Figure 4. 1,25(OH)₂D₃ promotes cholesterol efflux and M2 polarization in macrophage-derived foam cells via upregulating 27HOC.

Fig. 4A: THP-1 macrophages were treated with oxLDL (50 μg/ml) for 48 h, then cultured with apoA1(25 μg/ml) under 1,25(OH)₂D₃-deficient medium (Top) or 1,25(OH)₂D₃ (10nM)-supplement medium (bottom) for 24h. Macrophages were stained with Oil Red O. Fig. 4B: The intercellular free cholesterol (FC) was determined by the cholesterol quantitation kit. n = 3; ***p<0.001, **p<0.01, *p<0.05, two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test. Fig. 4C: Liquid scintillation counting was performed to determine the intercellular cholesterol efflux. Fig. 4D: THP-1 macrophage-derived foam cells were treated with 1,25(OH)₂D₃ (10nM) for 0, 6h, 12h and 24h, the expression of ABCA1 and ABCG1 were determined by western-blot. Fig. 4E: After cultured with 1,25(OH)₂D₃ (10 nM) for 24h, THP-1 macrophage-derived foam cells were treated with IL-4 (20ng/ml) for 6h. The M2 marker (CD206 and MGL-1) mRNA levels in cells were determined by quantitative real-time PCR. Fig. 4F: Representative histograms of CD206/mannose receptor (MR) in THP-1 macrophage-derived foam cells exposed to vehicle (Ctrl), 1,25(OH)₂D₃ (10 nM), IL-4 (20ng/ml), or IL-4+1,25(OH)₂D₃. Fig. 4G: After cultured with 1,25(OH)₂D₃ (10nM) for 24h, THP-1 macrophage-derived foam cells were treated with LPS (10ng/ml) for 12h. The level of TNFα was quantified by ELISA. n = 3; **p<0.01, *p<0.05, two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test. Fig. 4H: THP-1 macrophage derived foam cells were treated with either vehicle (ethanol) or 1,25(OH)₂D3 (10 nM) for 0, 6, 12, 24h. The expression of CYP27A1 was determined by Western-blot.