mol/L AngⅡ group showed no difference, between10-6and10-5mmol/L Angll group were significally different.2. The results of senescence associated β-galactosidase staining:compared to the control group, with the concentration of AngⅡ increased, the positive rate of SA-β-gal staining was significantly increased (P<0.05), suggesting that join AngⅡ on endothelial cells can enhance cell β-galactosidase activity. Conclusion:AngⅡ can induce senescence of human umbilical vein endothelial cells (HUVECs).Part Ⅱ Influence of atorvastatin on the autophagy and seneium of vaseular endothelial cellsObjective:we conducted the research of impact of atorvastatin on vascular endothelial cells seneium and autophagy possible signaling pathways.Methods:Experiment was divided into:(1) control group:no intervention of factors;(2)AngII group:add1×10-6mol/L of AngⅡ;(3)AngⅡ+atorvastatin group:the cells of which were incubated with10-6mol/L of atorvastatin for1hour before senescent induction;(4) AngⅡ+atorvastatin+Rapa group:the cells of which were incubated with10-6mol/L of atorvastatin and10-7mol/L of Rapa for1hours before senescent induction. Cells were collected48hours after the intervention. Senescence β-gal staining and cell cycle analysis were used to identify cell aging status.Western blot was used to detect the expression of LC3and beclin1.Results:1. Flow cytometry results of cell cycle analysis:Compared with AngⅡ group, AngⅡ+atorvastatin group showed G1phase decreased and S phase increased; Compared with AngⅡ+atorvastatin group, AngⅡ+atorvastatin+Rapa group in G0/G1cells increase and S phase cells decreased.2. The results of senescence associated β-galactosidase staining:Compared with AngⅡ group, adding atorvastatin showed β-galactosidase staining was significantly lower (6.48±3.52) P<0.05; Compared with AngⅡ+atorvastatin group, AngⅡ+atorvastatin+Rapa group (3-galactosidase positive cells were significantly increased (13.27±2.67), P<0.05.3. The results of Western blot:The expression of LC3Ⅱ/Ⅰ and beclinl in Angll+atorvastatin group were significantly lower than that of the Angll group (P<0.05).Compared with the Angll+atorvastatin group, the expression of LC3Ⅱ/Ⅰ in Angll+atorvastatin+Rapa group increased significantly(P<0.05).Conclusion:Atorvastatin can inhibits human umbilical vein endothelial cells senescence, which maybe related to the role of atorvastatin’s can reduce autophagy on vaseular endothelial cell. Akt/PKB may be one of the key signaling pathways.