Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway
Knockdown or inactivation of Src regulated lipid accumulation and cellular phenotype in VSMCs. (a, b) Tlr4(-) or NC were treated with or without oxLDL (50 μg/mL) for 1 hour; the activation of Src (Tyr-418, Y418) had been detected by western blot. ( per group, results were expressed as ;, as compared with oxLDL-untreated NC group; ##, as compared with oxLDL-treated NC group.) Src was knockdown by siRNA transfection within VSMCs. The Src-specific antagonist, PP2, was used to block Src activation, with PP3 serving as the negative control. The VSMCs were treated with and without 50 μg/mL oxLDL for 48 hours. (c, d) The Src-specific [Src (-)] and negative control (NC) siRNA were, respectively, transfected into VSMCs for 72 hours, and the knockdown efficiency was detected. ( per group, results were expressed as ;, as compared with the NC group.) (e) Nile Red (orange) was used to label the lipid, and the concentration of intracellular lipid was measured. ( per group, results were expressed as ;, as compared with the untreated group; @@, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ##, the oxLDL-treated PP2 group compared with the oxLDL-treated PP3 group.) (f, g) Western blot was conducted to measure the VSMC contractile phenotype markers (Myh11 and αSma) and foam cell markers (Mac2 and Cd68). All the western blot results were calculated using grayscale value, and β-actin served as an internal reference gene to normalize protein expression. ( per group, results were expressed as ;, as compared with the control group (untreated group); @@, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ##, the oxLDL-treated PP2 group compared with oxLDL-treated PP3 group).