AGEs/RAGE Promotes Differentiation Of Rat Endothelial Progenitor Cells Into Osteoblasts Through MAPK Signaling Pathway

Objective: To investigate whether endothelial progenitor cells o can differentiate into osteoblasts under the action of advanced-4-(EPCs)in vitro can differentiate into osteoblasts under the action of advanced glycation end products(AGEs),and to investigate the effect of AGEs on EPCs The relationship between bone morphogenetic membrane receptor protein RAGE and MAPK signaling pathway and its role in the development and progression of atherosclerosis 。 Methods :(1)The rats were sacrificed by cervical dislocation.The femoral and tibia of the lower extremities were separated and the bone marrow was collected.The mononuclear cells were obtained from the lower limb bone marrow by density gradient centrifugation and cultured in vitro using EGM-2MV medium.Morphological observation of adherent cells was observed at 3,7 and 14 days,respectively.FITC-UEA-I)and Di I-ac LDL were observed under fluorescence microscope at day 7 Low-density lipoprotein double-stained positive cells is the differentiation of EPCs.(2)The expression of calcified protein of EPCs after AGEs treatment was measured by adding different concentrations of AGEs for 7 days.After the concentration of20mg/ml AGEs was selected as the optimum concentration,10mmol/lβ-glycerophosphate + 20mg/ml AGEs were used to observe EPCs for 7 days,and the phenotype of cells was observed by immunofluorescence.The EGM-2MV medium containing 20mg/ml AGEs and EGM-2MV medium containing 10mmol/l β-glycerophosphate were added to the EGM-2MV medium supplemented with EGM-2MV medium,EGM-2MV medium with10 mmol / l β-glycerophosphoric acid + 20mg/ml AGEs was treated for 7 days.The expression of calcified protein was observed by western blot.(3)si RAGE was designed and packaged with lentiviral vector.The cells were added to the second generation EPCs.The transfection efficiency and the optimum MOIwere observed by fluorescence microscope.The cell protein was extracted and the transfected effect was observed by western blot.The cells were divided into control group,20mg/ml AGEs group,20mg/ml AGEs + empty virus transfection group,20mg/ml AGEs + virus treatment group,cultured for 7 days,and the changes of calcified protein were observed by western blot.(4)20mg/ml AGEs were added to the cells.The intervention was terminated at 0,5,15 and 30 min,and the protein was extracted.The effect of AGEs on phosphorylated MAPK pathway protein was observed by western blot.(PD98059 50umol/l,SB203580 20umol/l,SP600125 50umol/l),DMSO group was pretreated for 1h,the medium was replaced with the MAPK pathway inhibitor group(PD98059 50umol/l,SB203580 20umol/l,SP600125 50umol/l)Adding 20mg/ml of AGEs for 5min,observe the expression of related proteins to detect the effect of inhibitor.The cells were divided into control group,AGEs group,AGEs + PD98059 group,AGEs + SB203580 group and AGEs +SP600125 group.The inhibitor group was treated with inhibitor for 24 h,then the medium was replaced.AGEs were cultured for 7 days,Changes in related proteins.Results:(1)Mononuclear cells obtained from bone marrow were cultured in EGM-2MV medium in vitro.On the third day,more rounded cells with larger volume were observed.Colony growth occurred on day 7,Shaped,"spindle" morphology of the cells,identified by the double staining experiment is the differentiation of EPCs,12-14 days have been a typical paving stone-like morphology.(2)After treatment with β-glycerophosphate + AGEs,the phenotype of EPCs was significantly decreased and the osteoblast phenotype was significantly increased under EPCs immunofluorescence,and the expression of calcification-related proteins was also detected by western blotting increase.(3)The expression of RAGE protein was significantly increased after AGEs stimulation,and the expression of RAGE protein was significantly decreased by si RAGE lentivirus transfected cells.After treatment with AGEs,the expression of RAGE protein in transfection group wassignificantly higher than that in control group and empty vector transfection group Calcification protein was significantly reduced.(4)After treatment with AGEs,the expression of phosphorylated ERK1 / 2,p38 MAPK and JNK1 / 2 in EPCs was increased,and the expression of MAPK phosphorylated protein was significantly decreased by AGEs.Compared with the AGEs control group,the expression of calcification-related proteins treated with AGEs treated with SB203580 and SP600125 was significantly decreased,while the expression of calcified protein pretreated by PD98059 and AGEs group was not obvious.Conclusion:(1)EPCs can be induced to differentiate into osteoblasts under specific circumstances,and AGEs can promote this differentiation process.(2)AGEs promote the transformation of EPCs into osteoblast-like cells and is closely related to their membrane receptor RAGE.(3)EPCs were induced to differentiate into osteoblasts and related to p38 MAPK and JNK pathway in MAPK pathway,but not with ERK pathway.