Effect Of Advanced Glycation End Products Induce Autophagy In Human Periodontal Ligament Cells

Background:Advanced glyeation end products is produced under hyperglycemia condition in the long term,which is the harmful to various organs.Antophagy is a defense mechanism,is started when eukaryotic cells are in adverse environment.However,little is known about the effect of AGEs inducing autophagy in human periodontal ligament cells(hPDLCs).Methods:hPDLCs were isolated from non-carious human premolars extracting for orthodontic treatment from systemically and periodontally healthy patients with informed consents in Affiliated Stomatological Hospital of Nanjing Medical University.hPDLCs treated with different concentrations of AGEs for 0?1?3?6?9h were set as experiment groups,respectivelyisolated from the premolars.Untreated cells were set as control group.ntreated and treated with AGEs groups were carefuly seeded for 0?1?3?6?gh,The morphology of autophagosome were observed by transmission electron microscope(TEM)The expression of LC3 which is the autophagy protein were detected by immunofluorescence.The involvements of MAPKs and Akt/mTOR s signaling pathways were investigated by Western blot with specific inhibitor.Experiments then characterized the effects of AGEs on autophagy of hPDLCs,investigated whether MAPKs and Akt/mTOR signal pathways are relevant,using Western blot analysis,electron microscopy and immunofluorescence imaging.In following study,we used the ERKI/2 inhibitor U0126,the p38 inhibitor SB203580,the JNK inhibitor SP600125 and Akt/mTOR inhibitor LY294002 to examine the role of the ERK1/2,JNK and P38 pathway in AGE-induced autophagy.Results:The expression of LC3-? and the ratio of LC3-? to LC3-? were significantly descend at lh,then increased after treatment with AGEs,peaking at 3 h.In contrast,treatment with BSA after descend at lh,did not change the expression of LC3-? or the LC3-? to LC3-?ratio,we determined autophagic vacuoles by transmission electron microscopy analysis.We examined the colocalization of autophagosome-specific protein LC3 by immunoelectron microscopy.Cells were treated with AGEs for 3h.LC3 was confirmed in dots around the nucleus.However,in BSA-pretreated group,LC3 was distributed homogeneously in the cytoplasm.These results imply that the ERK1/2 and P38 pathway positively regulates autophagy.the Akt inhibitor,LY294002,could enhanced the effects of AGE-induced autophagy of hPDLCs.This result indicates that Aktand mTOR is involved in AGE-induced autophagy of hPDLCs.Conclusions:Our findings demonstrated tha treatment with AGEs could induce autophagy in a time-and dose-dependent manner in hPDLCs.The following experimental results tested the mechanism that AGEs impacted autophagy though ERKI/2 MAPK,p38MAPK and Akt/mTOR signaling pathways.