| Background and objective: Oxidized low density lipoprotein(ox-LDL) is closely related to the formation of atherosclerosis. Autophagy is involved in the degradation of ox-LDL in human umbilical vein endothelial cells(HUVECs). Sirtuin1(SIRT1), a new anti-atherosclerotic factor, is a mammalian homolog of Sir2 longevity factor. It has an important role in the regulation of metabolism, cellular survival, and organismal lifespan. More importantly, SIRT1 can induce autophagy. This study examined whether SIRT1 could regulate ox-LDL accumulation in HUVECs via the autophagy-lysosomal pathway.Methods: In the present study, we have used nicotinamide(NAM) as the inhibitor of SIRT1 and resveratrol(RSV) as the activator of SIRT1. We examined the ox-LDL content determined by flow cytometry. SIRT1 si RNA was used to knockdown the gene of SIRT1, then the ox-LDL content was examined. We used Atg5 si RNA to knockdown Atg5 and examined the ox-LDL content. Western blot was used to detect the protein level of LC3 and p62.The activation of autophagy was detected following transfection of cells with GFP-LC3. The presence of several intense fluorescent dots in cells is indicative of the accumulation of autophagosomes. We used immunofluorescence to examine the colocalization of Dil-ox-LDL and Lamp1 or Cathepsin D.Results: 1. Flow cytometry results showed the fluorescent intensity of Dil-ox-LDL in HUVECs increased by treated with SIRT1 si RNA or NAM. On the contrary, RSV decreased the fluorescent intensity of Dil-ox-LDL.2. Atg5 si RNA could increase the accumulation of Dil-ox-LDL in HUVECs. Compared with RSV and Dil-ox-LDL group, Atg5 si RNA with RSV and Dil-ox-LDL group decreased the effect of RSV reducing the accumulation of Dil-ox-LDL in HUVECs.3. In HUVECs with ox-LDL, expression of microtubule-associated protein 1 light chain 3(LC3)-II and LC3 puncta was decreased by treatment with SIRT1 si RNA or NAM, but increased by RSV; sequestosome 1(SQSTM1/P62) expression showed the opposite effects.4. The immunofluorescence results revealed that Dil-ox-LDL combined with SIRT1 si RNA or NAM showed a much smaller degree of overlap of Lamp1 or Cathepsin D with Dil-ox-LDL than in cells with Dil-ox-LDL alone, and RSV treatment resulted in a greater degree of overlap.Conclusions: The inhibitor of SIRT1 and SIRT1 si RNA could increase the accumulation of Dil-ox-LDL in HUVECs. The activator of SIRT1 could decrease the accumulation of Dil-ox-LDL in HUVECs. This effect is related to the autophagy-lysosomal pathway. |
