Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway
Overexpression of Sirt1 or Sirt3 inhibited oxLDL-induced ROS accumulation and altered cellular phenotype in VSMCs. All the western blot results were calculated using grayscale value, and β-actin served as an internal reference gene to normalize protein expression. (a, b) Tlr4(-) or NC VSMCs were treated with or without oxLDL (50 μg/mL) for 48 hours; western blot was conducted to measure Sirt1, Sirt3, Nox2, and Monsod. ( per group, results were expressed as ;, as compared with oxLDL-untreated NC group; ##, as compared with oxLDL-treated NC group.) The recombinant lentivirus of Sirt1 or Sirt3 has infected into VSMCs for Sirt1 and Sirt3 overexpression. The VSMCs were treated with or without 50 μg/mL oxLDL for 48 hours. (c, d) The VSMCs were infected by recombinant lentivirus of Sirt1 [Let-Sirt1(+)] or Sirt3 [Let-Sirt3(+)], respectively, for 72 hours; afterwards, the overexpression efficiency was detected. ( per group, results were expressed as ;, as compared with the Let-Blank group; ##, as compared with Let-Sirt1(+) group.) (e) ROS (DCFH-DA, green) and (f) mitochondrial superoxide (MitoSOX, red) were measured. ( per group, results were expressed as ;, as compared with the control group (untreated group); ##, as compared with Let-Blank group). (g, h) Western blot was conducted to measure the Nox2 and Mnsod (upper), as well as the VSMC contractile phenotype markers (Myh11 and αSma) and foam cell markers (Mac2 and Cd68) (bottom). ( per group, results were expressed as ;,, as compared with control group (untreated group); #,##, as compared with Let-Blank group).