| Objective: To explore the effects of ox-LDL on lipophagy in endothelial cells(ECs),and expect to provide scientific basis for ox-LDL leads to atherosclerosis.Method: The ECs were treated with oxidized low-density lipoprotein(ox-LDL)for 6 h,12 h,24 h,48 h.ECs activity was measured by MTT.The lipid accumulation of ECs was observed by oil red O staining.The formation of autolipophagosomes(ALPs)was determined by transmission electron microscopy.The ratio of LC3-II/LC3-I,p62,lysosome associated membrane protein 1(LAMP1)protein levels were analyzed by western blot.The colocalization of LC3 and Bodipy,and the colocalization of LAMP1 and Bodipy are exam by immunofluorescence.Results:1.The results of MTT showed that,compared with Control group,dealted with 25 μg/ml ox-LDL for 6 h,12 h,24 h and 48 h,there is no difference in cells activity.The group treated with 50 μg/ml ox-LDL for 48 h,the cells activity was inhibited.When treated with 100 μg/ml ox-LDL for 12 h,24 h,48 h,the cells activity was repressed obviously.2.the results of Oil Red O staining revealed treatment with ox-LDL for 6 h decreased lipid staining in ECs,Moreover,notably more lipid staining around the nucleus of ECs treated with ox-LDL for 24 h,48 h compared with Control.3.The results of transmission electron microscopy showed that,the number of ALPs in ox-LDL group for 6 h was more than that in Control group.Furthermore,this was ultrastructurally confirmed by the presence of less the formation of ALPs on exposure to ox-LDL(24 h and 48 h).Meanwhile,at the 48 h time point,those cells in ox-LDL group exhibited the features of damaged cells,characterized by abundant vacuoles.4.The results of western blot showed that,ox-LDL triggered autophagy in ECs after 6h.And ox-LDL up-regulated the ratio of LC3II/LC3 I and LAMP1 expression and down-regulation of p62 expression in ECs.However,ECs were treated with ox-LDL for 24 h,ox-LDL suppressed LAMP1 expression and increased p62 expression.Furthermor,on prolonged exposure to ox-LDL(48 h),there was a significantly decrease of LC3II/LC3 I and LAMP1 expression,and ox-LDL could markedly enhance p62 expression.5.The immunofluorescence results revealed treatment with ox-LDL for 6h increased co-localization between LC3-positive staining and Bodipy,also enhanced the co-localization between LAMP1-positive staining and Bodipy.However,treatment with ox-LDL for 24 h and 48 h not only significantly diminished co-localization between LC3-positive staining and Bodipy but also attenuated theco-localization between LAMP1-positive staining and Bodipy.Conclusion: Based on the above results,we deduced that,activation of the early phase of cytoprotective lipophagy,and subsequent attenuation lipid accumulation,this is followed by a late phase characterized by reduced lipophagy,and enhanced lipid accumulation in ECs.The increase of lipophagy by ox-LDL for 6 h may be related to the enhanced degradation of ox-LDL by autophagy/lysosome pathway,and the decrease of lipophagy by ox-LDL for at 24 h and 48 h may be related to the attenuated degradation of ox-LDL by autophagy/lysosome pathway. |
