Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway
oxLDL regulated expression of toll-like receptor family, ROS-related genes, and sirtuin family as well as activation of Src. After oxLDL stimulation, the mRNA or protein expression of genes was detected by real-time PCR or western blot in VSMCs. The phosphorylation of Src was detected using western blot. The untreated group served as control (Con), and β-actin served as an internal reference gene to normalize protein expression. All of the real-time PCR results were calculated by 2-ΔΔCT method; the western blot results were calculated using grayscale value. (a) The mRNA expression of the toll-like receptor family ranging from Tlr1 to Tlr13. ( per group, results were expressed as ;, as compared with the Con group.) (b) Upper: the phosphorylation of Src (Tyr-418, Y418) was detected after 1-hour stimulation by different doses (12.5, 25, and 50 μg) of oxLDL in VSMCs. Bottom: the protein expression of ROS generation (Nox2 and Nox4) and elimination-related (Mnsod) genes in VSMCs, after stimulating by different doses (12.5, 25, and 50 μg) of oxLDL for 48 hours. (c) After incubation with different doses (12.5, 25, and 50 μg) of oxLDL, the protein levels of the sirtuin family (Sirt1-7) were measured. ( per group, results were expressed as ; as compared with the Con group).