AGEs Promotes Calcification Of Human Aortic Vascular Smooth Muscle Cells Via Inducing Its Autophagy

Objective: This study aims to investigate the mechanism of autophagy in the AGEs-induced calcification of human aortic vascular smooth muscle cells.Methods : In this study,5th-8th generation aortic vascular smooth muscle cells were used for the experiment.First,human aortic vascular smooth muscle cells were divided into control group and calcification group,and calcification group was induced with 40 mg/L AGEs for 4 days.The expression levels of OPG,OPN,LC3 II,Beclin1 and P62 proteins in the two groups were detected by Western Blot.Secondly,the experiments were divided into A1 group(40 mg/L AGEs intervention for 1 day),A2 group(40 mg/L AGEs intervention for 2 days),A3 group(40 mg/L AGEs for 3 days),A4 group(40mg/L AGEs intervention for 4 days)according to the time of AGEs intervention.The Western Blot was used to detect the expression levels of OPG,LC3 II,Beclin1,P62 protein in each group of cells.Immunofluorescence was used to detect the difference in LC3 expression in each group.The ALP activity in each group was measured by ELISA.Then,the experiment was divided into control group,autophagy inhibition group and calcification group.The autophagy inhibition group was treated with 50 um wortmannin + 40mg/L AGEs for 4 days,and the calcification group was still treated with 40mg/L AGEs for 4 days.Western Blot was used to detect the expression levels of OPG,OPN and RUNX2 proteins in each group.The ALP activity in each group was measured by ELISA.The cytocalcification degree was measured by Von Kossa staining.Finally,in order to explore the relationship with the autophagy-related signaling pathways,the expression levels of PI3 K,p-AKT,AKT,p-mTOR and mTOR protein in the control group and AGEs group were detected by Western Blot.Results: 1.The expression levels of OPG,LC3 II,Beclin1 and P62 in calcification group were significantly higher than those in the control group(P<0.05).2.The levels of OPG,LC3 II,Beclin1 and P62 in A1 group,A2 group,A3 group and A4 group were increased in turn(P<0.05),and ALP activity was also increased in turn(P<0.05).The fluorescence intensity of LC3 in each group also increased with the intervention time.3.The expression levels of OPG,OPN,RUNX2 and LC3 II in the autophagy inhibition group were lower than those in the calcification group,but increased compared with the control group(P<0.05).The degree of cellular calcification detected by ALP activity and VON KOSSA staining also showed this trend.4.Compared with the control,the expression levels of PI3 K,p-AKT and p-mTOR protein in AGEs group were increased(P<0.05).Conclusion:1.AGEs induces calcification of human aortic vascular smooth muscle cells and induces autophagy.2.AGEs-induced vascular smooth muscle cell calcification and autophagy were time-dependent.3.Autophagy promotes AGEs-induced calcification of human aortic vascular smooth muscle cells.4.AGEs may inhibit the PI3K/AKT/mTOR pathway to promote autophagy and calcification of human aortic vascular smooth muscle cells.