ATO decreases serum lipid levels in ApoE-/-mice and CD36-mediated ox-LDL uptake in macrophages
Excessive cholesterol accumulation in macrophages results in the transformation of macrophages into foam cells, which eventually causes atherosclerosis (Akıl et al. , 2014; Kavurma et al. , 2017). Preventing excessive cholesterol accumulation in macrophages is one atheroprotective strategy. Indeed, high CD36 expression can facilitate foam cell formation (Zhao et al. , 2016). To investigate whether ATO regulates lipid metabolism by regulating CD36 expression in macrophages, we first determined the serum lipid levels of ApoE-/- mice treated with ATO and those left untreated (Figure 2A-E). After 4 weeks of ATO therapy, serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and ox-LDL levels decreased significantly compared with those in the controls. However, high-density lipoprotein (HDL) levels were slightly, but not significantly, lower in the ATO group (Figure 2A-E). These data indicate that the protective effect of ATO against atherosclerosis in ApoE-/- mice may be attributed to altered serum lipid metabolism. Compared with those in the untreated model group, CD36 mRNA and protein levels in the aortic tissue were significantly decreased by ATO treatment (Figure 2F, I, L). When we determined the CD36 expression in lesion areas by immunofluorescence staining, we observed that CD36 expression in macrophages (CD68+) was substantially reduced by ATO treatment (Figure 2G, H).
We also evaluated the ATO-mediated promotion of CD36-mediated ox-LDL internalization in vitro . Peritoneal macrophages of ApoE-/- mice were treated with ox-LDL (50 μg mL-1), and either 2.5 or 5.0 μΜ ATO for 24 h. Oil red O staining showed that both ATO doses significantly decreased ox-LDL uptake by peritoneal macrophages (Figure 2M, N). Moreover, both ATO doses significantly reduced CD36 protein and mRNA levels in RAW264.7 cells (Figure 2J, K, O). Taken together, these data suggest that ATO protects against foam cell formation by decreasing CD36-mediated cholesterol uptake in vivo and in vitro , which may be the mechanism by which ATO regulates blood lipid metabolism.