AGEs Promote Calcification Of Human Aortic Vascular Smooth Muscle Cells By PI3K/AKT GSK-3? Signaling Pathway

Objective: This study aims to investigate the effect of advanced glycation end products(AGEs)on the calcifition of human aorta vascular smooth muscle cells(HASMCs),and to explore whether it promote calcification of HASMCs by activating the wnt /?-catenin signaling pathway through the PI3 K / AKT GSK-3? signaling pathwa.It provide a new strategy for the prevention and treatment of diabetic vascular calcification.Methods:cultivating the primary human aortic vascular smooth muscle cells,to observe the cell morphology by immunofluorescence method for cell identification.The total protein was extracted after different concentrations of AGEs-BSA(0ug/ml?10ug/ml?20ug/ml?500ug/ml?100ug/ml?200ug/ml)to stimulate HASMCs for five days,to detect the expression of OPG,BMP and by western blot.Von Kossa staining and Alizarin red staining were used to detect the calcification of each group.The expression of P-AKT,AKT,P-GSK-3? and GSK-3? were detected by western blot after AKT-specific inhibitor LY294002 20mmol/ml and GSK-3? specific inhibitor TWS11910mmol/ml for 2h then adding AGEs for 15 min.From the experiments we concluded that the treatment with LY294002 can significantly inhibit the phosphorylation of AKT protein,but had no significant effect for the total amount of AKT protein,and it can reduce the expression of P-GSK-3?,indicating that inhibition of AKT protein can activate the downstream GSK-3?protein.In order to verify the influence by inhibiting the AKT and GSK-3?protein phosphorylation on HASMCs calcification,We added 40 ug /ml AGEs for 5 days after using LY294002 20mmol/ml pretreatment HASMC for 2 hours,TWS119 10mmol/ml pretreatment HASMCs 12 hours after,the total protein was extracted and detected the expression of OPG,BMP,and ?-catenin by western blot.In order to further explore the role of PI3 K / AKT signaling pathway in the calcifition of HASMCs induced by AGEs,We used AKT-siRNA to transfect HASMCs to establish the AKT gene silencing cell model.at the same time the GSK-3?-RNAi was used to transfect HASMCs to establish GSK-3? gene silencing cell model.AKT and GSK-3? protein were detected by western blot to confirm whether transfection was success and transfection efficiency.After establishing a stable low expression of AKT,GSK-3? cell model,then 5 days of intervention with 40ug/ml AGEs extraction of total cell protein,detection of calcification-related protein expression.Conclusion:1.AGEs can promote the calcification of HASMCs,and time and concentration-dependent,compared with the control group,the expression of calcification-related protein in AGEs group was significantly increased(P<0.05),at the concentration of 40 ug / ml AGEs is the most obvious to promote HASMCs calcification;The results showed that the number of calcified nodules in AGEs + calcified culture group was significantly higher than that in simple calcification culture group;2.AGEs can promote the phosphorylation of AKT and GSK-3? protein in HASMCs,Compared with the control group,the expression of P-AKT and P-GSK-3? protein in AGEs group was significantly increased(p <0.05),and it is time-and-concentration-dependent.and theconcentration of AGEs is 25 ug / ml and 15 min,the expression of P-AKT and P-GSK-3? is the highest in time and concentration.which will also be our next experiment the best concentration and time;3.The phosphorylated AKT protein expression was significantly reduced,but had no significant effect on the total amount of AKT protein after treatment with LY294002 of HASMCs,and it can reduce the downstream GSK-3? protein phosphorylation,indicating that inhibition of AKT protein phosphorylation can activate the downstream GSK-3?;The expression of calcification-related protein in AGEs + LY294002 group was significantly lower than that in AGEs group(p <0.05).4.The expression of AKT protein in AKT-siRNA transfected group was significantly lower than that in control group(p <0.05).The expression of calcification-related protein in AKT-si RNA transfected group was significantly higher than the simple AGEs intervention group;5.After transfected with GSK-3?-RNAi lentivirus 5 days,the stable expression of green fluorescence can be observed with fluorescence microscopy.Compared with the control group,GSK-3? protein expression in GSK-3?-RNAi lentivirus transfection group was significantly decreased(p <0.05).The expression of calcification-related proteins in GSK-3?-RNAi lentiviral transfection group was significantly higher than that in AGEs alone group after treating with AGEs.Conclusion:1.AGEs can promote the calcification of HASMCs;2.AGEs can activate the PIK3 / AKT signaling pathway and inhibit the activity of downstream GSK-3?;3.AGEs promote the calcifition of HASMCs may be mediated by PI3 K / AKT GSK-3? signaling pathways.