| Objective:Oxidized Low-density lipoprotein (ox-LDL) has a great implication in the initiation ofatherosclerosis. Our previous study reported that ox-LDL can activate autophagy in humanumbilical vein endothelial cells (HUVECs). Therefore studies designed to elucidate thepossible role of autophagy on ox-LDL accumulation in HUVECs and its associatemechanism.Methods:In the present study, we have examined the ox-LDL content determined by flowcytometry. The trafficking of ox-LDL within endothelial cells was analyzed byimmunofluorescence microscopy. Western blot was used to detect the protein level ofLC3,LAMP1,beclin1and P62.Results:(1) Flow cytometry results showed the fluorescent intensity of Dil-ox-LDL inHUVECs increased gradually as the time prolonged, implying an accumulative process ofox-LDL in HUVECs. Pharmacological induction of autophagy with rapacymin (rap)significantly decreased the fluorescent intensity of Dil-ox-LDL (P=0.003), indicating lessox-LDL content in HUVECs.(2) With confocal study, it is interesting to find that theox-LDL content in HUVECs (3h after ox-LDL exposure) treated with rapacymin wassignificantly lower than that treated with3MA. Moreover, treatment with rapacyminobviously enhanced the colocalization of Dil-ox-LDL/LC3compared to3MA group. Inline with this, it was further found that treatment with rapacymin also markedly increasedthe colocalization of Dil-ox-LDL/LAMP1, compared to ox-LDL alone treated group. After ox-LDL treatment for3h, a lot of LC3/LAMP1colocalization occurred in therapamycin-treated group. These data may indicate the decreased ox-LDL content inrapacymin group was due to the induction of autophagy.(3) Western blot analysis revealedthat LC3(P=0.024) and beclin1(P=0.005) levels are upregulated in response to ox-LDLstimulation in HUVECs. Treatment with rapamycin significantly increased both the LC3(P=0.045) and beclin1(P=0.001) level, indicating an enhanced autophagic flux. It was alsoobserved that there was a significant reduction of P62(P=0.030) after rapacymin treatment,indicating the finish of autophagy at this time point. This is consistent with the LC3andbeclin1results. There is no significant change on the protein levels of LAMP1aftertrentment of rapacymin.Conclusion:The content of ox-LDL in HUVECs accumulated as the time prolonged. Induction ofautophagy with rapacymin significantly reduced ox-LDL accumulation in HUVECs. Theautophagy flux in HUVECs is enhanced after treatment with rapacymin. It then promotedthe formation of autophgysome, which fused with lysosome and degredated ox-LDL. Thus,the ox-LDL in HUVECs is degredated through the autophagy-lysosome pathway. |
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